Suppression of neurodegeneration by zinc transporter protein 7

Abstract

Methods and compositions are provided for enhancing endoplasmic reticulum-associated degradation (ERAD) in cells and suppressing pathological accumulation of misfolded proteins by increasing the expression or activity of zinc transporter protein 7 (ZIP7). Methods of treating a subject for a disorder associated with protein misfolding are also provided, including gene therapy methods for expressing ZIP7 in vivo in an effective amount sufficient to suppress pathological accumulation of misfolded proteins.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of Provisional Application No. 63 / 243,590, filed September 13, 2021, which is incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grant No. R01 GM046425 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] INCORPORATION BY REFERENCE TO SEQUENCE LISTING The Sequence Listing is provided herewith as Sequence Listing XML file "UCSB-546WO_2022-758," created on September 9, 2022, and having a size of 8,811 bytes. The contents of the Sequence Listing XML file are incorporated herein by reference in their entirety.

[0004] Introduction Collective cell migration has emerged as a major driver of normal organ development, wound repair, and tumor metastasis (Mishra et al., Development 146, (2019); Friedl & Mayor, Cold Spring Harb. Perspect. Biol. 9, (2017); Friedl & Gilmour, Nat. Rev. Mol. Cell Biol. 10, 445-457 (2009); Friedl et al., Nat. Cell Biol. 14, 777-783 (2012)). Border cell migration in the Drosophila ovary provides a powerful in vivo model of collective cell migration that can be subjected to unbiased genetic screens. The Drosophila ovary is composed of ovarioles, connected egg chambers that progress through 14 stages of development into mature oocytes (Figure 1A). Each egg chamber is composed of 16 germ cells, including 15 nurse cells and one oocyte, surrounded by epithelial follicular cells. During stage 9 (Figure 1B), 4–8 border cells are identified at the anterior end of the egg chamber, detach from the follicular epithelium, and migrate posteriorly during developmental stage 9, reaching the anterior border of the oocyte by stage 10.

[0005] Genetic screens have revealed the molecular mechanisms that determine which of the approximately 850 follicular cells acquire the ability to migrate (Silver & Montell, Cell 107, 831-841 (2001); Bai & Montell, Development 129, 5377-5388 (2002)), the occurrence timing of their migration (Jang et al., Nat. Cell Biol. 11, 569-579 (2009); Bai & Montell, Cell 103, 1047-1058 (2000)), and direction sensing (McDonald et al., Development 130, 3469-3478 (2003); McDonald et al., Dev. Biol. 296, 94-103 (2006); Dai et al., Science 370(6519), 987-990(2020)), and cytoskeletal dynamics (McDonald et al. (2003), above, Murphy & Montell, J. Cell Biol. 133, 617-630 (1996), Kim et al., Genes Dev. 25, 730-741 (2011), Lee et al., Development 122,409-418(1996), Cai et al.,Cell 157,1146-1159(2014), McDonald,Curr.Biol.18,1659-1667(2008), Wang et al.,Nat.Cell Biol.12,591-597(2010), Duchek&Rorth,Science 291, 131-133 (2001); Fulga & Rorth, Nat. Cell Biol. 4, 715-719 (2002); Ramel, et al., Nat. Cell Biol. 15, 317-324 (2013); Assaker, et al., Proc Natl Acad Sci USA 107, 22558-22563 (2010)).In addition to the thermal conductivity, the thermodynamic properties of the particles have been studied (Dai et al., while Miao et al al.,Dev.Dev.Cell 54,501-515.e9(2020);Columbia et al.,Dev.Biol.423,12-18(2017);Chen et al.,elife 9,e52979(2020); Biol.198,57-67(2012);Ogienko et al.,Int.J.Mol.Sci.21,(2020);Berez et al.,Front.Physiol.11,803(2020);Wang et al.,iScience 23,101335(2020)、Wang, H., Guo, X., Wang, X., Wang, X. & Chen, J. Supracellular actomyosin mediates cell-cell communication and shapes collective migratory morphology.iScience 23,101204(2020), Fox et al.,Mol.Biol.Cell 31,1584-1594(2020);Plutoni et al.,Nat.Commun.10,3940(2019);Zeledon et al.,Cell Rep.28,3238-3248.e3(2019) Lamb et al al.,Dev.Dyn.249,961-982(2020)、Ghiglione,C.,Jouandin,P.,Cerezo,D.& Noselli,S.The Drosophila insulin pathway controls profilin expression and dynamic actin-rich protrusions during collective cell migration.Development 145,dev161117(2018);Sharma et al.,Development 145,(2018)).The gene Catsup was identified in both a large-scale ethyl methanesulfonate mutagenesis screen for defects in border cell migration in mosaic clones (Liu & Montell, Development 126, 1869-1878 (1999)) and in a genome-wide expression profile of border cells (Wang et al., Dev. Cell 10, 483-495 (2006)).

[0006] The name Catsup stands for "Catecholamines up," and its loss increases the synthesis of aromatic amines, including neurotransmitters such as epinephrine and dopamine (Stathakis et al., Genetics 153, 361-382 (1999)). Catsup is required for Drosophila tracheal morphogenesis, where it directly binds to and inhibits the Drosophila homolog of tyrosine hydroxylase Ple to limit dopamine synthesis (Hsouna et al., Dev. Biol. 308, 30-43 (2007)). In contrast, in wing imaginal disc cells, Catsup regulates the abundance and localization of Notch and EGFR (Groth et al., Development 140, 3018-3027 (2013)).

[0007] Catsup is Zn 2+ It shares 62% similarity and 53% identity with its mammalian homolog zinc transporter protein 7 (ZIP7), also known as SLC39A7 or HKE4 (Groth et al., supra), which is a member of one of the two major families of transporters (Kambe, Zinc Transport: Regulation in Encyclopedia of inorganic and bioinorganic chemistry (ed. Scott, RA) 1-9 (John Wiley & Sons, Ltd, 2011). ZIP7 is located within the endomembrane system, including the ER, and transports Zn 2+ (Taylor et al., Biochem. J. 377, 131-139 (2004)). Zn 2+Zn is a trace element essential for the functioning of many proteins. 2+ homeostasis of 24 Zn in humans 2+ Carefully maintained by the transporter, 14 of which are ZIP 43 ZIP7 is conserved throughout eukaryotes, and its loss causes ER stress in organisms as diverse as yeast, plants, and animals (Nguyen et al., Biosci. Biotechnol. Biochem. 77, 1337-1339 (2013); Tan et al., Transgenic Res. 24, 109-122 (2015); Zhang et al., Int. J. Mol. Sci. 15, 20413-20433 (2014); Adulcikas et al., Comput. Biol. Med. 100, 196-202 (2018); Tuncay et al., Diabetes 66, 1346-1358 (2017); Fauster et al., Cell Death Differ. 26, 1138-1155 (2019)). However, ER stress, Zn 2+ The relationships between trafficking, localization and activity of Notch and EGFR, and cell motility remain to be defined. Summary of the Invention

[0008] Methods and compositions are provided for enhancing endoplasmic reticulum-associated degradation (ERAD) in cells and suppressing pathological accumulation of misfolded proteins by increasing the expression or activity of ZIP7. Methods of treating a subject for a disorder associated with protein misfolding are also provided, including gene therapy methods for expressing ZIP7 in vivo in an effective amount sufficient to suppress pathological accumulation of misfolded proteins.

[0009] In one aspect, a method of treating a subject for a disorder associated with protein misfolding is provided, the method comprising administering a therapeutically effective amount of zinc transporter protein 7 (ZIP7) to the subject.

[0010] Disorders associated with protein misfolding that may be treated by the methods described herein include any condition that results in protein misfolding and / or protein aggregation, where misfolded proteins and / or protein aggregates accumulate in the endoplasmic reticulum (ER), causing ER stress and inhibiting ER-associated degradation (ERAD). Disorders associated with protein misfolding include, but are not limited to, retinitis pigmentosa, and neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), also known as Kennedy's disease, multiple system atrophy, and spinocerebellar ataxia (SCA).

[0011] In certain embodiments, the ZIP7 protein comprises an amino acid sequence having at least about 80-100% sequence identity to the sequence of SEQ ID NO:5, including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity.

[0012] In certain embodiments, the ZIP7 protein is administered locally to the eye or brain of a subject, hi some embodiments, the ZIP7 protein is administered locally to the photoreceptors of the eye.

[0013] In certain embodiments, the ZIP7 protein is administered according to a daily dosing regimen or intermittently.

[0014] In certain embodiments, the ZIP7 protein is provided by an expression vector, such as a viral vector.

[0015] In certain embodiments, the misfolded protein is variant rhodopsin (e.g., Rh1G69D), Vap33, or amyloid b42.

[0016] In another aspect, a method of providing ZIP7 to a subject to inhibit pathological accumulation of misfolded proteins is provided, the method comprising introducing into a cell an expression vector comprising a promoter operably linked to a coding sequence encoding ZIP7, wherein the cell expresses ZIP in vivo in the subject in an effective amount sufficient to inhibit pathological accumulation of misfolded proteins in the cell.

[0017] In certain embodiments, the cell is a retinal cell or a brain cell, hi some embodiments, the retinal cell is a photoreceptor cell.

[0018] In certain embodiments, the misfolded protein is a misfolded rhodopsin protein.

[0019] In certain embodiments, the expression vector is introduced into cells ex vivo or in vivo.

[0020] In another aspect, a method of treating a subject for a disorder associated with protein misfolding is provided, the method comprising administering to the subject an expression vector comprising a promoter operably linked to a nucleotide sequence encoding ZIP7, wherein ZIP7 is expressed in vivo in the subject in a therapeutically effective amount sufficient to inhibit pathological accumulation of misfolded proteins.

[0021] In another aspect, a method of enhancing endoplasmic reticulum (ER)-associated degradation (ERAD) or proteosome-associated degradation in a cell is provided, the method comprising increasing expression or activity of ZIP7 in the cell.

[0022] In certain embodiments, the cell is a retinal cell, hi some embodiments, the retinal cell is a photoreceptor cell.

[0023] In certain embodiments, expression of ZIP7 is increased sufficiently to increase the degradation of misfolded rhodopsin protein and inhibit the accumulation of misfolded rhodopsin protein in retinal cells.

[0024] In certain embodiments, increasing the expression of ZIP7 comprises transfecting a cell with a recombinant polynucleotide comprising a coding sequence encoding ZIP7. In some embodiments, the recombinant polynucleotide further comprises a viral vector comprising a promoter operably linked to the coding sequence encoding ZIP7. In some embodiments, the coding sequence encoding ZIP7 is integrated into a chromosomal locus of the transfected cell, and an endogenous promoter is operably linked to the integrated coding sequence encoding ZIP7 at the chromosomal locus.

[0025] In another aspect, a method is provided for inhibiting accumulation of misfolded proteins in an organ, cell, or tissue of a subject, the method comprising increasing expression or activity of the zinc transporter ZIP7 in the organ, cell, or tissue.

[0026] In certain embodiments, the organ is the eye or the brain.

[0027] In certain embodiments, the tissue is neural tissue.

[0028] In certain embodiments, the tissue is retinal tissue.

[0029] In certain embodiments, the cell is a retinal cell, hi some embodiments, the retinal cell is a photoreceptor cell.

[0030] In certain embodiments, the misfolded protein is a rhodopsin protein.

[0031] In certain embodiments, the subject has retinitis pigmentosa.

[0032] In certain embodiments, the disorder associated with protein misfolding is retinitis pigmentosa, Huntington's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, or frontotemporal dementia.

[0033] In certain embodiments, expression of ZIP7 is increased sufficiently to reduce the pathological accumulation of misfolded proteins and increase cell survival.

[0034] In certain embodiments, expression of ZIP7 is increased by providing an organ, cell, or tissue with a recombinant polynucleotide comprising a coding sequence encoding ZIP7, such that ZIP7 is expressed in the organ, cell, or tissue.

[0035] In certain embodiments, the recombinant polynucleotide further comprises a viral vector comprising a promoter operably linked to a coding sequence encoding ZIP7.

[0036] In certain embodiments, the coding sequence encoding ZIP7 is integrated into a chromosomal locus. In some embodiments, the endogenous promoter is operably linked to the integrated coding sequence encoding ZIP7 at the chromosomal locus.

[0037] In another aspect, a composition is provided for use in a method for treating a disorder associated with protein misfolding, the composition comprising ZIP7 or an expression vector comprising a promoter operably linked to a coding sequence encoding ZIP7. In some embodiments, the composition further comprises a pharma- ceutically acceptable excipient. [Brief description of the drawings]

[0038] [Figure 1A]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1B]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1C]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1D]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1E]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1F]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1G]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1H]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1I]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1J]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1K]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1L]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1M]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1N]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1O]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1P]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1Q]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1R]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1S]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 1T]Catsup expression and subcellular localization in Drosophila ovaries. (Figures 1A, 1B) Egg chambers developed from germarium to developmental stage 10 were expressed Catsup::GFP and stained for DNA with Hoechst (blue) and F-actin with phalloidin (magenta). Border cells (white arrowheads) migrate during stage 9 (Figure 1B) and complete their migration by stage 10 (Figure 1A). (A', B') Single Catsup::GFP channel (grayscale). (Figures 1C-1G) Higher magnification of a border cell cluster showing overexpressed CatsupV5 (yellow), anti-PDI staining for ER (green), phalloidin (magenta), and Hoechst (blue) localization. (Figures 1H-1J) 2D intensity histograms of two selected channels showing colocalization of CatsupV5 to ER, F-actin, and DNA. The regression Pearson coefficients of colocalization are displayed in the upper right corner. (Fig. 1K) Comparison of Pearson coefficients (average of four border cell clusters). **P value < 0.01. (Fig. 1L-1P) Higher magnification of border cell clusters showing the localization of tagged Catsup::GFP expressed under endogenous regulatory sequences (green), ER (PDI, yellow), F-actin (phalloidin, magenta), and nuclear DNA (Hoechst, blue). (Fig. 1Q-1T) Two-dimensional intensity histograms of two selected channels showing the colocalization and Pearson coefficients of Catsup::GFP relative to ER, F-actin, nucleus, and ER relative to F-actin. Scale bar, 20 μm. [Figure 2A]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2B]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2C]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2D]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2E]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2F]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2G]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2H]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2I]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 2J]Border cells require Catsup for normal migration. (Figure 2A, 2B) Confocal micrographs of stage 10 egg chambers in which fruitlessGal4 drives expression UAS-Catsup RNAi and (Figure 2A) UAS-GFPnls or (Figure 2B) UAS-CatsupV5 in the outer migratory border cells. (Figure 2C-2D) Expression of Catsup::GFP in control border cells (Figure 2C) or knocked down by c306Gal4>CatsupRNAi (Figure 2D). (Figure 2E) Quantification of defective migration at stage 10 in fruitlessGal4 (blue) and c306Gal4 (magenta) driving the indicated transgenes. Experiments were repeated three times independently. (Figure 2F) Egg chamber with GFP-negative (homozygous Catsup mutant) cells. Both the polar cell (p) and two border cells (b) are mutant. (Fig. 2G) Egg chambers in which all outer border cells are GFP negative (homozygous Catsup mutant). (Fig. 2H) Migration distance expressed as a percentage of the migration path of the mosaic border cell clusters as a function of the proportion of homozygous mutant cells in each cluster. (Fig. 2I) Higher magnification view showing the spatial distribution of Catsup+ (GFP+) and Catsup- / - (GFP- / -) cells in the migrating clusters. (Fig. 2J) Quantification of the percentage of Catsup+ vs. Catsup- / - border cells at the front, side, or back of the border cell cluster, showing that Catsup- / - cells are more likely to occupy a posterior position. "p" indicates polar cells, "b" indicates border cells, green labels control cells, yellow labels mutant cells. **P≦0.01, ***P≦0.001, ****P≦0.0001. Scale bars, 20 μm. [Figure 3A]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3B]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3C]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3D]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3E]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3F]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3G]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3H]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 3I]Changes in the abundance and localization of Notch and EGFR in cells expressing CatsupRNAi. (Figure 3A, 3B) Differential interference contrast images of stage 10 egg chambers stained for Ple (magenta) in w1118 control (Figure 3A) or egg chambers expressing UAS-Ple with c306Gal4 (Figure 3B). (Figure 3C) Dissected fly brains stained for endogenous Ple: dopaminergic neurons are Ple positive. (Figure 3D-3E) CatsupRNAi-expressing clones (GFP positive, green) accumulate intracellular Notch protein in epithelial follicular cells (Figure 3D) and border cells (Figure 3E) relative to adjacent wild-type cells. (Figure 3F) CatsupRNAi-expressing border cells (GFP positive, green) show reduced Notch signaling indicated by Notch-responsive elements driving RFP (white) relative to adjacent wild-type cells. (Figures 3G-3H) Accumulation of EGFR (magenta) in CatsupRNAi-expressing border cells (Figure 3G) and epithelial follicular cells (Figure 3H). (Figure 3I) c306Gal4>CatsupRNAi reduces Catsup::GFP expression but does not cause intracellular accumulation of E-cadherin (magenta). Scale bar, 20 μm. [Figure 4A]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4B]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4C]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4D]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4E]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4F]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4G]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4H]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4I]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4J]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4K]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 4L]ER stress in Catsup mutant border cells. (Figure 4A) Mosaic border cell cluster consisting of a mixture of control cells (RFP positive, magenta, which could be Catsup+ / + or Catsup- / +) and homozygous Catsup mutant cells (RFP negative, outlined). Polar cells (p) express higher levels of RFP compared to outer border cells. Xbp1::EGFP (green), a marker of ER stress. (Figure 4B) Mosaic follicle cell clones expressing CatsupRNAi and GFPnls caused ER expansion indicated by PDI (magenta). (Figure 4C) Mosaic clones expressing CatsupRNAi and CatsupV5 and RFP (magenta). (Figure 4D) Expression of the misfolded rhodopsin protein Rh1G69D (magenta) in border cells induced ER stress (Xbp1::EGFP in green) and blocked border cell migration. (Figure 4E) Co-expression of CatsupV5 reduced Rh1G69D protein levels (magenta) and Xbp1::EGFP and rescued migration. (Figure 4F) Percentage of border cells expressing RH1G69D that are Xbp1 positive in the absence (blue dots) or presence (pink dots) of CatsupV5 rescue. mIFR is a control unrelated fluorescent protein. (Figure 4G) CatsupV5 rescue of the RH1G69D migration defect. (Fig. 4H-4L) Mosaic clones expressing RH1G69D, GFP, show comparable Notch intensity in epithelial clones (Fig. 4H) and border cell clones (I, I'), and EGFR intensity in epithelial clones (Fig. 4J) and border cell clones (Fig. 4K), a Notch-responsive element reporter, show Notch transcriptional activity (white) in wild-type but not in border cells expressing Catsup RNAi (Fig. 4L). **P ≤ 0.01, ****P ≤ 0.0001. Scale bar, 20 μm. [Figure 5A]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5B] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5C]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5D] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5E]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5F] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5G]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5H] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5I]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5J] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5K]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5L] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5M]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5N] Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 5O]Point mutations suggest a requirement for Zn2+ transport in ER homeostasis and cell motility. (Fig. 5A) Schematic of Catsup transmembrane domains and topology. Point mutations H183A and H187A reside within the second transmembrane domain, whereas H315A and H344A reside within the HELP domain required for Zn2+ transport. (Fig. 5B-5E) Expression and colocalization of V5-tagged RNAo-resistant Catsup mutants with the ER marker PDI (green) in border cells. (Fig. 5F) Quantification of defective migration at stage 10 in egg chambers expressing CatsupRNAi together with the indicated mutants. Experiments were repeated three times independently. (Fig. 5G-5N) Mosaic expression of CatsupRNAi together with the indicated mutants of Catsup marked by RFPnls (magenta) and stained green for Notch or EGFR, as indicated. Scale bar, 20 μm. (Figure 5O) Model of Catsup / ZIP7 function. [Figure 6A] ZIP7 prevents Rh1G69D-induced photoreceptor cell death leading to rough eye. (FIG. 6A) Percentage of flies with the Rh1G69D genotype that develop rough eye in the presence and absence of ZIP7 expression. (FIG. 6B) Comparison of eye morphology of flies with the Rh1G69D genotype in the presence and absence of ZIP7 expression. [Figure 6B] ZIP7 prevents Rh1G69D-induced photoreceptor cell death leading to rough eye. (FIG. 6A) Percentage of flies with the Rh1G69D genotype that develop rough eye in the presence and absence of ZIP7 expression. (FIG. 6B) Comparison of eye morphology of flies with the Rh1G69D genotype in the presence and absence of ZIP7 expression. [Figure 7] Overexpression of ZIP7 alleviates ER stress and neuronal cell death in protein aggregation diseases. [Figure 8] Screening of aggregate-prone proteins for Zip7 rescue. [Figure 9] Overexpression of ZIP7 suppresses neurodegeneration caused by Vap33 expression. [Figure 10] Overexpression of ZIP7 suppresses amyloid b42-induced neurodegeneration. [Figure 11] Schematic showing the role of Zn2+. Ubiquitin ligases are RING finger domain proteins that require Zn2+. Many protein components of the proteasome require Zn2+. Overexpression of ZIP7 enhances proteasome activity even in the presence of proteasome inhibitors. [Figure 12A] Overexpression of ZIP7 reduces ubiquitinated protein levels. (Figure 12A) Egg chambers with and without ZIP7 overexpression (OE) were incubated with medium for 5 hours. (Figure 12B) Egg chambers with and without ZIP7 overexpression (OE) were treated with 10 mM MG132 proteasome inhibitor for 5 hours. (Figure 12C) Comparison of FK2 / DNA ratios of egg chambers with or without ZIP7 overexpression and incubated with medium or 10 mM MG132 proteasome inhibitor for 5 hours. [Figure 12B] Overexpression of ZIP7 reduces ubiquitinated protein levels. (Figure 12A) Egg chambers with and without ZIP7 overexpression (OE) were incubated with medium for 5 hours. (Figure 12B) Egg chambers with and without ZIP7 overexpression (OE) were treated with 10 mM MG132 proteasome inhibitor for 5 hours. (Figure 12C) Comparison of FK2 / DNA ratios of egg chambers with or without ZIP7 overexpression and incubated with medium or 10 mM MG132 proteasome inhibitor for 5 hours. [Figure 12C] Overexpression of ZIP7 reduces ubiquitinated protein levels. (Figure 12A) Egg chambers with and without ZIP7 overexpression (OE) were incubated with medium for 5 hours. (Figure 12B) Egg chambers with and without ZIP7 overexpression (OE) were treated with 10 mM MG132 proteasome inhibitor for 5 hours. (Figure 12C) Comparison of FK2 / DNA ratios of egg chambers with or without ZIP7 overexpression and incubated with medium or 10 mM MG132 proteasome inhibitor for 5 hours. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Methods and compositions are provided for enhancing endoplasmic reticulum-associated degradation (ERAD) in cells and suppressing pathological accumulation of misfolded proteins by increasing the expression or activity of zinc transporter protein 7 (ZIP7). Methods of treating a subject for a disorder associated with protein misfolding are also provided, including gene therapy methods for expressing ZIP7 in vivo in an effective amount sufficient to suppress pathological accumulation of misfolded proteins.

[0040] Before describing exemplary embodiments of the invention, it is to be understood that the invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0041] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limits of that range is also specifically disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated value or intervening value within a stated range and any other stated value or intervening value within that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may be independently included or excluded in the range, and each range in which either, neither, or both limits are included in the smaller range is also encompassed within the invention, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are likewise included in the invention.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and exemplary methods and materials can be described here. Any and all publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials for which the publications are cited. In case of conflict, it should be understood that the present disclosure supersedes any disclosure of the incorporated publication.

[0043] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of such proteins, and reference to "the protein" includes a reference to one or more peptides and equivalents thereof known to those skilled in the art, e.g., polypeptides and peptides.

[0044] It is further noted that the claims may be drafted to exclude any element that may be optional. Accordingly, this specification is intended to serve as a predicate basis for using exclusive terminology, such as "solely," "only," or "negative" limitations in connection with the recitation of claim elements.

[0045] Publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Further, the dates of publications provided may be different from the actual publication dates, which may need to be independently confirmed. To the extent that such publications may provide definitions of terms that contradict the express or implied definitions of the present disclosure, the definitions of the present disclosure shall control.

[0046] As will be apparent to those skilled in the art upon reading this disclosure, each of the separate embodiments described and illustrated herein has separate components and features which may be readily separated from or combined with the features of any of the other various embodiments without departing from the scope or spirit of the invention. Any recited method may be carried out in the order of events recited or in any other order which is logically possible.

[0047] definition The term "protein misfolding-associated disorder" is used herein to refer to any condition that results in protein misfolding and / or protein aggregation, where misfolded proteins and / or protein aggregates accumulate in the endoplasmic reticulum (ER), causing ER stress and inhibiting ER-associated degradation (ERAD). Misfolded protein aggregation can further cause cellular dysfunction, loss of synaptic connections, neuronal death, eye damage, and / or brain damage. Protein misfolding-associated disorders include, but are not limited to, retinitis pigmentosa, and neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), also known as Kennedy's disease, multiple system atrophy, and spinocerebellar ataxia (SCA).

[0048] The terms "treatment", "treating", "treating" and the like are used herein generally to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventative in that it completely or partially prevents the disease or its symptom(s) and / or may be therapeutic in that it partially or completely stabilizes or cures the disease and / or adverse effects caused by the disease. The term "treatment" encompasses any treatment of disease in a mammal, particularly a human, and includes (a) preventing the onset of the disease and / or condition in a subject who may have a predisposition to the disease or condition(s) but has not yet been diagnosed as having the disease or condition, (b) inhibiting the disease and / or condition(s), i.e., arresting its onset, or (c) alleviating the disease symptom(s), i.e., causing regression of the disease and / or condition(s). Those in need of treatment include those already affected (e.g., those having a protein misfolding associated disorder) as well as those in which prevention is desired (e.g., those having an increased susceptibility to or genetic predisposition to developing a protein misfolding associated disorder).

[0049] A therapeutic treatment is one that the subject is administered prior to administration, and a prophylactic treatment is one that the subject is suffering from prior to administration. In some embodiments, the subject has an increased likelihood of suffering or is suspected of suffering prior to the treatment. In some embodiments, the subject is suspected of having an increased likelihood of suffering.

[0050] The term "subject" as used herein refers to a patient in need of the treatment disclosed herein. The patient may be a mammal, such as a rodent, cat, dog, primate, or human, e.g., an adult, such as a child, adolescent, young, middle-aged, or elderly human. The patient may have been diagnosed with a protein misfolding-related disorder or may be suspected of suffering from a protein misfolding-related disorder.

[0051] A "pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in a composition of the present invention and that causes no significant adverse toxicological effects to a patient.

[0052] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salt, salt prepared with inorganic acid, such as chloride, sulfate, phosphate, diphosphate, bromide and nitrate, or salt prepared from any of the above corresponding inorganic acid forms, such as hydrochloride, or salt prepared with organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, paratoluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate.Similarly, salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium (including substituted ammonium).

[0053] As used herein, the term "survival" refers to the time from the start of treatment to death.

[0054] A "therapeutically effective dose or amount" of a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein, as described herein, contemplates an amount that, when administered, results in a positive therapeutic response, such as improved recovery from disorders associated with protein misfolding. Improved recovery may include improved ERAD function resulting in increased degradation of misfolded proteins and reduced formation of protein aggregates, restored neurological function, improved cognition, improved memory, and / or increased survival. In the case of retinitis pigmentosa, a therapeutically effective dose or amount of a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein may reduce the accumulation of misfolded rhodopsin and rhodopsin protein aggregates in the ER and photoreceptors in the retina of the eye, reduce ER stress, improve survival of photoreceptor cells, and prevent or delay eye damage and vision loss. In the case of amyloid beta aggregation-related diseases (e.g., Alzheimer's disease), a therapeutically effective dose or amount of a ZIP7 protein may reduce amyloid beta aggregation and reduce the formation of amyloid plaques in the brain. In addition, the therapeutically effective dose or amount can slow the loss of cerebellar Purkinje neurons and the loss of brain cells.In the case of diseases related to the aggregation of alpha-synuclein or synucleinopathies (e.g., Parkinson's disease), the therapeutically effective dose or amount of ZIP protein can reduce the aggregation of alpha-synuclein.In addition, the therapeutically effective dose or amount can reduce the accumulation of alpha-synuclein aggregates in neurons, nerve fibers, and / or glial cells, and reduce the formation of Lewy bodies.

[0055] The terms "peptide", "oligopeptide", and "polypeptide" refer to any compound that contains naturally occurring or synthetic amino acid polymers or amino acid-like molecules, including, but not limited to, compounds that contain amino and / or imino molecules. No particular size is implied by the use of the terms "peptide", "oligopeptide", or "polypeptide", and these terms are used interchangeably. Included within this definition are, for example, polypeptides that contain one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, and other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic). Thus, synthetic oligopeptides, dimers, multimers (e.g., tandem repeats, linearly linked peptides), cyclized, branched molecules, and the like. The term also includes molecules that contain one or more peptoids (e.g., N-substituted glycine residues) and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005, 5,877,278, and 5,977,301, Nguyen et al. (2000) Chem Biol. 7(7):463-473, and Simon et al. (1992) Proc. Natl. Acad. Sci. USA 89(20):9367-9371 for a description of peptides.) Non-limiting lengths of peptides suitable for use in the present invention include peptides 3-5 residues long, 6-10 residues long (or any integer in between), 11-20 residues long (or any integer in between), 21-75 residues long (or any integer in between), 75-100 residues long (or any integer in between), or polypeptides greater than 100 residues long. Typically, polypeptides useful in the present invention can have a maximum length suitable for the intended application. Preferably, the polypeptide is about 3 to 100 residues in length. Generally, one of skill in the art can readily select a maximum length in view of the teachings herein. Additionally, the peptides and polypeptides described herein, e.g., synthetic peptides, can include additional molecules such as labels or other chemical moieties.

[0056] Thus, reference to a polypeptide or peptide also includes derivatives of the amino acid sequences of the invention that contain one or more non-naturally occurring amino acids. A first polypeptide or peptide is "derived from" a second polypeptide or peptide if it (i) is encoded by a first polynucleotide that is derived from a second polynucleotide encoding the second polypeptide or peptide, or (ii) exhibits sequence identity to the second polypeptide or peptide described herein. Sequence (or percent) identity may be determined as described below. Preferably, derivatives exhibit at least about 50% identity to the sequences from which they are derived, more preferably at least about 80%, even more preferably about 85%-99% (or any value therebetween). Such derivatives include post-expression modifications of the polypeptide or peptide, such as glycosylation, acetylation, phosphorylation, and the like.

[0057] Amino acid derivatives may also include modifications to the native sequence, such as deletions, additions and substitutions (generally conservative in nature), so long as the protein (or fragment thereof) maintains the desired activity (e.g., biological activity of ZIP7, ability to improve ERAD function, increase degradation of misfolded proteins, reduce aggregation of misfolded proteins, and / or reduce ER stress). These modifications may be deliberate, via site-directed mutagenesis, or accidental, via errors resulting from mutations of the host producing the protein or PCR amplification. Additionally, modifications may be made that have one or more of the following effects: enhancing ERAD, increasing degradation of misfolded proteins, and / or increasing the ability to inhibit protein aggregation, or facilitating purification, delivery, or cell processing. The protein or biologically active fragments thereof may be produced recombinantly, synthetically, or in tissue culture.

[0058] As used herein, the term "zinc transporter protein 7" or "ZIP7" encompasses all forms of ZIP7, as well as biologically active fragments, variants, analogs, and derivatives thereof that retain biological activity (e.g., the ability to improve ERAD function, increase degradation of misfolded proteins, and / or inhibit protein aggregation).

[0059] ZIP7 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule does not have to be physically derived from an organism, but can be synthetically or recombinantly produced. Many ZIP7 nucleic acid and protein sequences are known. A representative sequence of ZIP7 protein from Drosophila melanogaster is presented in SEQ ID NO: 4, and a representative sequence of human ZIP7 protein is presented in SEQ ID NO: 5. Additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, the following NCBI entries: Accession Nos. NM_001077516, NM_006979, NM_001288777, NM_130931, XM_015449564, XM_015449563, XM_005553337, XM_005553336, XM_040983171, XM_040983170, XM_040983169, XM_011976665, XM_011976664, XM_011976663, XM_017522703, XM_017522702. , XM_017522700, XM_017522701, XM_021185695, XM_021185696, NM_001048100, XM_038682747, XM_038682746, XM_036766199, XM_036766198, XM_036766197, AQY77122, AQY77121, NP_001070984, NP_008910, and NP_001275706, all of which sequences (entered by the filing date of this application) are incorporated herein by reference. Any of these sequences or variants thereof (including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto) can be used to produce ZIP7 proteins, or recombinant polynucleotides comprising coding sequences encoding ZIP7 proteins, for use in the methods described herein.

[0060] By "fragment" is intended a molecule consisting of only a portion of the intact full-length sequence and structure. Fragments may include C-terminal deletions, N-terminal deletions, and / or internal deletions of the polypeptide. An active fragment of a particular protein or polypeptide will generally contain at least about 5-14 contiguous amino acid residues of the full-length molecule, but may contain at least about 15-25 contiguous amino acid residues of the full-length molecule, at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity (e.g., ZIP7 biological activity, the ability to enhance ERAD, increase degradation of misfolded proteins, inhibit protein aggregation, and / or reduce ER stress).

[0061] "Substantially purified" generally refers to the isolation of a substance (compound, polynucleotide, protein, polypeptide, peptide composition) such that the substance constitutes the majority percentage of the sample in which it is present. Typically, the substantially purified component of a sample comprises 50%, preferably 80%-85%, and more preferably 90%-95% of the sample. Techniques for purifying polynucleotides and polypeptides of interest are well known in the art and include, for example, ion exchange chromatography, affinity chromatography, and sedimentation by density.

[0062] "Isolated," when referring to a protein, polypeptide, or peptide, means that the indicated molecule is separated and separate from the whole organism in which it is found in nature, or exists in the substantial absence of other biological macromolecules of the same type. With reference to polynucleotides, the term "isolated" refers to a nucleic acid molecule that lacks all or part of a sequence that is normally associated with it in nature, or a sequence that is naturally occurring but has heterologous sequences associated with it, or a molecule that is dissociated from the chromosome.

[0063] The term "derived from" is used herein to identify the original source of a molecule, but is not intended to limit the manner in which the molecule is made, which may be done, for example, by chemical synthesis or recombinant means.

[0064] The terms "variant," "analog," and "mutein" refer to biologically active derivatives of a reference molecule that retain a desired activity, such as the ability to improve ZIP7 activity, ERAD function, increase degradation of misfolded proteins, inhibit pathological accumulation of misfolded proteins in cells, and / or reduce ER stress, for use in the treatment of disorders associated with protein misfolding aggregation, as described herein. Generally, the terms "variant" and "analog" refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature), and / or deletions relative to the native molecule, and are "substantially homologous" to the reference molecule, as defined below, so long as the modifications do not destroy the biological activity. Generally, the amino acid sequence of such analogs will have a high degree of sequence homology to the reference sequence, e.g., greater than 50%, generally greater than 60%-70%, even more particularly 80%-85% or more, at least 90%-95% or more amino acid sequence homology, when the two sequences are aligned. Often, analogs contain the same number of amino acids as described herein, but contain substitutions. The term "mutein" further includes compounds containing only amino and / or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, and other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules, etc. The term also includes molecules containing one or more N-substituted glycine residues ("peptoids") and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005, 5,877,278, and 5,977,301, Nguyen et al., Chem Biol. (2000) 7:463-473, and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371, for a description of peptides.) Preferably, the analog or mutein has at least the same biological activity as the native molecule.Methods for making polypeptide analogs and muteins are known in the art and are further described below.

[0065] As explained above, analogs generally include substitutions that are conservative in nature, i.e., substitutions that occur within a family of amino acids that are related in their side chains. Specifically, amino acids are generally classified into four families: (1) acidic-aspartic acid and glutamic acid, (2) basic-lysine, arginine, histidine, (3) non-polar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine. Phenylalanine, tryptophan, and tyrosine may be classified as aromatic amino acids. For example, a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or a similar conservative substitution of an amino acid with a structurally related amino acid can be reasonably expected not to significantly affect biological activity. For example, a polypeptide of interest can contain up to about 5-10 conservative or non-conservative amino acid substitutions, or up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5 and 25, so long as the desired function of the molecule is intact. One of skill in the art can readily determine regions of a molecule of interest that can tolerate change by reference to Hopp / Woods plots and Kyte-Doolittle plots, which are well known in the art.

[0066] By "derivative" is intended any suitable modification of a naturally occurring polypeptide of interest, a fragment of a naturally occurring polypeptide, or their respective analogs such as glycosylation, phosphorylation, polymer conjugation (such as polyethylene glycol), or other addition of foreign moieties, so long as the desired biological activity of the naturally occurring polypeptide is retained. Methods for making polypeptide fragments, analogs, and derivatives are generally available in the art.

[0067] "Homology" refers to the percent identity between two polynucleotides or two polypeptide molecules. Two nucleic acid or two polypeptide sequences are "substantially homologous" to each other if the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences that exhibit complete identity to a specified sequence.

[0068] In general, "identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence, respectively, of two polynucleotide or polypeptide sequences. Percent identity can be determined by directly comparing the sequence information between the two molecules by aligning the sequences, counting the number of exact matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. For peptide analysis, readily available computer programs can be used to assist in the analysis, such as ALIGN, Dayhoff, MO, in Atlas of Protein Sequence and Structure MO Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which applies the local homology algorithm of Smith and Waterman Advances, Appl. Math. 2:482 489, 1981. Programs for determining nucleotide sequence identity, such as the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm, are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, WI).These programs are easily used with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package mentioned above.For example, the percent identity of a particular nucleotide sequence to a reference sequence can be determined using the Smith and Waterman homology algorithm with a default scoring table and a gap penalty of 6 nucleotide positions.

[0069] Another method for establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this set of packages, the Smith-Waterman algorithm can be used with default parameters (e.g., gap opening penalty of 12, gap extension penalty of 1, and gap of 6) used in the scoring table. From the data generated, the "match" value reflects the "sequence identity". Other programs suitable for calculating percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST used with default parameters. For example, BLASTN and BLASTP can be used with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; description=50 sequences; sort=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+Swiss Protein+Spupdate+PIR. Details of these programs are readily available.

[0070] Alternatively, homology can be determined by hybridization of polynucleotides under conditions that form stable double strands between homologous regions, followed by digestion with single-strand specific nuclease(s) and sizing of digested fragments. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, for example, Sambrook et al. (supra); DNA Cloning (supra); Nucleic Acid Hybridization (supra).

[0071] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin that is not associated by its origin or manipulation with all or a portion of a polynucleotide with which it is naturally associated. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. Generally, a gene of interest is cloned and then expressed in a transformed organism, as further described below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0072] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, regardless of the method used for the insertion, including, for example, direct uptake, transduction, or f-mating. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0073] "Recombinant host cell," "host cell," "cell," "cell line," "cell culture," and other such terms referring to microorganisms or higher eukaryotic cell lines cultured as unicellular entities, refer to cells that can be, or have been, used as recipients of recombinant vectors or other transferred DNA, including the original progeny of the original cell that was transfected.

[0074] A "coding sequence" or a sequence "encoding" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. Coding sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence can be located 3' to the coding sequence.

[0075] Exemplary "control elements" include, but are not limited to, transcriptional promoters, transcriptional enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), translation initiation optimization sequences (located 5' to the coding sequence), and translation termination sequences.

[0076] "Operably linked" refers to an arrangement of elements such that the components so described are configured to perform their normal functions. Thus, a given promoter operably linked to a coding sequence can affect the expression of the coding sequence if the appropriate enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to induce expression of the coding sequence. Thus, for example, there may be intervening untranslated but transcribed sequences between the promoter sequence and the coding sequence, and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0077] "Encoded by" refers to a nucleic acid sequence that encodes a polypeptide sequence, where the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3-5 amino acids, more preferably at least 8-10 amino acids, and even more preferably at least 15-20 amino acids from the polypeptide encoded by the nucleic acid sequence.

[0078] "Expression cassette" or "expression construct" refers to an assembly capable of inducing the expression of a sequence(s) or gene(s) of interest. An expression cassette generally includes a control element such as a promoter operably linked (to induce transcription) with a sequence(s) or gene(s) of interest, as described above, and often also includes a polyadenylation sequence. In certain embodiments of the invention, the expression cassettes described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include one or more selectable markers, a signal that allows the plasmid construct to exist as single-stranded DNA (e.g., M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., SV40 or adenovirus origin of replication).

[0079] A "purified polynucleotide" refers to a polynucleotide of interest or a fragment thereof that is substantially free of the proteins with which the polypeptide is naturally associated, e.g., contains less than about 50%, preferably less than about 70%, and more preferably at least about 90% of the proteins. Techniques for purifying a polynucleotide of interest are well known in the art and include, for example, disruption of cells containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion exchange chromatography, affinity chromatography, and sedimentation according to density.

[0080] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell is "transfected" when exogenous DNA is introduced into the cell membrane. Several transfection techniques are generally known in the art. See, for example, Graham et al. (1973) Virology, 52:456; Sambrook et al. (2001) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York; Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill; and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into a suitable host cell. The term refers to both stable and transient uptake of genetic material, including uptake of peptide- or antibody-bound DNA.

[0081] A "vector" is capable of transferring a nucleic acid sequence into a target cell (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector constructs", "expression vectors", and "gene transfer vectors" refer to any nucleic acid construct that can induce expression of a nucleic acid of interest and transfer a nucleic acid sequence into a target cell. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0082] The terms "variant," "analog," and "mutein" refer to biologically active derivatives of a reference molecule that retain a desired activity, such as the ability to increase ZIP7 activity, ERAD, increase degradation of misfolded proteins, inhibit pathological accumulation of misfolded proteins in cells, and / or reduce ER stress. In general, the terms "variant" and "analog" refer to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature), and / or deletions relative to the native molecule, and are "substantially homologous" to the reference molecule, as defined below, so long as the modifications do not destroy the biological activity. Generally, the amino acid sequence of such an analog will have a high degree of sequence homology to the reference sequence, such as, for example, greater than 50%, generally greater than 60%-70%, even more particularly 80%-85% or more, at least 90%-95% or more amino acid sequence homology, when the two sequences are aligned. Often, an analog will contain the same number of amino acids, but with substitutions, as described herein. The term "mutein" further includes compounds containing only amino and / or imino molecules, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids, etc.), polypeptides with substituted linkages, and other modifications known in the art, including, but not limited to, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules, etc. The term also includes molecules containing one or more N-substituted glycine residues ("peptoids") and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos. 5,831,005, 5,877,278, and 5,977,301, Nguyen et al., Chem Biol. (2000) 7:463-473, and Simon et al., Proc. Natl. Acad. Sci. USA (1992) 89:9367-9371 for a description of peptides.) Methods for making polypeptide analogs and muteins are known in the art and are further described below.

[0083] As explained above, analogs generally include substitutions that are conservative in nature, i.e., substitutions that occur within a family of amino acids that are related in their side chains. Specifically, amino acids are generally classified into four families: (1) acidic-aspartic acid and glutamic acid, (2) basic-lysine, arginine, histidine, (3) non-polar-alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar-glycine, asparagine, glutamine, cysteine, serine threonine, and tyrosine. Phenylalanine, tryptophan, and tyrosine may be classified as aromatic amino acids. For example, a single substitution of leucine with isoleucine or valine, a single substitution of aspartic acid with glutamic acid, a single substitution of threonine with serine, or a similar conservative substitution of an amino acid with a structurally related amino acid can be reasonably expected not to significantly affect biological activity. For example, a polypeptide of interest can contain up to about 5-10 conservative or non-conservative amino acid substitutions, or up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5 and 25, so long as the desired function of the molecule is intact. One of skill in the art can readily determine regions of a molecule of interest that can tolerate change by reference to Hopp / Woods plots and Kyte-Doolittle plots, which are well known in the art.

[0084] "Gene transfer" or "gene delivery" refers to a method or system for reliably inserting DNA or RNA of interest into a host cell. Such methods may result in transient expression of unintegrated transferred DNA, extrachromosomal replication and expression of the transferred replicon (e.g., episome), or integration of the transferred genetic material into the genomic DNA of the host cell. Gene delivery expression vectors include, but are not limited to, bacterial plasmid vectors, viral vectors, non-viral vectors, vectors derived from adenoviruses, lentiviruses, alphaviruses, poxviruses, and vaccinia viruses.

[0085] A polynucleotide "derived from" a specified sequence refers to a polynucleotide sequence that contains a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides, that corresponds to, i.e., is identical to or complementary to, a region of the specified nucleotide sequence. A derived polynucleotide need not necessarily be physically derived from the nucleotide sequence of interest, but may be generated in any manner, including but not limited to chemical synthesis, replication, reverse transcription, or transcription, based on information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. Thus, it may represent either the sense or antisense orientation of the original polynucleotide.

[0086] ZIP7 promotes ERAD and prevents accumulation of misfolded proteins The present disclosure provides methods and compositions for enhancing ERAD and suppressing pathological accumulation of misfolded proteins by increasing the expression or activity of zinc transporter protein 7 (ZIP7). Methods of treating a subject for a disorder associated with protein misfolding are also provided, including gene therapy methods for expressing ZIP7 in vivo in an effective amount sufficient to suppress pathological accumulation of misfolded proteins.

[0087] Many neurodegenerative diseases are associated with the pathological accumulation of misfolded proteins and protein aggregates in specific regions of the eye, brain, and spinal cord. Without being bound to a particular theory, protein misfolding and aggregation cause ER stress and disrupt endoplasmic reticulum-associated degradation (ERAD). Misfolded protein aggregation can also cause neuronal and brain damage leading to cellular dysfunction, loss of synaptic connections, and pathological progression of neurodegenerative diseases. The accumulation of misfolded proteins can be suppressed by promoting ERAD with ZIP7.

[0088] Protein misfolding-related disorders that may be treated with the compositions and methods disclosed herein include any condition that results in protein misfolding and / or protein aggregation, where misfolded proteins and / or protein aggregates accumulate in the endoplasmic reticulum (ER), causing ER stress and inhibiting ER-associated degradation (ERAD). Protein misfolding-related disorders include, but are not limited to, retinitis pigmentosa, Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), also known as Kennedy's disease, multiple system atrophy, and spinocerebellar ataxia (SCA).

[0089] Production of ZIP7 ZIP7 proteins (or biologically active fragments thereof) can be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, etc.) and in various forms (e.g., native, fused, labeled, lipidated, amidated, acetylated, PEGylated, etc.). ZIP7 proteins can include naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means of preparing proteins are well understood in the art. Proteins are preferably prepared in substantially pure form (i.e., substantially free of other host cell or non-host cell proteins).

[0090] ZIP7 nucleic acid and protein sequences can be from any source. Many ZIP7 nucleic acid and protein sequences are known. Representative ZIP7 sequences are shown in SEQ ID NO: 4 for ZIP7 from Drosophila melanogaster and SEQ ID NO: 5 for ZIP7 from human, and additional representative sequences are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, the following NCBI entries: Accession Nos. NM_001077516, NM_006979, NM_001288777, NM_130931, XM_015449564, XM_015449563, XM_005553337, XM_005553336, XM_040983171, XM_040983170, XM_040983169, XM_011976665, XM_011976664, XM_011976663, XM_017522703, XM_017522702. , XM_017522700, XM_017522701, XM_021185695, XM_021185696, NM_001048100, XM_038682747, XM_038682746, XM_036766199, XM_036766198, XM_036766197, AQY77122, AQY77121, NP_001070984, NP_008910, and NP_001275706, all of which sequences (entered by the filing date of this application) are incorporated herein by reference. Any of these sequences or variants thereof (including sequences having at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto) can be used to produce ZIP7 proteins, or recombinant polynucleotides comprising coding sequences encoding ZIP7 proteins, for use in the methods described herein.In certain embodiments, the ZIP7 protein used to promote ERAD comprises or consists of the amino acid sequence of SEQ ID NO:5, or a sequence exhibiting at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto), or a biologically active fragment thereof, and ZIP7 is capable of increasing ERAD in cells and suppressing the pathological accumulation of misfolded proteins.

[0091] In one embodiment, the ZIP7 protein is produced using recombinant techniques. One of skill in the art can readily determine the nucleotide sequence encoding the desired protein using standard methods and the teachings herein. Oligonucleotide probes can be devised based on the known sequence and used to probe genomic or cDNA libraries. The sequence can then be further isolated using standard techniques and, for example, a restriction enzyme used to truncate the gene at the desired portion of the full-length sequence. Similarly, the sequence of interest can be directly isolated from cells and tissues containing it using known techniques such as phenol extraction and the sequence further manipulated to produce the desired truncation. See, for example, Sambrook et al., supra, for a description of techniques used to obtain and isolate DNA.

[0092] Protein coding sequences can also be produced synthetically, for example, based on known sequences. Nucleotide sequences can be designed with the appropriate codons for the specific amino acid sequence desired. Complete sequences are generally assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence. See, for example, Edge (1981) Nature 292:756; Nambair et al. (1984) Science 223:1299; Jay et al. (1984) J.Biol.Chem.259:6311; Stemmer et al. (1995) Gene 164:49-53.

[0093] Recombinant techniques are readily used to clone protein-encoding sequences, which can then be mutagenized in vitro by replacement of the appropriate base pair(s) to result in the codon for the desired amino acid. Such changes may involve as little as one base pair, resulting in a single amino acid change, or may encompass several base pair changes. Alternatively, mutations can be achieved using mismatched primers that hybridize to the parent nucleotide sequence (generally a cDNA corresponding to an RNA sequence) at a temperature below the melting temperature of the mismatched duplex. Primers can be made specific by keeping the length and base composition of the primer within relatively narrow limits and centrally positioning the mutant base. See, for example, Innis et al, (1990) PCR Applications: Protocols for Functional Genomics; Zoller and Smith, Methods Enzymol. (1983) 100:468. Primer extension is achieved using a selected DNA polymerase, the cloned product, and a clone containing the mutated DNA derived by separation of the primer extension strand. Selection can be achieved using mutant primers as hybridization probes. This technique is also applicable to generate multiple point mutations. See, for example, Dalbie-McFarland et al. Proc. Natl. Acad. Sci USA (1982) 79:6409.

[0094] Once the coding sequences have been isolated and / or synthesized, they can be cloned into any suitable vector or replicon for expression. (See also the Examples.) As will be apparent from the teachings herein, a wide variety of vectors encoding modified proteins can be produced by creating expression constructs that operably link polynucleotides encoding proteins having deletions or mutations therein in various combinations.

[0095] Many cloning vectors are known to those of skill in the art, and selection of an appropriate cloning vector is a matter of choice. Examples of recombinant DNA vectors for cloning and host cells which they may transform include bacteriophage λ (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (Gram negative bacteria), pGV1106 (Gram negative bacteria), pLAFR1 (Gram negative bacteria), pME290 (non-E. coli Gram negative bacteria), pHV14 (E. coli and Bacillus subtilis), pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCp19 (Saccharomyces), and bovine papilloma virus (mammalian cells). See generally, DNA Cloning: Vols. I & II, supra; Sambrook et al., supra; B. Perbal, supra.

[0096] Insect cell expression systems such as the baculovirus system may also be used and are known to those of skill in the art and described, for example, in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus / insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego Calif. ("MaxBac" kit).

[0097] Plant expression systems can also be used to produce ZIP7 proteins. Generally, such systems use virus-based vectors to transfect heterologous genes into plant cells. For a description of such systems, see, for example, Mol. Biotech. (1996) 5:209-221, and Hackland et al., Arch. Virol. (1994) 139:1-22.

[0098] Viral systems such as the vaccinia-based infection / transfection system described in Tomei et al., J. Virol. (1993) 67:4017-4026 and Selby et al., J. Gen. Virol. (1993) 74:1103-1113 may also be used with the present invention. In this system, cells are first transfected in vitro with a vaccinia virus recombinant encoding the bacteriophage T7 RNA polymerase. This polymerase exhibits exquisite specificity in that it only transcribes templates that carry the T7 promoter. After infection, cells are transfected with the DNA of interest driven by the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the host translation machinery. This method provides high-level, transient cytoplasmic production of large amounts of RNA and its translation product(s).

[0099] The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "control" elements) so that the DNA sequence encoding the desired polypeptide is transcribed into RNA in a host cell transformed with a vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Both naturally occurring signal peptides or heterologous sequences may be used in the present invention. Leader sequences may be removed by the host in post-translational processing. See, for example, U.S. Patent Nos. 4,431,739, 4,425,437, and 4,338,397. Such sequences include, but are not limited to, the TPA leader and the honey bee melittin signal sequence.

[0100] Other regulatory sequences that allow for regulation of the expression of the protein sequence relative to the growth of the host cell may also be desirable. Such regulatory sequences are known to those skilled in the art and include, for example, those that turn the expression of the gene on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Other types of regulatory elements, such as enhancer sequences, may also be present in the vector.

[0101] The control sequences and other regulatory sequences can be ligated to the coding sequence prior to its insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.

[0102] In some cases, it may be necessary to modify the coding sequence so that it may be attached to the control sequences with the proper orientation, i.e., to maintain the proper reading frame. Mutants or analogs may be prepared by deleting portions of the protein-encoding sequence, inserting sequences, and / or substituting one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those of skill in the art. See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, supra.

[0103] The expression vector is then used to transform a suitable host cell. Many mammalian cell lines are known in the art, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), such as Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), Vero293 cells, and others. Similarly, bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp. may be used with the present expression constructs. Yeast hosts useful in the present invention include, among others, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Insect cells for use with baculovirus expression vectors include, among others, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.

[0104] Depending on the expression system and host selected, the fusion protein of the present invention is produced by growing a host cell transformed with the above expression vector under conditions in which the protein of interest is expressed. The selection of appropriate growth conditions is within the skill of the art.

[0105] In one embodiment, the transformed cells secrete the ZIP7 protein product into the surrounding medium. Certain regulatory sequences can be included in the vector to enhance secretion of the protein product, for example, using the tissue plasminogen activator (TPA) leader sequence, interferon (gamma or alpha) signal sequence, or other signal peptide sequences from known secreted proteins. The secreted ZIP7 protein product can then be isolated by a variety of techniques described herein, for example, using standard purification techniques, including, but not limited to, hydroxyapatite resin, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunoadsorption techniques, affinity chromatography, immunoprecipitation, and the like.

[0106] Alternatively, transformed cells are disrupted using chemical, physical, or mechanical means that lyse the cells but still keep the recombinant peptide or polypeptide substantially intact. Intracellular proteins can also be obtained by removing components from the cell wall or membrane, for example by the use of detergents or organic solvents, resulting in leakage of the polypeptide. Such methods are known to those skilled in the art and are described, for example, in Protein Purification Applications: A Practical Approach, (Simon Roe, Ed., 2001).

[0107] For example, methods of disrupting cells for use according to the invention include, but are not limited to, sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thawing; drying; catastrophic vacuum; osmotic shock; treatment with lytic enzymes, including proteases such as trypsin, neuraminidase, and lysozyme; alkaline treatment; and the use of detergents and solvents such as bile salts, sodium dodecyl sulfate, Triton, NP40, and CHAPS. The particular technique used to disrupt cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, the culture conditions, and any pretreatments used.

[0108] After cell disruption, cell debris is generally removed by centrifugation, and the peptide or polypeptide produced within the cells is further purified using standard purification techniques, including, but not limited to, column chromatography, ion exchange chromatography, size exclusion chromatography, electrophoresis, HPLC, immunoadsorption techniques, affinity chromatography, immunoprecipitation, and the like.

[0109] For example, one method for obtaining intracellular peptides or polypeptides of the invention includes affinity purification, such as by immunoaffinity chromatography using antibodies (e.g., previously generated antibodies) or by lectin affinity chromatography. Particularly preferred lectin resins are those that recognize mannose moieties, such as, but not limited to, resins derived from snowdrop agglutinin (GNA), lentil agglutinin (LCA or lentil lectin), pea agglutinin (PSA or pea lectin), daffodil agglutinin (NPA), and ramson agglutinin (AUA). Selection of suitable affinity resins is within the skill of the art. After affinity purification, the peptide or polypeptide can be further purified using conventional techniques well known in the art, for example, by any of the techniques described above.

[0110] ZIP7 proteins can be conveniently chemically synthesized, for example, by any of a number of techniques known to those of skill in the peptide art. See, for example, Fmoc Solid Phase Peptide Synthesis: A Practical Approach (W.C. Chan and Peter D. White eds., Oxford University Press, 1999). st edition, 2000), N. Leo Benoiton, Chemistry of Peptide Synthesis (CRC Press; 1 stedition,2005), Peptide Synthesis and Applications(Methods in Molecular Biology,John Howl ed.,Humana Press,1 st ed., 2005) and Pharmaceutical Formulation Development of Peptides and Proteins (The Taylor&Francis Series in Pharmaceutical Sciences, Lars Hovgaard, Sven Frokjaer, and Marco van de Weert eds., CRC Press; 1 st edition, 1999), which is incorporated herein by reference.

[0111] In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid can then either be attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence, which has a suitably protected complementary (amino or carboxyl) group, under conditions that allow for the formation of an amide bond. The protecting group is then removed from the newly added amino acid residue and the next (suitably protected) amino acid is added. After the desired amino acids have been linked in the proper sequence, any remaining protecting groups (and any solid support, if solid phase synthesis techniques are used) are removed sequentially or simultaneously to provide the final peptide or polypeptide. Simple modifications of this general procedure allow the addition of more than one amino acid at a time to the growing chain, for example, by coupling a protected tripeptide (under conditions that do not racemize the chiral centers) with a suitably protected dipeptide to form a pentapeptide after deprotection. For solid phase peptide synthesis techniques, see, for example, JM Stewart and JD Young, Solid Phase Peptide Synthesis (Pierce Chemical Co., Rockford, IL 1984) and G. Barany and RB Merrifield, The Peptides: Analysis, Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, (Academic Press, New York, 1980), pp. 3-254, and for classical solution synthesis, see M. Bodansky, Principles of Peptide Synthesis, (Springer-Verlag, Berlin 1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, Vol. 1.These methods are typically used for relatively small polypeptides, ie, up to about 50-100 amino acids in length, but are also applicable to larger polypeptides.

[0112] Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz), p-toluenesulfonyl (Tx), 2,4-dinitrophenyl, benzyl (Bzl), biphenylisopropyloxy-carbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o-nitrophenylsulfonyl, and the like.

[0113] Exemplary solid supports are crosslinked polymer supports. These can include divinylbenzene crosslinked styrene-based polymers, such as divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers, and divinylbenzene-benzhydrylaminopolystyrene copolymers.

[0114] ZIP7 proteins can also be prepared chemically by other methods, such as simultaneous multiple peptide synthesis methods, see, e.g., Houghten Proc. Natl. Acad. Sci. USA (1985) 82:5131-5135, U.S. Patent No. 4,631,211.

[0115] Pharmaceutical Compositions ZIP7 protein or recombinant polynucleotides comprising coding sequences encoding ZIP7 protein can be formulated into pharmaceutical compositions, optionally including one or more pharma- ceutically acceptable excipients. Exemplary excipients include, but are not limited to, carbohydrates, inorganic salts, antibacterial agents, antioxidants, surfactants, buffers, acids, bases, and combinations thereof. Excipients suitable for injectable compositions include water, alcohols, polyols, glycerin, vegetable oils, phospholipids, and surfactants. Carbohydrates such as sugars, derivatized sugars, e.g., alditols, aldonic acids, esterified sugars, and / or sugar polymers can be present as excipients. Specific carbohydrate excipients include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), pyranosyl sorbitol, myo-inositol, etc. The excipient can also include an inorganic salt or buffer, such as citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.

[0116] The compositions can also include antimicrobial agents to prevent or inhibit microbial growth. Non-limiting examples of antimicrobial agents suitable for the present invention include benzalkonium chloride, benzethonium chloride, benzyl alcohol, cetylpyridinium chloride, chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, thimersol, and combinations thereof.

[0117] Antioxidants can also be present in the composition.Antioxidants are used to prevent oxidation, thereby preventing the deterioration of ZIP7 protein or other components of preparations.Suitable antioxidants for use in the present invention include, for example, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and combinations thereof.

[0118] Surfactants can be present as excipients.Exemplary surfactants include polysorbates such as "Tween 20" and "Tween 80" and pluronics such as F68 and F88 (BASF, Mount Olive, New Jersey); sorbitan esters; lipids such as phospholipids such as lecithin and other phosphatidylcholines, phosphatidylethanolamines (but preferably not in liposomal form), fatty acids, and fatty esters; steroids such as cholesterol; chelating agents such as EDTA; and zinc and other such suitable cations.

[0119] Acid or base can be present in the composition as an excipient.Non-limiting examples of acids that can be used include the acid selected from the group consisting of hydrochloric acid, acetic acid, phosphoric acid, citric acid, malic acid, lactic acid, formic acid, trichloroacetic acid, nitric acid, perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, and combinations thereof.Examples of suitable bases include, but are not limited to, the base selected from the group consisting of sodium hydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide, ammonium acetate, potassium acetate, sodium phosphate, potassium phosphate, sodium citrate, sodium formate, sodium sulfate, potassium sulfate, potassium fumarate, and combinations thereof.

[0120] The amount of ZIP7 protein in the composition (e.g., when contained in a drug delivery system) will vary depending on many factors, but will optimally be a therapeutically effective dose when the composition is in a unit dosage form or container (e.g., vial). The therapeutically effective dose can be determined empirically by repeatedly administering increasing amounts of the composition to determine which amount produces a clinically desired endpoint.

[0121] The amount of any individual excipient in the composition will vary depending on the nature and function of the excipient and the specific needs of the composition. Typically, the optimal amount of any individual excipient is determined through routine experimentation, i.e., by preparing compositions containing various amounts of the excipient (ranging from low to high amounts), examining stability and other parameters, and then determining the range in which optimal performance is achieved without significant side effects. Generally, however, the excipient(s) will be present in the composition in an amount of about 1% to about 99% by weight of the excipient, preferably about 5% to about 98% by weight, more preferably about 15% to about 95% by weight, and most preferably the concentration is less than 30% by weight. These aforementioned pharmaceutical excipients, along with other excipients, are described in “Remington: The Science & Practice of Pharmacy”, 19th ed., Williams & Williams, (1995), the “Physician's Desk Reference”, 52nd ed., Medical Economics, Montvale, NJ (1998), and Kibbe, AH, Handbook of Pharmaceutical Excipients, 3rd Edition, American Pharmaceutical Association, Washington, DC, 2000.

[0122] The compositions include all kinds of formulations, especially formulations suitable for injection, such as powders or lyophilized products that can be reconstituted with a solvent before use, as well as ready-to-inject solutions or suspensions, dry insoluble compositions for combining with a vehicle before use, and emulsions and liquid concentrates for dilution before administration.Examples of suitable diluents for reconstituting solid compositions before injection include bacteriostatic water for injection, 5% dextrose in water, phosphate buffered saline, Ringer's solution, saline, sterile water, deionized water, and combinations thereof.For liquid pharmaceutical compositions, solutions and suspensions are envisioned.Further preferred compositions include compositions for oral, ocular, or topical delivery.

[0123] The pharmaceutical formulations herein can also be housed in syringes, implantation devices, etc., depending on the intended mode of delivery and use. Preferably, the compositions comprising ZIP7 protein are in unit dosage form, meaning the amount of the conjugate or composition appropriate for a single dose, in premeasured or prepackaged form.

[0124] The compositions herein may optionally include one or more additional agents, such as other drugs for treating disorders associated with protein misfolding, or other pharmaceuticals used to treat a subject for a condition or disease. The combination formulations include ZIP7 protein and tetrabenazine, amantadine, neuroleptics (e.g., butyrophenones, diphenylbutylpiperidines, phenothiazines, thioxanthenes, benzamides, tricyclics, and benzisoxazoles / benzisothiazoles), benzodiazepines (e.g., alprazolam, flunitrazepam, chlordiazepoxide, clonazepam, diazepam, lorazepam, midazolam, ozone ... azepam, azepam, and prazepam), cholinesterase inhibitors (e.g., Razadyne (galantamine), Exelon (rivastigmine), Aricept (donepezil), and Cognex (tacrine)), N-methyl-D-aspartate (NMDA) antagonists (e.g., Namenda (memantine), remacemide), selective serotonin reuptake inhibitors (e.g., citalopram, escitalopram, fluoxetine, , fluvoxamine, paroxetine, and sertraline), anticonvulsants (e.g., barbiturates such as paraldehyde, stiripentol, phenobarbital, methylphenobarbital, and barbexaclone, carboxamides such as carbamazepine, oxcarbazepine, and eslicarbazepine acetate, fatty acids such as valproate, vigabatrin, progabide, and tiagabine, fructose derivatives such as topiramate, gamma-aminobutyric acid (GABA) analogs such as gabapentin, pregabalin, vigabatrin, and progabide, and hydantoins such as ethotoin, phenytoin, mephenytoin, and fosphenytoin), vitamin A, docosahexaenoic acid (DHA), and lutein, or other pharmaceutical agents for treating disorders associated with protein misfolding. Alternatively, such agents can be contained in a composition separate from the composition comprising the ZIP7 protein and co-administered simultaneously with, prior to, or after the composition comprising the ZIP7 protein.

[0125] Nucleic acid encoding ZIP7 The nucleic acid encoding ZIP7 can be used to treat disorders related to protein misfolding.The nucleic acid described herein can be inserted into an expression vector to generate an expression cassette that can produce ZIP7 in suitable host cells.The ability of the construct to produce ZIP7 can be empirically determined.

[0126] An expression cassette typically includes control elements operably linked to the coding sequence that allow expression of the gene in vivo in the target species. For example, typical promoters for mammalian cell expression include, among others, the SV40 early promoter, CMV promoters such as the CMV immediate early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter (Ad MLP), and herpes simplex virus promoter. Other non-viral promoters, such as the promoter from the mouse metallothionein gene, are also used for mammalian expression. Typically, transcription termination and polyadenylation sequences located 3' to the translation stop codon are also present. A sequence for optimizing translation initiation, preferably located 5' to the coding sequence, is also present. Examples of transcription terminator / polyadenylation signals include those from SV40 as described in Sambrook et al., supra, and the bovine growth hormone terminator sequence.

[0127] Enhancer elements may also be used herein to increase expression levels of mammalian constructs. Examples include the SV40 early gene enhancer described in Dijkema et al., EMPOJ. (1985) 4:761, the enhancer / promoter derived from the long terminal repeat (LTR) of Rous sarcoma virus described in Gorman et al., Proc. Natl. Acad. Sci. USA (1982b) 79:6777, and elements derived from human CMV described in Boshart et al., Cell (1985) 41:521, such as elements contained in the CMV intron A sequence.

[0128] Once complete, the construct encoding ZIP7 can be administered to a subject using standard gene delivery protocols. Methods for gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466. Genes can be delivered either directly to a subject, or alternatively, delivered ex vivo to cells derived from the subject and reimplanted into the subject.

[0129] Many virus-based systems for gene transfer into mammalian cells have been developed, including adenoviruses, retroviruses (γ-retroviruses and lentiviruses), poxviruses, adeno-associated viruses, baculoviruses, and herpes simplex viruses (see, e.g., Warnock et al. (2011) Methods Mol. Biol. 737:1-25; Walther et al. (2000) Drugs 60(2):249-271; and Lundstrom (2003) Trends Biotechnol. 21(3):117-122, which are incorporated herein by reference).

[0130] For example, retroviruses provide a convenient platform for gene delivery systems. Selected sequences can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of a subject, either in vivo or ex vivo. Many retroviral systems have been described (U.S. Pat. No. 5,219,740; Miller and Rosman (1989) BioTechniques 7:980-990; Miller, AD (1990) Human Gene Therapy 1:5-14; Scarpa et al. (1991) Virology 180:849-852; Burns et al. (1993) Proc. Natl. Acad. Sci. USA 90:8033-8037; Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3:102-109; and Ferry et al. (2011) Curr Pharm Des. 17(24):2516-2527). Lentiviruses are a class of retroviruses that are particularly useful for delivering polynucleotides to mammalian cells because they are capable of infecting both dividing and non-dividing cells (see, e.g., Lois et al. (2002) Science 295:868-872; Durand et al. (2011) Viruses 3(2):132-159, incorporated herein by reference).

[0131] A number of adenoviral vectors have also been described. Unlike retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomally, thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-274; Bett et al., J. Virol. (1993) 67:5911-5921; Mittereder et al., Human Gene Therapy (1994) 5:717-729; Seth et al., J. Virol. (1994) 68:933-940; Barr et al., Gene Therapy (1994) 1:51-58; Berkner, KL BioTechniques (1988) 6:616-629; and Rich et al., Human Gene Therapy (1993) 4:461-476). In addition, various adeno-associated virus (AAV) vector systems have been developed for gene delivery. Such constructs can be designed using techniques well known in the art. See, for example, U.S. Patent Nos. 5,173,414 and 5,139,941, International Publication Nos. WO 92 / 01070 (published January 23, 1992) and WO 93 / 03769 (published March 4, 1993), Lebkowski et al., Molec. Cell. Biol. (1988) 8:3988-3996, Vincent et al., Vaccines 90 (1990) (Cold Spring Harbor Laboratory Press), Carter, BJ Current Opinion in Biotechnology (1992) 3:533-539, Muzyczka, N. Current Topics in Microbiol. and Immunol. (1992) 158:97-129, Kotin, R Human Gene Therapy (1994) 5:793-801, Shelling and Smith, Gene Therapy (1994) 1:165-169, and Zhou et al., J. Exp. Med. (1994) 179:1867-1875.

[0132] Another vector system useful for delivering a polynucleotide encoding ZIP7 is the enterally administered recombinant poxvirus vaccine described by Small, Jr., PA, et al. (U.S. Patent No. 5,676,950, issued October 14, 1997, incorporated herein by reference).

[0133] Additional viral vectors used to deliver nucleic acid molecules encoding ZIP7 include those derived from the pox family of viruses, including vaccinia virus and avian poxvirus. By way of example, a vaccinia virus recombinant expressing ZIP7 can be constructed as follows: DNA encoding a particular ZIP7 coding sequence is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and adjacent vaccinia DNA sequences, such as sequences encoding thymidine kinase (TK). This vector is then used to transfect cells and simultaneously infect them with vaccinia. Homologous recombination serves to insert the vaccinia promoter plus a gene encoding the coding sequence of interest into the viral genome. The resulting TK- recombinants can be selected by culturing cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant to it.

[0134] Alternatively, avipox viruses, such as fowlpox and canarypox viruses, can also be used to deliver genes. Recombinant avipox viruses expressing immunogens from mammalian pathogens are known to confer protective immunity when administered to non-avian species. The use of avipox vectors is particularly desirable in humans and other mammalian species, since members of the avipox genus can only productively replicate in susceptible avian species and are therefore not infectious in mammalian cells. Methods for producing recombinant avipox viruses are known in the art and use genetic recombination, as described above for the production of vaccinia viruses. See, for example, WO91 / 12882, WO89 / 03429, and WO92 / 03545.

[0135] Molecular conjugate vectors can also be used for gene delivery, such as the adenovirus chimeric vectors described in Michael et al., J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al., Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103.

[0136] Members of the alphavirus genus, including but not limited to vectors derived from Sindbis virus (SIN), Semliki Forest virus (SFV), and Venezuelan equine encephalitis virus (VEE), are also used as viral vectors to deliver the polynucleotides of the present invention. For a description of Sindbis virus-derived vectors useful in practicing the present methods, see Dubensky et al. (1996) J. Virol. 70:508-519, and WO 95 / 07995, WO 96 / 17072, as well as U.S. Patent No. 5,843,723 issued December 1, 1998 to Dubensky, Jr., TW, et al., and U.S. Patent No. 5,789,245 issued August 4, 1998 to Dubensky, Jr., TW, both of which are incorporated herein by reference. Particularly preferred are chimeric alphavirus vectors consisting of sequences derived from Sindbis virus and Venezuelan equine encephalitis virus. See, e.g., Perri et al. (2003) J. Virol. 77:10394-10403, as well as WO 02 / 099035, WO 02 / 080982, WO 01 / 81609, and WO 00 / 61772, which are incorporated by reference in their entireties.

[0137] A vaccinia-based infection / transfection system can be conveniently used to provide inducible, transient expression of a coding sequence of interest (e.g., a ZIP7 expression cassette) in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase exhibits exquisite specificity in that it only transcribes templates that carry the T7 promoter. After infection, cells are transfected with a polynucleotide of interest driven by the T7 promoter. The polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA, which is then translated into protein by the host translation machinery. This method provides high-level, transient, cytoplasmic production of large amounts of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al., Proc. Natl. Acad. Sci. USA (1986) 83:8122-8126.

[0138] As an alternative approach to infection with vaccinia or avipox virus recombinants, or delivery of genes using other viral vectors, an amplification system can be used that will result in high levels of expression after introduction into a host cell. In particular, a T7 RNA polymerase promoter preceding the coding region of the T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase, which will then transcribe more templates. In conjunction, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplified template RNA will result in transcription of the desired gene. Since some T7 RNA polymerase is required to initiate amplification, T7 RNA polymerase can be introduced into the cell along with the template(s) to prime the transcription reaction. The polymerase can be introduced as a protein or on a plasmid encoding the RNA polymerase. For further discussion of T7 systems and their use to transform cells, see, e.g., WO 94 / 26911, Studier and Moffatt, J. Mol. Biol. (1986) 189:113-130, Deng and Wolff, Gene (1994) 143:245-249, Gao et al., Biochem. Biophys. Res. Commun. (1994) 200:1201-1206, Gao and Huang, Nuc. Acids Res. (1993) 21:2867-2872, Chen et al., Nuc. Acids Res. (1994) 22:2114-2120, and U.S. Patent No. 5,135,855.

[0139] The synthetic expression cassette of interest can also be delivered without a viral vector. For example, the synthetic expression cassette can be packaged as DNA or RNA in liposomes before delivery to a subject or cells derived therefrom. Lipid encapsulation is generally achieved using liposomes that can stably bind or entrap and retain nucleic acid. The ratio of condensed DNA to lipid preparation can vary, but is generally about 1:1 (mg DNA:micromoles lipid), or more lipid. For a review of the use of liposomes as carriers for delivery of nucleic acids, see Hug and Sleight, Biochim. Biophys. Acta. (1991.) 1097:1-17; Straubinger et al., in Methods of Enzymology (1983), Vol. 101, pp. 512-527.

[0140] Liposome preparations for use in the present invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes being particularly preferred.Cationic liposomes have been shown to mediate the intracellular delivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416), mRNA (Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081), and purified transcription factors (Debs et al., J. Biol. Chem. (1990) 265:10189-10192) in a functional form.

[0141] Cationic liposomes are readily available. For example, N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin from GIBCO BRL, Grand Island, NY (see also Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416). Other commercially available lipids include (DDAB / DOPE) and DOTAP / DOPE (Boerhinger). Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, for example, Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198, PCT Publication No. 90 / 11092, for a description of the synthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes.

[0142] Similarly, anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include, among others, phosphatidylcholine, cholesterol, phosphatidylethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylethanolamine (DOPE). These materials can be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.

[0143] Liposomes may comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). The various liposome-nucleic acid complexes are prepared using methods known in the art. For example, Straubinger et al., in Methods of Immunology (1983), Vol. 101, pp. 512-527, Szoka et al., Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198, Papahadjopoulos et al. al., Biochim. Biophys. Acta (1975) 394:483, Wilson et al., Cell (1979) 17:77), Deamer and Bangham, Biochim. Biophys. Acta (1976) 443:629, Ostro et al. al.,Biochem.Biophys.Res.Commun.(1977)76:836, Fraley et al.,Proc.Natl.Acad.Sci.USA(1979)76:3348), Enoch See, for example, Fraley et al., J. Biol. Chem. (1980) 255:10431, Szoka and Papahadjopoulos, Proc. Natl. Acad. Sci. USA (1978) 75:145, and Schaefer-Ridder et al., Science (1982) 215:166.

[0144] The DNA and / or peptide(s) can also be delivered in cochleate lipid compositions similar to those described by Papahadjopoulos et al., Biochem. Biophys. Acta (1975) 394:483-491. See also U.S. Patent Nos. 4,663,161 and 4,871,488.

[0145] The expression cassette of interest can also be encapsulated, adsorbed or associated with particulate carriers. Examples of particulate carriers include those derived from polymethylmethacrylate polymers, and microparticles derived from poly(lactide) and poly(lactide-co-glycolide), known as PLG. See, for example, Jeffery et al., Pharm.Res.(1993)10:362-368; McGee J.P.,et al., J Microencapsul.14(2):197-210,1997; O'Hagan DT,et al., Vaccine 11(2):149-54,1993.

[0146] In addition, other particle systems and polymers can be used for in vivo or ex vivo delivery of the nucleic acid of interest. For example, polymers such as polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules are useful for transferring the nucleic acid of interest. Similarly, DEAE-dextran-mediated transfection, calcium phosphate precipitation, or precipitation using other insoluble inorganic salts such as strontium phosphate, aluminum silicate including bentonite and kaolin, chromium oxide, magnesium silicate, talc, etc., can be used in the present method. For example, for a review of delivery systems useful for gene transfer, see Felgner, PL, Advanced Drug Delivery Reviews (1990) 5:163-187. Peptoids (U.S. Patent No. 5,831,005, issued November 3, 1998 to Zuckerman, RN, et al., incorporated herein by reference) can also be used for delivery of the constructs of the present invention.

[0147] In addition, biological delivery systems using particulate carriers such as gold and tungsten are particularly useful for delivering synthetic expression cassettes encoding ZIP7. The particles are coated with the synthetic expression cassette(s) to be delivered and accelerated to high speeds, typically under reduced atmosphere, using gun powder discharge from a "gene gun". Thus, for a description of such techniques and useful devices, see, for example, U.S. Patent Nos. 4,945,050, 5,036,006, 5,100,792, 5,179,022, 5,371,015, and 5,478,744. Also, needleless injection systems can be used (Davis, HL, et al, Vaccine 12:1503-1509, 1994; Bioject, Inc., Portland, Oreg.).

[0148] The recombinant vector carrying the synthetic expression cassette encoding ZIP7 is formulated into a composition for delivery to a vertebrate subject. These compositions can be either prophylactic (to prevent accumulation and / or aggregation of misfolded proteins) or therapeutic (to treat disorders associated with protein misfolding). The composition will contain a "therapeutically effective amount" of the nucleic acid of interest such that an amount of ZIP7 protein (or a biologically active fragment thereof) can be produced in vivo such that ERAD is increased and accumulation of misfolded proteins is reduced in the individual to whom it is administered. The exact amount required will vary depending, among other things, on the subject being treated, the age and general condition of the subject being treated, the degree of protection desired, the severity of the condition being treated, the particular ZIP7 protein being produced and its mode of administration. An appropriate effective amount can be readily determined by one of ordinary skill in the art. Thus, a "therapeutically effective amount" will fall within a relatively broad range that can be determined through routine testing.

[0149] The compositions will generally contain one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, ethanol, etc. In addition, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, surfactants, etc., may be present in such vehicles. Certain enhancers of nucleic acid uptake and / or expression may also be included in the composition or co-administered.

[0150] Once formulated, the composition can be administered directly to the subject (e.g., as described above) using the method described above, or alternatively, can be delivered ex vivo to cells derived from the subject.For example, the method for ex vivo delivery and re-implantation of transformed cells into the subject is known in the art, and can include, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, lipofetamine and LT-1-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) into liposomes, and direct microinjection of DNA into nucleus.

[0151] Direct delivery of the synthetic expression cassette compositions in vivo, with or without the viral vectors described above, is generally accomplished by injection using either a conventional syringe, a needle-free device such as Bioject™, or a gene gun such as the Accell™ Gene Delivery System (PowderMed Ltd, Oxford, England).

[0152] Administration At least one therapeutically effective cycle of treatment with ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding ZIP7 is administered to the subject for the treatment of disorders associated with protein misfolding, including but not limited to retinitis pigmentosa, Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), also known as Kennedy's disease, multiple system atrophy, and spinocerebellar ataxia (SCA).

[0153] A "therapeutically effective dose or amount" of a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein, as described herein, contemplates an amount that, when administered, results in a positive therapeutic response, such as improved recovery from disorders associated with protein misfolding. Improved recovery may include improved ERAD function resulting in increased degradation of misfolded proteins and reduced formation of protein aggregates, restored neurological function, improved cognition, improved memory, or increased survival. In the case of retinitis pigmentosa, a therapeutically effective dose or amount of a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein may reduce the accumulation of misfolded rhodopsin and rhodopsin protein aggregates in the ER and photoreceptors in the retina of the eye, reduce ER stress, improve survival of photoreceptor cells, and prevent or delay eye damage and vision loss. In the case of amyloid beta aggregation-related diseases (e.g., Alzheimer's disease), a therapeutically effective dose or amount of a ZIP7 protein may reduce amyloid beta aggregation and reduce the formation of amyloid plaques in the brain. In addition, the therapeutically effective dose or amount can slow the loss of cerebellar Purkinje neurons and the loss of brain cells.In the case of diseases related to the aggregation of alpha-synuclein or synucleinopathies (e.g., Parkinson's disease), the therapeutically effective dose or amount of ZIP protein can reduce the aggregation of alpha-synuclein.In addition, the therapeutically effective dose or amount can reduce the accumulation of alpha-synuclein aggregates in neurons, nerve fibers, and / or glial cells, and reduce the formation of Lewy bodies.

[0154] In certain embodiments, a composition is administered in multiple therapeutically effective doses containing a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding ZIP7, and / or one or more other therapeutic agents, such as other drugs or other pharmaceuticals for treating disorders associated with protein misfolding. The compositions of the present invention are typically, but not necessarily, administered orally, by injection (subcutaneous, intravenous, or intramuscular), infusion, or topical administration. Additional modes of administration are also contemplated, such as intraventricular, intracerebral, intraneuronal, intraspinal, intralesional, intraparenchymal, pulmonary, rectal, transdermal, transmucosal, intrathecal, pericardial, intraarterial, intraocular, intraperitoneal, etc. In certain embodiments, the composition is administered to the subject's eye, brain, spinal cord, or cerebrospinal fluid.

[0155] The preparation is also suitable for localized treatment. In certain embodiments, the composition is used for localized delivery of ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding ZIP7 for the treatment of disorders associated with protein misfolding. For example, the composition can be administered directly to photoreceptors or neurons, or administered by stereotactic injection into the brain. A particular preparation and a suitable administration method are selected to target ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding ZIP7 protein to the abnormal accumulation site of misfolded protein or protein aggregates (e.g., misfolded and dysfunctional rhodopsin in photoreceptors, insoluble polyQ protein aggregates in the nucleus of neurons, or amyloid beta plaques in the brain).

[0156] The pharmaceutical preparations may be in the form of a liquid solution or suspension immediately prior to administration, but may also take other forms such as syrups, creams, ointments, tablets, capsules, powders, gels, matrices, suppositories, etc. Pharmaceutical compositions containing ZIP7 proteins and other agents may be administered according to any medically acceptable method known in the art, using the same or different routes of administration.

[0157] In another embodiment, pharmaceutical compositions containing ZIP7 proteins or recombinant polynucleotides comprising coding sequences encoding ZIP7 proteins, and / or other agents are administered prophylactically, for example, to prevent the accumulation of misfolded proteins and / or protein aggregation (e.g., rhodopsin, polyQ, or amyloid beta accumulation or aggregation). Such prophylactic use is particularly valuable for subjects with a genetic predisposition to developing disorders associated with protein misfolding. In another embodiment, pharmaceutical compositions containing ZIP7 proteins or recombinant polynucleotides comprising coding sequences encoding ZIP7 proteins, and / or other agents are administered therapeutically to subjects with symptoms such as vision loss, dementia, loss of mental agility, or loss of muscle coordination caused by disorders associated with protein misfolding.

[0158] In another embodiment, the pharmaceutical composition comprising the ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding the ZIP7 protein, and / or other agents is a sustained release formulation or a formulation administered using a sustained release device. Such devices are well known in the art and include, for example, transdermal patches and miniature implantable pumps that can provide continuous steady-state drug delivery over time at various doses to achieve a sustained release effect in non-sustained release pharmaceutical compositions.

[0159] The present disclosure also provides a method for administering a conjugate containing a ZIP7 protein to a patient suffering from a disorder associated with protein misfolding or a condition that responds to treatment with a ZIP7 protein contained in the conjugate or composition. The method includes administering a therapeutically effective amount of the conjugate or drug delivery system, preferably provided as part of a pharmaceutical composition, by any of the modes described herein. The administration method can be used to treat any condition that responds to treatment with a ZIP7 protein.

[0160] Those skilled in the art will understand which conditions a particular ZIP7 protein can effectively treat. The actual dose administered will vary depending on the age, weight, and general condition of the subject, as well as the particular disorder associated with protein misfolding being treated, the severity of the condition being treated, the judgment of the medical professional, and the ZIP7 protein or conjugate being administered. The therapeutically effective amount can be determined by those skilled in the art and will be adjusted according to the specific requirements of each particular case.

[0161] In certain embodiments, multiple therapeutically effective doses of ZIP7 protein will be administered according to a daily dosing regimen or intermittently. For example, a therapeutically effective dose can be administered one day per week, two days per week, three days per week, four days per week, or five days per week, etc. By "intermittent" administration, it is intended that a therapeutically effective dose can be administered, for example, every other day, every two days, every three days, every week, every other week, etc. For example, in some embodiments, a composition comprising a ZIP7 protein is administered once per week, twice per week, or three times per week for an extended period of time, such as 1, 2, 3, 4, 5, 6, 7, 8...10...15...24 weeks. By "twice per week" or "twice per week" it is intended that two therapeutically effective doses of the agent are administered to a subject within a seven day period, starting on the first day of the first week of administration, with a minimum of 72 hours between doses and a maximum of 96 hours between doses. By "three times a week" or "three times per week" is intended that three therapeutically effective doses are administered to a subject within a seven day period, allowing a minimum of 48 hours between doses and a maximum of 72 hours between doses. For the purposes of this disclosure, this type of administration is referred to as "intermittent" therapy. According to the methods described herein, a subject can receive intermittent therapy (i.e., once a week, twice a week, or three times a week administration of a therapeutically effective dose) for one or more weekly cycles until a desired therapeutic response is achieved. The agent can be administered by any acceptable route of administration as described herein below. The dosage will depend on the potency of the particular ZIP7 protein, the particular disorder associated with protein misfolding being treated, the magnitude of the desired effect, and the route of administration.

[0162] Purified ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein (again, preferably provided as part of a pharmaceutical formulation) can be used alone or in combination with other drugs, such as tetrabenazine, amantadine, neuroleptics (e.g., butyrophenones, diphenylbutylpiperidines, phenothiazines, thioxanthenes, benzamides, tricyclics, and benzisoxazoles / benzisothiazoles), benzodiazepines (e.g., alprazolam, flunilis), and the like. cyclosporine (e.g., cyclosporine, cyclosporine), cyclosporine (e.g. ... anticonvulsants (e.g., barbiturates such as paraldehyde, stiripentol, phenobarbital, methylphenobarbital, and barbexaclone; carboxamides such as carbamazepine, oxcarbazepine, and eslicarbazepine acetate; fatty acids such as valproate, vigabatrin, progabide, and tiagabine; fructose derivatives such as topiramate; gabapentin; It may be administered in combination with one or more other therapeutic agents, such as gamma-aminobutyric acid (GABA) analogs, such as pentin, pregabalin, vigabatrin, and progabide, and hydantoins, such as ethotoin, phenytoin, mephenytoin, and fosphenytoin, vitamin A, docosahexaenoic acid (DHA), and lutein, or other agents used to treat a particular condition or disease according to various dosing schedules, depending on the judgment of the clinician, the needs of the patient, etc. The particular dosing schedules will be known by those of skill in the art or can be determined empirically using routine methods.Exemplary dosing schedules include, but are not limited to, 5 times a day, 4 times a day, 3 times a day, 2 times a day, once a day, 3 times a week, twice a week, once a week, twice a month, once a month, once a month, and any combination thereof. Preferred compositions are those requiring administration no more than once a day.

[0163] The ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein may be administered prior to, simultaneously with, or subsequent to the other agent. When provided simultaneously with the other agent, the ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein may be provided in the same or different compositions. Thus, the ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein and / or the other agent may be presented to an individual by combination therapy. By "combination therapy" is intended administration to a subject such that the therapeutic effect of the combination of substances is caused in the subject undergoing the therapy. For example, combination therapy may be achieved by administering a dose of a pharmaceutical composition comprising a ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein and a dose of a pharmaceutical composition comprising at least one other agent, such as another drug for treating a disorder associated with protein misfolding, in a combination comprising a therapeutically effective dose according to a particular dosing regimen. Similarly, the ZIP7 protein or recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein and one or more other therapeutic agents may be administered in at least one therapeutic dose. The administration of the separate pharmaceutical compositions can occur at the same time or at different times (i.e., on the same or different days, sequentially in any order), so long as the therapeutic effect of the combination of these agents is provided to the subject undergoing therapy.

[0164] kit Also provided is a kit for treating a patient for a disorder associated with protein misfolding with a ZIP7 protein or a recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein as described herein. The ZIP7 protein or the recombinant polynucleotide comprising a coding sequence encoding a ZIP7 protein, and optionally other therapeutic agents, can be contained in separate compositions or in the same composition. The kit can include a unit dose, e.g., tablet or injectable dose(s), of a formulation comprising a ZIP7 protein suitable for use in the therapeutic methods described herein. In such a kit, in addition to a container containing the unit dose, an informational package insert is provided that describes the use of the treatment for a disorder associated with protein misfolding and the associated benefits. The kit can include, for example, an administration regimen for the ZIP7 protein.

[0165] Formulations suitable for intravenous administration are of particular interest, and in such embodiments, the kit may further comprise a syringe or other device for accomplishing such administration, which syringe or device may be pre-filled with the ZIP7 protein. The instructions may be printed on a label affixed to the container or may be a package insert associated with the container.

[0166] In certain embodiments, the kit comprises a ZIP7 protein comprising or consisting of an amino acid sequence of SEQ ID NO:5, or a sequence exhibiting at least about 80-100% sequence identity thereto (including any percent identity within this range, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% sequence identity thereto), or a biologically active fragment thereof, wherein ZIP7 can increase ERAD in cells and inhibit pathological accumulation of misfolded proteins. The subject kits may include at least one container comprising a solution comprising a unit dose of ZIP7 protein and a pharma- ceutically acceptable excipient, and instructions for administering the unit dose according to a desired regimen or exemplary regimens depending on the particular disorder associated with protein misfolding to be treated, age, weight, etc.

[0167] Utilities The compositions and methods of the present disclosure are used in a variety of different applications, including the treatment of conditions resulting in protein misfolding and / or protein aggregation (i.e., protein misfolding-associated disorders), where misfolded proteins and / or protein aggregates accumulate in the endoplasmic reticulum (ER), causing ER stress and inhibiting ER-associated degradation (ERAD). Protein misfolding-associated disorders include, but are not limited to, retinitis pigmentosa, and neurodegenerative diseases such as Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, amyotrophic lateral sclerosis (ALS), dentatorubral-pallidoluysian atrophy (DRPLA), spinal-bulbar muscular atrophy (SBMA), also known as Kennedy's disease, multiple system atrophy, and spinocerebellar ataxia (SCA). Misfolded protein aggregation can cause cellular dysfunction, loss of synaptic connections, neuronal death, and / or brain damage. Treatment with ZIP7, or a recombinant polynucleotide containing a coding sequence encoding ZIP7, may reduce or prevent the accumulation or aggregation of misfolded proteins, improve cellular function, and slow or prevent loss of synaptic connections, neuronal death, vision loss, or brain damage.

[0168] Examples of Non-Limiting Aspects of the Disclosure Aspects including embodiments of the subject matter described above may be useful alone or in combination with one or more other aspects or embodiments. Without limiting the above description, certain non-limiting aspects of the present disclosure, numbered 1-47, are provided below. As will be apparent to one of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or succeeding individually numbered aspects. This is intended to provide support for all such combinations of aspects, and is not limited to the combinations of aspects explicitly provided below. 1. A method for providing zinc transporter protein 7 (ZIP7) to a subject to inhibit pathological accumulation of misfolded proteins, the method comprising introducing into a cell an expression vector comprising a promoter operably linked to a coding sequence encoding ZIP7, wherein the cell expresses ZIP in vivo in the subject in an effective amount sufficient to inhibit pathological accumulation of misfolded proteins in the cell. 2. The method of embodiment 1, wherein the cell is a retinal cell or a brain cell. 3. The method of embodiment 2, wherein the retinal cells are photoreceptor cells. 4. The method of embodiment 2 or 3, wherein the misfolded protein is a misfolded rhodopsin protein. 5. The method of any one of aspects 1 to 4, wherein the expression vector is introduced into the cell ex vivo or in vivo. 6. The method of any one of aspects 1-5, wherein the subject has a disorder associated with protein misfolding. 7. The method of any one of aspects 6, wherein the protein misfolding associated disorder is retinitis pigmentosa. 8. The method of embodiment 6, wherein the disorder associated with protein misfolding is a neurodegenerative disease. 9. The method of any one of aspects 1-8, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42. 10. A method of treating a subject for a disorder associated with protein misfolding, the method comprising administering to the subject an expression vector comprising a promoter operably linked to a nucleotide sequence encoding zinc transporter protein 7 (ZIP7), wherein ZIP7 is expressed in vivo in the subject in a therapeutically effective amount sufficient to inhibit pathological accumulation of misfolded proteins. 11. The method of embodiment 10, wherein the disorder associated with protein misfolding is retinitis pigmentosa or a neurodegenerative disease. 12. The method of embodiment 10 or 11, wherein the expression vector is administered locally to the eye or brain of the subject. 13. The method of embodiment 10 or 11, wherein the expression vector is locally administered to the retina or photoreceptors of the subject. 14. The method of any one of aspects 10-13, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42. 15. A method of treating a subject for a disorder associated with protein misfolding, the method comprising administering to the subject a therapeutically effective amount of zinc transporter protein 7 (ZIP7) protein. 16. The method of embodiment 15, wherein the ZIP7 protein comprises or consists of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:5. 17. The method of embodiment 15 or 16, wherein the disorder associated with protein misfolding is retinitis pigmentosa or a neurodegenerative disease. 18. The method of any one of aspects 15-17, wherein the ZIP7 protein is administered locally to the eye or brain of the subject. 19. The method of any one of aspects 15-18, wherein the ZIP7 protein is administered locally to the retina or photoreceptors of the subject. 20. The method of any one of aspects 15-19, wherein the ZIP7 protein is administered according to a daily dosing regimen or intermittently. 21. The method of any one of aspects 15-20, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42. 22. A method for enhancing endoplasmic reticulum (ER)-associated degradation (ERAD) or proteosome-associated degradation in a cell, the method comprising increasing the expression or activity of zinc transporter protein 7 (ZIP7) in the cell. 23. The method of embodiment 22, wherein the cell is a retinal cell. 24. The method of embodiment 23, wherein the retinal cells are photoreceptor cells. 25. The method of embodiment 23 or 24, wherein increasing the expression or activity of said ZIP7 is sufficient to increase degradation of misfolded rhodopsin protein and inhibit accumulation of misfolded rhodopsin protein in retinal cells. 26. The method of any one of aspects 22-25, wherein increasing the expression of ZIP7 comprises transfecting the cell with a recombinant polynucleotide comprising a coding sequence encoding ZIP7. 27. The method of embodiment 26, wherein the recombinant polynucleotide is provided by a viral vector comprising a promoter operably linked to a coding sequence encoding ZIP7. 28. The method of embodiment 27, wherein the coding sequence encoding ZIP7 is integrated into a chromosomal locus of the transfected cell. 29. The method of embodiment 28, wherein the endogenous promoter is operably linked to the integrated coding sequence encoding ZIP7 at a chromosomal locus. 30. A method for inhibiting accumulation of misfolded proteins in an organ, cell, or tissue of a subject, the method comprising increasing the expression or activity of zinc transporter protein 7 (ZIP7) in the organ, cell, or tissue. 31. The method of embodiment 30, wherein the organ is an eye or a brain. 32. The method of embodiment 30, wherein the tissue is neural tissue. 33. The method of embodiment 30, wherein the tissue is retinal tissue. 34. The method of embodiment 30, wherein the cell is a retinal cell. 35. The method of embodiment 34, wherein the retinal cells are photoreceptor cells. 36. The method of embodiment 35, wherein the misfolded protein is a rhodopsin protein. 37. The method of any one of aspects 30-36, wherein the subject has retinitis pigmentosa. 38. The method of any one of aspects 30-37, wherein the subject has a disorder associated with protein misfolding. 39. The method of embodiment 38, wherein the disorder associated with protein misfolding is a neurodegenerative disease. 40. The method of any one of aspects 30-39, wherein expression of ZIP7 is increased sufficiently to reduce pathological accumulation of misfolded proteins and increase cell survival. 41. The method of any one of aspects 30-40, wherein increasing the expression of ZIP7 comprises providing to an organ, cell, or tissue a recombinant polynucleotide comprising a coding sequence encoding ZIP7, and ZIP7 is expressed in the organ, cell, or tissue. 42. The method of embodiment 41, wherein the recombinant polynucleotide further comprises a viral vector comprising a promoter operably linked to the coding sequence encoding ZIP7. 43. The method of embodiment 42, wherein the coding sequence encoding ZIP7 is integrated into a chromosomal locus. 44. The method of embodiment 43, wherein the endogenous promoter is operably linked to the integrated coding sequence encoding ZIP7 at a chromosomal locus. 45. The method of any one of aspects 30-44, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42. 46. ​​A composition for use in a method for treating a disorder associated with protein misfolding, the composition comprising zinc transporter protein 7 (ZIP7), or an expression vector comprising a promoter operably linked to a coding sequence encoding ZIP7. 47. The composition according to aspect 46, further comprising a pharma- ceutically acceptable excipient. EXAMPLES

[0169] As can be understood from the disclosure provided above, the present disclosure has a wide variety of applications. Therefore, the following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as the invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, dimensions, etc.), but some experimental error and deviation should be accounted for. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to obtain essentially similar results.

[0170] Example 1: Collective border cell migration induces Zn2+ binding to promote endoplasmic reticulum-associated protein degradation 2+ Requires the transporter Catsup Here we study Catsup function in border cells. Together with published results, our data indicate that Zn-Catsup is rate-limiting for the degradation of ER-localized misfolded proteins. 2+ Our results suggest a unified model for Catsup / ZIP7 providing ER signaling pathways that mediate ER activation and thereby alleviate ER stress to promote cell survival, migration, and Notch transcriptional responses.

[0171] result To study the role of Catsup in the ovary, we first examined its expression using a Catsup::GFP fusion protein expressed under endogenous genomic regulatory sequences. 50 Catsup::GFP was expressed throughout oogenesis, including in all follicular cells (Figures 1A-1B). Mammalian ZIP7 is primarily localized to the ER. 42 Both overexpressed CatsupV5 (Fig. ​(Fig.1C–1K)) and Catsup::GFP (Fig.1L–1T) significantly colocalized with the ER-resident protein folding enzyme protein disulfide isomerase (PDI), but not with DNA or F-actin, consistent with previous findings in wing imaginal discs. 40 .

[0172] The border cell cluster is composed of 4–6 migratory cells that surround and carry two non-migratory polar cells. fruitlessGal4 51 Expression of a Catsup RNAi strain in the outer migratory border cells using c306Gal4 inhibited migration (Figure 2A). The defect was rescued by co-expression of UAS-CatsupV5 (Figure 2B). Reduction of Catsup::GFP confirmed the efficacy of the RNAi (Figure 2C,C' and 2D,D'). When Catsup RNAi was driven by c306Gal4 (Figure 2E) and expressed in both polar and migratory cells, border cell migration was also impaired. FruitlessGal4-driven RNAi impaired border cell migration at least as extensively as c306Gal4, indicating that Catsup was required primarily for the outer migratory cells (Figure 2E).

[0173] As a further test of cell autonomy, we used the FLP-FRT system to generate mosaic egg chambers with clones of homozygous Catsup mutant cells. In homozygous mutant polar cells, migration was not detected (Figure 2F). However, migration was impaired when border cell clusters contained homozygous mutant outer border cells. Moreover, the size of the defect correlated with the proportion of mutant cells (Figures 2G-2H). In clusters containing both heterozygous and homozygous mutant cells, homozygous mutant cells tended to occupy a posterior position (Figures 2I-2J), which is typical of mutations in genes that are autonomously required for motility. 52 We conclude that Catsup is required in outer migratory border cells for motility.

[0174] Catsup mutant wing disc epithelial cells are more susceptible to apoptosis 40We observed that 33% (112 / 337) of Catsup mutant follicular epithelial cells were positive for cleaved and activated caspases, indicating that the cells were undergoing apoptosis. However, we did not detect cleaved caspases in Catsup mutant border cells (0 / 32), indicating that border cells are more resistant to death, which is consistent with our previous report that border cell mutants of thread, which encodes the Drosophila inhibitor of apoptosis protein (DIAP1), are viable. 53 .

[0175] One known function of Catsup is the direct binding and inhibition of tyrosine hydroxylase Ple, the rate-limiting enzyme in catecholamine synthesis. 54 Ple and Catsup are expressed in embryonic tracheal cells and contribute to achieving adequate dopamine levels regulating Breathless (fibroblast growth factor receptor) endocytosis and signaling 39 To test whether Catsup functions similarly in border cell migration, we used an antibody to assess the expression of Ple in wild-type egg chambers. In contrast to tracheal cells, we did not detect Ple protein in wild-type egg chambers (Figure 3A). The antibody was effective because it was able to detect ectopically expressed Ple using c306Gal4 (Figure 3B), as well as endogenous expression of Ple in neurons of the adult brain (Figure 3C). 55 Thus, negative regulation of Ple activity is unlikely to be a critical function of Catsup in border cells, suggesting that its role in border cell migration is distinct from its role in tracheal development.

[0176] In the wing imaginal disc epithelium, multiple transmembrane receptor proteins, including Notch and EGFR, abnormally accumulate in Catsup mutant cells 40Similarly, upon Catsup knockdown, we found abnormal intracellular accumulation of Notch in follicular cells in general (Fig. 3D,D') and specifically in border cells (Fig. 3E,E'). Cells lacking Catsup also showed defective Notch transcriptional activity, as detected by a Notch-responsive element reporter. 56 (Fig. 3F,F'). Notch signaling is essential for border cell migration, and expression of constitutively active Notch (Notch intracellular domain, NICD), which does not require intracellular trafficking, ligand binding, or processing, rescues loss of Notch signaling in border cells. 57 To determine whether NICD could rescue Catsup knockdown, we investigated whether NICD expression or overexpression of the Notch-specific chaperone O-fucosyltransferase-1 rescued Catsup knockdown. 58 Neither of these was sufficient to rescue Catsup RNAi. Similar to imaginal discs, EGFR also aberrantly accumulated in Catsup RNAi-expressing border cells (Fig. 3G,G') and epithelial follicular cells (Fig. 3H,H'), whereas E-cadherin was unaffected (Fig. 3I,I'). From these observations, we conclude that Catsup prevents the intracellular accumulation of specific transmembrane proteins and promotes Notch signaling, migration, and survival in multiple cell types and organisms.

[0177] The abnormal intracellular accumulation of Notch and EGFR suggested a possible disruption of ER protein folding homeostasis (a condition commonly referred to as "ER stress") and activation of the unfolded protein response (UPR). To test whether Catsup knockdown induces the UPR in follicular cells, we cultured heterozygous Catsup + / - and homozygous Catsup - / - Expression of the ER stress marker Xbp1::EGFP in mosaic border cell clusters composed of mutant cells 59 Compared. Catsup + / - Catsup, not border cells - / -We detected high levels of Xbp1 in border cells (Figure 4A-A"). Consistent with this result, we observed increased expression of PDI (Figure 4B-B"). Accumulation of XBP1 and PDI indicates induction of an adaptive UPR. 60 This effect was rescued by co-expression of CatsupV5 (Figure 4C-C”). These results indicate that cells lacking Catsup experience ER stress and loss of migration, suggesting that ER stress itself may inhibit motility.

[0178] To test whether ER stress impairs border cell migration, we used the misfolded rhodopsin protein Rh1, which is known to induce ER stress. G69D The 61 As expected, Rh1 G69D induced Xbp1 expression in border cells (Fig. 4D inset). G69D Expression of CatsupV5 also blocked migration (Figure 4D), indicating that high levels of misfolded proteins in the ER and subsequent ER stress are sufficient to inhibit motility. Because loss of Catsup caused ER stress, we wondered whether overexpression of Catsup might suppress ER stress. Interestingly, overexpressing CatsupV5 reduced the expression of Rh1. G69D Inducible Xbp1 expression (Figures 4E, 4F) and border cell migration (Figures 4E and 4G) were rescued. These results suggest that Catsup is a limiting factor to prevent ER stress that impedes cell motility.

[0179] The UPR restores ER homeostasis by upregulating the protein folding capacity of the ER and increasing its proteolytic capacity 62 During ERAD, misfolded proteins are extruded from the ER, ubiquitinated, and degraded by the proteasome. 60 To test whether overexpression of Catsup could enhance ERAD, we first isolated Rh1 in cells overexpressing Catsup. G69DProtein abundance was examined. Catsup overexpression increased Rh1 G69D The protein was reduced to undetectable levels (Figures 4D, 4E), suggesting that the function of Catsup is restrictive for ERAD.

[0180] To distinguish whether ER stress disrupts Notch and EGFR trafficking, we used Rh1 G69D The abundance and localization of Notch and EGFR in Rh1-expressing cells were examined. G69D Expression was normal in epithelial follicular cells (Fig. 4H, H') and border cells (Fig. 4I, I'), as was EGFR expression and localization (Fig. 4J-K'). This result suggests that ER stress itself does not disrupt Notch or EGFR trafficking, as previously suggested as an explanation for the Catsup mutant phenotype.

[0181] Interestingly, despite normal localization and abundance of Notch in these cells, Notch signaling was nevertheless impaired (Fig. 4L,L'). Thus, ER stress induced by accumulation of misfolded ER client proteins does not affect Notch or EGFR proteostasis but impairs Notch transcriptional activity. This finding is consistent with previous studies that identified pharmacological inhibitors of ZIP7 in a screen for compounds that block transcriptional responses to overexpressed NICD that do not require Notch trafficking via the ER, cell surface expression, ligand binding, or proteolytic activity. 63 Consistent with this, we conclude that ZIP7 promotes Notch transport. 63 Our results suggest that ER stress is a potent regulator of the Catsup and Rh1 G69D These results suggest that ZIP7 reduces NICD transcriptional activity, whether caused by expression or by pharmacological inhibition of ZIP7.

[0182] ZIP7 is present in the ER membrane and binds Zn 2+ Transports from the ER to the cytoplasm 64 Zn in Catsup2+ To test whether transporter activity is important for border cell migration, Zn 2+ We introduced point mutations, H315A and H344A, which replace histidine residues that are required for transport and are conserved among Catsup, ZIP7, and the more distant family from Arabidopsis IRT1. 2+ Catsup does not affect transportation H187A and Catsup H183A Engineered mutants 65 We generated transgenic flies expressing the mutants under Gal4 / UAS control and included a V5 tag to allow monitoring of protein abundance and localization. We then co-expressed each of these RNAi-resistant transgenes with CatsupRNAi and assessed protein expression and border cell migration (Figures 5B-5E). 2+ A point mutation that does not disrupt transport, Catsup H187A and Catsup H183A was able to rescue border cell migration to nearly wild-type levels (Figure 5F), whereas Castup H344A Or Catsup H315A None of the proteins provided significant rescue (Figure 5F). All proteins were stably expressed and correctly localized to the ER (Figures 5B-5E), and therefore the lack of rescue is likely the result of a loss of transporter activity rather than a loss of expression, localization, or another function. Similarly, Zn 2+ Trafficking-defective proteins failed to rescue Notch and EGFR accumulation (Figures 5G-5J'), whereas Catsup H187A and Catsup H183A (Figures 5K-5N') were rescued. 2+ We conclude that trafficking is an essential function of Catsup in promoting ERAD.

[0183] Consideration A model for Catsup function: local Zn 2+ Trafficking limits ERAD and relief of ER stress In this study, we explored the role of the multifunctional protein Catsup in the Drosophila ovary. Catsup is a conserved protein with names including ZRT1 in yeast, IRT1 in plants, and SLC39a7 / Zip7 / Ke4 in mammals. Prior to this study, diverse functions have been attributed to ZIP7 orthologues at the biochemical, cellular, tissue, and organ levels. Integrating previous studies with the results presented herein, we propose a research model (Figure 5O) for the conserved function of Catsup in ERAD, which relieves ER stress and promotes cell migration and survival.

[0184] ERAD requires a complex machinery involving dozens of proteins responsible for recognizing misfolded proteins in the ER, extruding them into the cytoplasm by retrotranslocation, ubiquitinating them, and degrading them via the proteasome. 66 . They reside in the ER membrane and require cytoplasmic Zn for their catalytic activity. 2+ The ERAD E3 RING finger ubiquitin ligase, which requires 66 Most of the Zn 2+ is bound to proteins, so there is no free Zn in the cells. 2+ is almost nonexistent 67 The estimate of the cytoplasmic concentration of free Ca 2+ Thus, an attractive possibility is that Catsup mediates the binding of Zn to the ER / cytoplasmic interface of ERAD E3 ubiquitin ligases. 2+ Consistent with this idea, overexpression of either of the two ERAD E3 ubiquitin ligases, SORDD1 / 2, inhibits the expression of ectopically expressed misfolded Rh1 in border cells, whereas Catsup overexpression inhibits the expression of ectopically expressed misfolded Rh1 in border cells. G69D Reducing the levels of the protein and alleviating all of the associated phenotypes, including ER stress and defects in border cell migration, also reduced Rh1 expression in the Drosophila eye. G69D Suppress the proteotoxic effects of expression 68 Rh1 G69DThis similarity between overexpression of Catsup / ZIP7 and ERAD E3 ubiquitin ligases in phenotypic rescue supports the idea that they function in a common pathway.

[0185] The abnormal accumulation of Notch and EGFR in Catsup mutant border cells resembles the Catsup phenotype in Drosophila wing imaginal discs. Notch and EGFR regulate cell fate and border cell migration in imaginal disc cells, and our results support a common role for Catsup in both tissues. Groth et al. 40 used live-cell labeling with an antibody against the extracellular domain to show that normal levels of Notch receptor protein were present on the plasma membrane of Catsup mutant cells and were endocytosed normally, despite their abnormal accumulation in the ER. This result is more consistent with our proposed role for Catsup in ERAD than the conclusion drawn by others that Catsup / ZIP7 affects Notch trafficking through the secretory pathway. Our results suggest that Catsup / ZIP7 localizes Zn2+ to the ERAD E3 ubiquitin ligase. 2+ An interesting implication of this idea is that it provides a source of Zn 2+ Transporters also transport the total free Zn in the cytoplasm or in specific organelles. 2+ In addition to or in addition to regulating Zn localized to specific protein partners, 2+ This is mainly due to the fact that it can be specialized to provide Zn 2+ Such a scenario would be the case for 24 Zn in humans. 2+ Explain the need for transporters.

[0186] Our observations also raise the interesting question of why some proteins, such as Notch and EGFR, are more prone to accumulate in the ER in Catsup mutant cells than others that also traverse the secretory pathway, such as E-cadherin. All of these are single-pass transmembrane proteins. Notch is a particularly large protein with 36 EGF-like repeats and three cysteine-rich LIN12 / Notch repeats in its extracellular domain, all of which require multiple disulfide bonds. Thus, Notch may be particularly prone to misfolding. In contrast, the EGFR extracellular domain is not as large or complex, but like Notch, it contains two cysteine-rich domains and multiple disulfide bonds. 69 In addition to affecting ERAD, loss of Catsup / ZIP function also reduces the expression of excess Zn 2+ In principle, excess Zn can cause protein folding in the ER lumen. 2+ Catsup can interact with cysteine ​​residues, disrupting proper disulfide bond formation. In this case, Catsup mutant cells may be particularly susceptible to misfolding of newly synthesized proteins in the ER. Nolin et al. found that upon inhibition of ZIP7, free Zn in the ER lumen was reduced. 2+ measured a rapid increase of 2.5-fold 63 Free Zn in the ER lumen 2+ Whether such a modest increase in is sufficient to prevent protein folding is not known.

[0187] Our results show that Rh1 G69DOur results indicate that ER stress impairs Notch signaling independently of abnormal protein accumulation, since ZIP7 induces ER stress and inhibits Notch signaling without aberrant Notch or EGFR protein accumulation in the ER lumen. Although it is unclear how ER stress or the UPR inhibits Notch signaling, the observation that a pharmacological inhibitor of ZIP7 was identified as an inhibitor of Notch signaling by NICD in cultured U2OS cells suggests that ZIP7 inhibits Notch signaling independently of abnormal protein accumulation. 63 suggest that the requirement for Catsup / ZIP7 for Notch transcriptional activity is remarkably conserved. Nolin et al. 63 showed that ZIP7 inhibition leads to accumulation of full-length Notch and a decrease in NICD, and concluded that Notch activation by proteolysis becomes deregulated upon ZIP7 inhibition. An alternative interpretation is that inhibition of ERAD leads to accumulation of full-length Notch in the ER lumen and that NICD is degraded independently and more rapidly as part of a global ER stress response.

[0188] Rh1 G69D The ability of Catsup overexpression to alleviate ER stress and cell defects caused by opsin expression has several general biomedical implications. Dominant mutations in opsins are the most common cause of retinal degeneration in human patients, for which there is currently no effective prevention or therapy. Ectopic expression of proteins that enhance ERAD may be a new therapeutic strategy to consider. In addition, toxic protein aggregates have been proposed to kill neurons by inhibiting ERAD in many neurodegenerative diseases, including Huntington's disease, Alzheimer's disease, Parkinson's disease, frontotemporal dementia, etc., even when the toxic proteins are not localized to the ER. 70 Therefore, strategies to enhance ERAD may be useful in treating these diseases as well.

[0189] Suppression of ER stress and border cell migration by Catsup overexpression is consistent with the observation that ZIP7 is overexpressed in many cancers where it promotes survival, proliferation, and migration, and correlates with disease progression, invasion, and metastasis. The functional and phenotypic similarities of Catsup / ZIP7 across different cells, tissues, and organisms suggest that the border cell system provides an excellent model for deciphering fundamental and conserved effects of this protein in vivo.

[0190] Materials and Methods Drosophila Genetics Catsup mutant flies were generated by ethyl methanesulfonate (EMS) mutagen. 37 The mutation results in a glycine (G) to aspartic acid (D) substitution at amino acid 178. Using the FLP / FRT system, FRT40A-Catsup G178D and hsFLP12,yw;ubi:GFPnls,FRT40A or hsFLP12,yw;ubi:RFPnls,FRT40A / (CyO) to produce Catsup G178DHomozygous mutant clones were generated. Catsup::GFP expression patterns visualized by strains from VDRC 318542 in the fTRG stock library. UAS-CatsupRNAi transgene strains are derived from VDRC 100095 P{KK103630}VIE-260B. Wild-type rescued w[*];sna[Sco] / CyO;P{w[+mC]=UAS-Catsup.V5}6 Bloomington 63229. Additional transgenic Drosophila stocks used: UAS-wRNAi / Cyo was a laboratory stock, UAS-PleRNAi Bloomington 25796 y[1]v[1];P{y[+t7.7]v[+t1.8]=TRiP.JF01813}attP2, UAS-Ple was Bloomington 37539 w[*];P{w[+mC]=UAS-ple.T}331f2, and O-fucosyltransferase 1 Bloomington 9376 P{UAS-O-fut1.O}11.1, Notch.Intracellular.Domain on chromosome 3 was a gift from the Artavanis-Tsakonas lab. 71 ER stress marker UAS-Xbp1-EGFP.HG Bloomington 60731 w[*];P{w[+mC]=UAS-Xbp1.EGFP.HG}3, UAS-HSC70-3 Bloomington 5843 w

[0126] ;P{w[+mC]=UAS-Hsc70-3.WT}B. Catsup point mutations were cloned into vector pUASt-attb using forward primer ctctgaatagggaattgggATGGCCAAACAAGTGGCTGA (SEQ ID NO:1) and reverse primer ccgcagatctgttaacgtcaCGTAGAATCGAGACCGAGGAGAG (SEQ ID NO:2). Vectors were injected into attp2 flies y1 w67c23;P{CaryP}attP2 by BestGene Inc.

[0191] Engineering UAS-RNAi-resistant Catsup point mutations To generate UAS-Catsup-point mutations, constructs were designed to be unable to be targeted by the Catsup RNAi sequence by replacing redundant codons of the same amino acid within the region targeted by RNAi. The RNAi resistance sequence is as follows, with lowercase letters being nucleic acid substitutions: (SEQ ID NO:3)

[0192] Point mutations were designed by changing the codons at the following sites: CatsupH183A (CAC to GCC), CatsupH187A (CAT to GCT), CatsupH315A (cat to GCT), and CatsupH344A (GCT to CAT).

[0193] Immunostaining and confocal imaging Female flies were fattened in yeast for 2 days at 29 °C. Egg chambers were dissected from the ovaries of female flies in Schneider's medium with 10% FBS (pH = 6.85-6.95) as previously described. 72 Freshly dissected egg chambers were fixed in 4% paraformaldehyde and then incubated overnight in 1xPBS with 0.4% Triton and the following primary antibodies: mouse anti-PDI (1:200) (ADI-SPA-891-D Enzo Life Sciences, Inc.), chicken anti-GFP (1:200) (ab13970 Abcam plc.), Ple (anti-TH) antibody (a gift from Craig Montell lab), mouse anti-Notch intracellular domain (1:100) C17.9C6 DSHB, rat E-cadherin antibody DCAD2 (1:50) DSHB, V5 tag monoclonal antibody-Alexa Fluor 555 (2F11F7) Invitrogen, mouse anti-dEGFR (1:2000) E2906 Sigma Aldrich. O-fut1 antibody was used to confirm overexpression of O-fut1 and was provided by Kenneth D. Irvine lab. 73 The secondary antibodies were kindly provided by Sigma-Aldrich. Secondary antibodies were incubated for 2 hours along with Hoechst staining for nuclei and phalloidin staining for F-actin. Mouse anti-PDI and mouse anti-V5-555 co-staining was performed by first staining with PDI primary and secondary, followed by thorough washing and application of anti-V5-555 overnight. Immunostained samples were mounted in VECTASHIELD mounting medium from Vector Laboratories. Images were acquired using Zeiss LSM780 and LSM800 confocal microscopes. Images were processed using FIJI and rotated and cropped for display.

[0194] Sequence alignment The amino acid sequences of Catsup and ZIP7 were obtained from NCBI in FASTA format. The files were input into T-coffee tcoffee.crg.cat / apps / tcoffee / do:regular to generate a multiple sequence alignment. The output was fed into Boxshade ch.embnet.org / software / BOX_form.html to generate a sequence alignment in black and grey shades to show conserved sequence regions.

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[0268] Example 2: ZIP7 prevents RH1G69D-induced photoreceptor cell death Overexpression of ZIP7 prevented photoreceptor cell death in the eyes of flies expressing mutant forms of rhodopsin. As shown in Figures 6A and 6B, ZIP7 prevented Rh1G69D-induced photoreceptor cell death, which leads to rough eyes.

[0269] Example 3: ZIP7 overexpression prevents neuronal cell death in flies expressing Vapor33 or Amyloid-β42 Many neurodegenerative diseases involve abnormal protein aggregation that triggers the unfolded protein response (UPR). These include Alzheimer's disease (Ab42), Huntington's disease (mutant Htt), mutant alpha-synuclein (Parkinson's disease and other dementias), TDP-43 (amyotrophic lateral sclerosis (ALS) and other dementias), and VapB (ALS). ER stress caused by these proteins can be rescued by ZIP7 expression.

[0270] Overexpression of ZIP7 may alleviate ER stress and neuronal death in protein aggregation diseases (Figure 7). We screened proteins prone to aggregation for ZIP7 rescue (Figure 8). ZIP7 overexpression suppressed neurodegeneration due to the expression of Vap33 (Figure 9) and amyloid b42 (Figure 10).

[0271] Example 4: Overexpression of ZIP7 reduces ubiquitinated protein levels Egg chambers with and without ZIP7 overexpression (OE) were incubated with medium in the presence or absence of MG132 proteasome inhibitor (10 mM) for 5 h (Figures 12A and 12B). ZIP7 overexpression reduced ubiquitinated protein levels and FK2 / DNA ratios (Figure 12C).

[0272] Although the foregoing inventions have been described in some detail by way of illustration and example for clarity of understanding, it will be readily apparent to those skilled in the art that, in light of the teachings of the present invention, certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0273] Thus, the above merely illustrates the principles of the invention. It will be appreciated that those skilled in the art can devise various arrangements that embody the principles of the invention and are included within its spirit and scope, although not expressly described or shown herein. Furthermore, all examples and conditional language described herein are intended primarily to aid the reader in understanding the principles of the invention and the concepts the inventors contribute to furthering the art, and should not be construed as being limited to such specifically described examples and conditions. Furthermore, all statements herein that describe the principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, regardless of structure, i.e., any elements developed to perform the same function, regardless of structure. Thus, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the invention is embodied by the appended claims.

Claims

1. 1. A method of providing zinc transporter protein 7 (ZIP7) to a subject to inhibit pathological accumulation of misfolded proteins, the method comprising introducing into a cell an expression vector comprising a promoter operably linked to a coding sequence encoding the ZIP7, wherein the cell expresses the ZIP7 in vivo in the subject in an effective amount sufficient to inhibit the pathological accumulation of the misfolded protein in the cell.

2. The method of claim 1 , wherein the cell is a retinal cell or a brain cell.

3. The method of claim 2 , wherein the retinal cells are photoreceptor cells.

4. The method of claim 2 or 3, wherein the misfolded protein is a misfolded rhodopsin protein.

5. The method according to any one of claims 1 to 4, wherein the expression vector is introduced into the cell ex vivo or in vivo.

6. The method of any one of claims 1 to 5, wherein the subject has a disorder associated with protein misfolding.

7. The method of claim 6, wherein the protein misfolding associated disorder is retinitis pigmentosa.

8. 7. The method of claim 6, wherein the protein misfolding associated disorder is a neurodegenerative disease.

9. The method of any one of claims 1 to 8, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42.

10. 1. A method of treating a subject for a disorder associated with protein misfolding, the method comprising administering to the subject an expression vector comprising a promoter operably linked to a nucleotide sequence encoding zinc transporter protein 7 (ZIP7), wherein the ZIP7 is expressed in vivo in the subject in a therapeutically effective amount sufficient to inhibit pathological accumulation of the misfolded protein.

11. 11. The method of claim 10, wherein the protein misfolding associated disorder is retinitis pigmentosa or a neurodegenerative disease.

12. The method of claim 10 or 11, wherein the expression vector is administered locally to the eye or brain of the subject.

13. The method of claim 10 or 11, wherein the expression vector is administered locally to the retina or photoreceptors of the subject.

14. The method of any one of claims 10 to 13, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42.

15. 1. A method of treating a subject for a disorder associated with protein misfolding, the method comprising administering to the subject a therapeutically effective amount of zinc transporter protein 7 (ZIP7) protein.

16. 16. The method of claim 15, wherein the ZIP7 protein comprises or consists of an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO:

5.

17. 17. The method of claim 15 or 16, wherein the protein misfolding associated disorder is retinitis pigmentosa or a neurodegenerative disease.

18. The method of any one of claims 15 to 17, wherein the ZIP7 protein is administered locally to the eye or brain of the subject.

19. The method of any one of claims 15 to 18, wherein the ZIP7 protein is administered locally to the retina or photoreceptors of the subject.

20. The method of any one of claims 15 to 19, wherein the ZIP7 protein is administered according to a daily dosing regimen or intermittently.

21. The method of any one of claims 15 to 20, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42.

22. A method for enhancing endoplasmic reticulum (ER)-associated degradation (ERAD) or proteosome-associated degradation in a cell, the method comprising increasing the expression or activity of zinc transporter protein 7 (ZIP7) in the cell.

23. 23. The method of claim 22, wherein the cell is a retinal cell.

24. 24. The method of claim 23, wherein the retinal cells are photoreceptor cells.

25. The method of claim 23 or 24, wherein increasing the expression or activity of ZIP7 is sufficient to increase the degradation of misfolded rhodopsin protein and inhibit the accumulation of the misfolded rhodopsin protein in the retinal cells.

26. The method of any one of claims 22 to 25, wherein increasing the expression of ZIP7 comprises transfecting the cell with a recombinant polynucleotide comprising a coding sequence encoding ZIP7.

27. The method of claim 26, wherein the recombinant polynucleotide is provided by a viral vector comprising a promoter operably linked to the coding sequence encoding the ZIP7.

28. 28. The method of claim 27, wherein the coding sequence encoding the ZIP7 is integrated into a chromosomal locus of the transfected cell.

29. The method of claim 28, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the ZIP7 at the chromosomal locus.

30. 1. A method for inhibiting accumulation of misfolded proteins in an organ, cell, or tissue of a subject, the method comprising increasing the expression or activity of zinc transporter protein 7 (ZIP7) in the organ, cell, or tissue.

31. 31. The method of claim 30, wherein the organ is an eye or a brain.

32. 31. The method of claim 30, wherein the tissue is neural tissue.

33. 31. The method of claim 30, wherein the tissue is retinal tissue.

34. 31. The method of claim 30, wherein the cell is a retinal cell.

35. 35. The method of claim 34, wherein the retinal cells are photoreceptor cells.

36. 36. The method of claim 35, wherein the misfolded protein is a rhodopsin protein.

37. The method of any one of claims 30 to 36, wherein the subject has retinitis pigmentosa.

38. The method of any one of claims 30 to 37, wherein the subject has a disorder associated with protein misfolding.

39. 39. The method of claim 38, wherein the protein misfolding associated disorder is a neurodegenerative disease.

40. 40. The method of any one of claims 30 to 39, wherein expression of ZIP7 is increased sufficiently to reduce pathological accumulation of misfolded proteins and increase cell survival.

41. 41. The method of any one of claims 30 to 40, wherein increasing expression of ZIP7 comprises providing to the organ, cell, or tissue a recombinant polynucleotide comprising a coding sequence encoding ZIP7, wherein the ZIP7 is expressed in the organ, cell, or tissue.

42. The method of claim 41 , wherein the recombinant polynucleotide further comprises a viral vector comprising a promoter operably linked to the coding sequence encoding the ZIP7.

43. 43. The method of claim 42, wherein the coding sequence encoding the ZIP7 is integrated into a chromosomal locus.

44. The method of claim 43, wherein an endogenous promoter is operably linked to the integrated coding sequence encoding the ZIP7 at the chromosomal locus.

45. 45. The method of any one of claims 30 to 44, wherein the misfolded protein is Rh1G69D rhodopsin, Vap33, or amyloid b42.

46. 1. A composition for use in a method for treating a disorder associated with protein misfolding, the composition comprising zinc transporter protein 7 (ZIP7) or an expression vector comprising a promoter operably linked to a coding sequence encoding ZIP7.

47. 47. The composition of claim 46, further comprising a pharma- ceutically acceptable excipient.

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