Compositions and methods for treating c-terminus of HSP70 interacting protein-related neurodegenerative diseases

Abstract

Disclosed herein are methods for treating or reducing symptoms of a neurodegenerative disease in a subject. This disclosure relates to a method of restoring the tertiary structure of a mutant C terminus of HSP70-Interacting Protein (CHIP) to the tertiary structure of wild-type CHIP. Also disclosed herein are methods of increasing ubiquitination of a protein by a mutant C terminus of HSP70-Interacting Protein (CHIP) and methods of treating a neurodegenerative disorder comprising administering a therapeutically effective amount of a small molecule or a peptide to a subject.

Description

MBF Ref.028193-0010-WO01 COMPOSITIONS AND METHODS FOR TREATING C-TERMINUS OF HSP70 INTERACTING PROTEIN-RELATED NEURODEGENERATIVE DISEASES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No.63 / 591,712, filed October 19, 2023, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under grants R01 NS112191, R35GM119544, and S10OD024999 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING

[0003] This application is filed with a Computer Readable Form of a Sequence Listing in accord with 37 C.F.R. § 1.821(c). The text file submitted by EFS, “028193-0010-WO01 Sequence Listing.xml,” was created on October 17, 2024, has a file size of 49486605 bytes, and is hereby incorporated by reference in its entirety. FIELD

[0004] This disclosure relates to a method of restoring the tertiary structure of a mutant C terminus of HSP70-Interacting Protein (CHIP) to the tertiary structure of wild-type CHIP. Described herein are methods of increasing ubiquitination of a protein by a mutant C terminus of HSP70-Interacting Protein (CHIP). This disclosure also relates to methods of treating a neurodegenerative disorder comprising administering a therapeutically effective amount of a small molecule or a peptide to a subject. INTRODUCTION

[0005] The maintenance of proteostasis is essential for cellular health. In many diseases, including most neurodegenerative diseases, a loss of proteostasis contributes to cellular toxicity. To maintain proteostasis, cells utilize protein quality control pathways including the ubiquitin- proteosome system (UPS) to target misfolded proteins for degradation. In the UPS, E3 ubiquitin ligases play an essential role in selecting substrates to be targeted for ubiquitination and proteasomal degradation. One E3 ubiquitin ligase, C-terminus of Hsc70 Interacting ProteinMBF Ref.028193-0010-WO01 (CHIP), plays an important role in maintaining proteostasis. CHIP is thought to protect against cellular stress by binding chaperones via its tetratricopeptide repeat (TPR) domain and targeting chaperone bound misfolded proteins for degradation. This prevents the accumulation of toxic, misfolded proteins and prevents protein aggregation. In addition to ubiquitinating chaperone client proteins, CHIP is thought to regulate the translocation and activation of the master regulator of the heat shock response factor Heat Shock Factor 1 (HSF1) during heat stress. Together, through these proposed functions CHIP plays a critical role in regulating proteostasis.

[0006] In vivo, CHIP plays a critical role in regulating viability during stress. Deletion of CHIP results in postnatal lethality in approximately 20% of CHIP knockout (CHIP-KO) mice. In addition, mice that survive have decreased longevity and accelerated aging phenotypes including the accumulation of misfolded proteins. Moreover, CHIP-KO mice are unable to survive heat stress with no mice surviving after 15 minutes of hyperthermia at 42°C. In cell culture, deletion of CHIP also decreases viability in response to stressors including heat stress and the induction of protein misfolding. Interestingly, during heat stress CHIP-KO cells fail to properly upregulate the production of HSP70 protein, presumably through dysregulation of HSF1 signaling. Thus, CHIP is important for responding to cellular stress and for some stressors, including heat stress, CHIP is essential to maintain viability.

[0007] In addition to general stressors like heat stress, CHIP is especially important to respond to stressors that cause neurodegenerative diseases. In disease models of many neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and polyglutamine diseases, decreasing CHIP levels accelerates disease phenotypes while increasing CHIP levels is protective. In addition, mutations in CHIP have also been identified as the cause of two neurodegenerative diseases. Recessive mutations in CHIP cause spinocerebellar ataxia autosomal recessive type 16 (SCAR16), while dominant mutations cause spinocerebellar ataxia 48 (SCA48).

[0008] Thus, there remains a considerable unmet need for safe and effective therapeutics that target CHIP for the treatment of neurodegenerative diseases. SUMMARY

[0009] In an aspect, the disclosure relates to a method of restoring the tertiary structure of a mutant C terminus of HSP70-Interacting Protein (CHIP) to the tertiary structure of wild-type CHIP in cells, the method comprising contacting the cells with a small molecule or a peptide. InMBF Ref.028193-0010-WO01 an embodiment, the mutant CHIP comprises a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. In another embodiment, the TPR domain is partially unfolded. In another embodiment, the small molecule or the peptide bind to the mutant CHIP. In another embodiment, the cells are in a subject or in vitro. In another embodiment, the subject has a neurodegenerative disorder.

[0010] In a further aspect, the disclosure relates to a method of increasing ubiquitination of a protein by a mutant C terminus of HSP70-Interacting Protein (CHIP) or a wild-type CHIP in cells, the method comprising contacting the cells with a small molecule or a peptide. In an embodiment, the small molecule or the peptide induces a conformational change in the mutant CHIP or the wild-type CHIP. In another embodiment, the mutant CHIP comprises a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. In another embodiment, the cells are in a subject or in vitro. In another embodiment, the subject has a neurodegenerative disorder. In another embodiment, the small molecule or the peptide bind to the mutant CHIP or the wild-type CHIP. In another embodiment, affinity between the protein and the mutant CHIP or the wild-type CHIP is increased. In another embodiment, the protein is a binding partner of the TPR domain of the mutant CHIP or the wild-type CHIP. In another embodiment, the protein is heat shock protein 70 (HSP70), transcription factor II A (TFIIA), charged multivesicular body protein 2B (CHMP2B), or a combination thereof.

[0011] Another aspect of the disclosure provides a method of treating a neurodegenerative disorder, the method comprising administering a therapeutically effective amount of a small molecule or a peptide to a subject in need thereof. In an embodiment, the subject comprises a mutant C terminus of HSP70-Interacting Protein (CHIP) comprising a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain or a wild-type CHIP. In another embodiment, the neurodegenerative disorder is Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s disease (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). In another embodiment, the small molecule or the peptide bind to the mutant CHIP or the wild-type CHIP. In another embodiment, the mutation is a mutation in the TPR domain of CHIP, coiled-coil domain of CHIP, the U-box domain of CHIP, or a combination thereof.MBF Ref.028193-0010-WO01

[0012] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIGS.1A-H show that heat stress and SCA48 mutations result in partial unfolding of the TPR domain of CHIP. FIG.1A is a diagram showing that residues in the TPR domain of CHIP that are mutated in SCA48 are highly conserved. Multiple sequence alignments of CHIP from the indicated organisms was performed utilizing clustal omega. Mutations that cause SCA48 are shown in grey. FIG.1B is a diagram showing the eight mutations in the TPR domain of CHIP that cause SCA48 cluster to the interface between the first and second TPR repeats of CHIP. The crystal structure of CHIP (PDB: 2C2L) was utilized to visualize the residues in the TPR domain of CHIP that have been identified to cause SCA48 (dark grey). FIG.1C is a blot showing that SCA48 mutant CHIP is more susceptible to cleavage by trypsin compared to wild-type CHIP. Partial proteolysis experiments were performed and analyzed by SDS-PAGE and Coomassie staining. The asterisk indicates a band that more rapidly appears with SCA48 mutant CHIP compared to wild-type CHIP. FIG.1D is a blot showing that heat stressed CHIP is more rapidly proteolyzed by trypsin. Trypsin digest was performed at the temperature indicated. FIG.1E is a diagram showing a representation of the region of CHIP that becomes more unstructured and accessible to proteolytic cleavage. The crystal structure of CHIP was utilized to show the region of CHIP that becomes more unstructured. Residues mutated in SCA48 are shown in dark grey and the region of CHIP that corresponds to the lost peptides from E is shown as partially transparent. FIG.1F is a plot showing that the SCA48 mutant TPR domain has decreased structure of the initial helices that comprise the TPR domain. 2D1H / 15N-HSQC NMR spectra were collected using wild-type or SCA48 mutant CHIP TPR domain. Spectra were overlayed (wild-type CHIP is black, SCA48 mutant CHIP is grey). FIG.1G is a plot showing that increased temperature results in a partial unfolding of the TPR domain of CHIP. The 2D1H / 15N-HSQC NMR spectra of wild-type CHIP were collected at 37°C (black) and at 45°C (grey). Spectra were overlayed and peaks that were lost are labeled. FIG. 1H is a structural representation of the loss of structure for SCA48 mutant TPR domain and heat stressed TPR domain. The crystal structure of CHIP (PBD: 2C2L) was utilized to visualize the peaks that were lost upon SCA48 mutation and with heat stress. Residues that lost intensity are partially transparent.MBF Ref.028193-0010-WO01

[0014] FIGS.2A-B show optimization of partial proteolysis of CHIP, CHIPA52G, heat stressed CHIP. FIG.2A is a blot showing that CHIPA52Gis digested by trypsin more rapidly than wild-type CHIP. Partial proteolysis experiments were performed with either wild-type or SCA48 mutant CHIP for the time points indicated. Results were analyzed by SDS-PAGE and Coomassie staining. FIG.2B is a blot showing that CHIP has an increased sensitivity to proteolysis when heat stressed. Partial proteolysis was performed with the ratio of CHIP / trypsin indicated. Results were analyzed by SDS-PAGE and Coomassie staining.

[0015] FIGS.3A-H show that both heat stress and SCA48 mutations result in a loss of affinity of CHIP for chaperones but do not disrupt E3 ligase activity. FIG.3A is a blot showing mutations in the TPR domain of CHIP that cause SCA48 disrupt ubiquitination of HSP70. In vitro ubiquitination assays were performed for one hour with the indicated proteins. Samples were analyzed by SDS-PAGE and Western blot with the indicated antibodies (n=3). FIG.3B is a blot showing that increased temperature results in inhibition of CHIP mediated ubiquitination of HSP70. In vitro ubiquitination assays were performed with the proteins listed at the temperatures indicated. Samples were analyzed by SDS-PAGE and Western blot analysis with the indicated antibody. FIG.3C is a graph showing that increased temperature reduces the interaction between CHIP and chaperones. FP analysis was performed at the indicated temperatures with increasing concentrations of CHIP (n=3). FIG.3D is a graph showing that heat stress results in a decrease in the CHIP / HSP70 interaction in cells. HEK293 cells were transfected with smBiT tagged CHIP and lgBiT tagged HSP70. Forty-eight hours later cells were incubated at either 37°C or 43°C for one hour prior to measurement of luciferase signal (n=3; p-value: ** = 0.0091; **** = <0.0001). FIG.3E is a graph showing that SCA48 mutant CHIP has decreased interaction with HSP70 in cells. HEK293 cells were transfected with smBiT tagged CHIP (wild-type or SCA48 mutant as indicated) and lgBiT tagged HSP70 for 24 hours prior to measurement of luciferase signal (n=3; p-value: ** = 0.0014; *** = 0.0003; **** = <0.0001). FIG.3F is a blot showing that SCA48 mutations do not disrupt CHIP autoubiquitination. Reactions from FIG.3A were blotted for CHIP. FIG.3G is a blot showing that mutations to the TPR domain of CHIP that cause SCA48 do not disrupt E3 ligase activity. Thioester discharge assays were performed for 30 minutes in the presence of free lysine with wild-type CHIP or the indicated CHIP mutant. Samples were analyzed by SDS-PAGE and Coomassie staining (n=3). FIG.3H is a blot showing that heat-stressed CHIP retains E3 ligase activity. Thioester discharge assays were performed for 30 minutes with the proteins indicatedMBF Ref.028193-0010-WO01 at different temperatures. Samples were analyzed by SDS-PAGE and Coomassie staining (n=3).

[0016] FIGS.4A-G show that the structural changes induced by SCA48 mutations and heat stress are reversible. FIG.4A is a plot that demonstrates that the TPR domain of CHIP partially unfolds (dark grey) at 45°C but returns back to its proper fold (black) upon shifting back to 25°C (light grey) using protein NMR. FIG.4B is a graph showing that CHIP regains the ability to bind HSP70 after heat shock. Fluorescence polarization assays were performed with rhodamine labeled peptide that corresponds to the C-terminus of HSP70 and increasing concentrations of CHIP at 37°C (light grey), after increasing the temperature to 42°C for 30 minutes (dark grey) and upon returning that same sample to 37°C for 5 minutes (black). FIG.4C is a blot showing that CHIP retains the ability to ubiquitinate substrate following heat stress. In vitro ubiquitination assays were performed at the indicated temperatures. After heat stress returning CHIP to lower temperatures resulted in a restoration of HSP70 ubiquitination. FIG.4D is the 42°C and 45°C portions of the blot shown in FIG.4C. FIG.4E is a graph showing that CHIP and HSP70 binding in cells is restored after heat shock. NanoBiT assays were performed at the indicated temperatures, heat stress (HS) was performed for 1 hour. FIG.4F is a plot showing that structural changes induced by heat stress are reversible. The TPR domain of CHIP was incubated in the NMR tube at 45°C prior to addition of 4-fold molar excess C-terminal HSP70 peptide. CHIP could refold after addition of peptide. FIG.4G is a plot showing that structural changes induced by SCA48 mutation are reversible. 4-fold molar excess C-terminal HSP70 peptide was added to the TPR domain of SCA48 mutant CHIP in an NMR tube. CHIP could refold after addition of peptide.

[0017] FIGS.5A-C show that ubiquitin, Ube1, and UbcH5 are fully function at elevated temperatures. FIG.5A is a graph of differential scanning fluorimetry (DSF) revealing that CHIP, but not ubiquitin, Ube1, and UbcH5 has a TM near physiological temperature. Recombinant CHIP, Ube1, UbcH5, ubiquitin were assessed by DSF. CHIP had an unfolding temperature near physiological temperature while ubiquitin, Ube1, and UbcH5 did not. Ubiquitin is not pictured because it did not unfold up to 99°C. FIG.5B is a blot showing that UbcH5 can be form a thioester with ubiquitin at elevated temperatures. Thioester assays were performed at the indicated temperatures. Samples were analyzed by SDS-PAGE and Coomassie staining. Sample without b-ME were also not boiled. FIG.5C is a blot showing that UbcH5 can discharge ubiquitin at elevated temperature. Thioester discharge assays were performed at theMBF Ref.028193-0010-WO01 temperatures indicated in the presence of free lysine. Results were analyzed by SDS-PAGE and Coomassie staining.

[0018] FIGS.6A-D shows that mutations to the TPR domain of CHIP that cause SCA48 and heat stress result in localization of CHIP to the nucleus. FIG.6A is fluorescent images showing that heat stress results in nuclear localization of CHIP. CHIP-KO MEFs were electroporated withGFPCHIP. Twenty-four hours later cells were either maintained at 37°C or heat shocked at 43°C for 30 minutes prior to imaging by confocal microscopy. Representative cells are shown (scale bar = 20 ^m). FIG.6B is fluorescent images showing that mutations in the TPR domain of CHIP that cause SCA48 result in aberrant localization of CHIP to the nucleus. GFP-tagged wild-type or SCA48 mutant CHIP was electroporated into CHIP knockout MEFs and localization was visualized 24 hours later. Hoechst stain was utilized to visualize nuclei (scale bar = 20 ^m). FIG.6C is fluorescent images showing that mutations in the TPR domain of CHIP that cause SCA48 result in aberrant localization of CHIP to the nucleus. GFP-tagged SCA48 mutant CHIP was electroporated into CHIP knockout MEFs and localization was visualized 24 hours later. Hoechst stain was utilized to visualize nuclei (scale bar = 20 ^m). FIG.6D is blots showing that SCA48 mutant CHIP localizes to the nucleus in HEK293 cells. HEK293 cells were transfected with either wild-type or SCA48 mutant Myc-His tagged CHIP (MHCHIP orMHCHIPA52G) for 48 hours prior to fractionation. Samples were then analyzed by SDS-PAGE and Western blot with the indicated antibodies (WCL = Whole Cell Lysate; Cyt = Cytosol; Nuc = Nucleus).

[0019] FIGS.7A-F show that SCA48 mutant CHIP is in proximity to proteins involved in RNA biosynthesis and splicing. FIG.7A is a plot showing that proximity labeling with quantitative proteomics reveals proteins that are nearby wild-type and SCA48 mutant CHIP. Miniturbo tagged CHIPA52G(MTCHIPA52G) differentially labels protein compared toMTCHIP. A volcano plot to represent the comparison of proteins labeled withMTCHIPA52GcomparedMTCHIP. FIG.7B is a graph showing the major GO molecular function terms that have decreased proximity toMTCHIPA52Gcompared toMTCHIP. Protein-protein interaction enrichment analysis was performed using ShinyGO v0.76.2 (Ge et al., Bioinformatics, 2020.36(8): p.2628- 2629). FIG.7C is a chart showing that SCA48 mutations decrease the proximity of CHIP to chaperones and CHIP inhibitors. Using geneontology GO biological processes were analyzed and proteins that were hits for the indicated pathway are listed. FIG.7D is a plot showing that SCA48 mutations cause increased proximity to proteins with the GO molecular function term RNA binding. ShinyGO was utilized to analyze proteins with increased proximity to SCA48 mutant CHIP by GO molecular function. FIG.7E and FIG.7F are charts showing that SCA48MBF Ref.028193-0010-WO01 mutations increase the proximity of CHIP to proteins that regulate RNA. Using geneontology GO biological processes were analyzed and proteins that were hits for the indicated pathway are listed.

[0020] FIGS.8A-H show that heat stress and SCA48 mutation induces CHIP dependent changes to the ubiquitinome. FIG.8A is a diagram showing an overview of the proteomic pipeline for enrichment of ubiquitinated peptides. CHIP-KO MEFs stably expressing either mCherry (negative control) or CHIP at 37°C or at 43°C, or SCA48 mutant CHIP at 37°C were collected for analysis. FIG.8B is a graph and FIG.8C is a chart showing that heat stress induces CHIP-dependent changes to the ubiquitinome. Proteins that had increased ubiquitination in cells expressing CHIP versus CHIP-KO cells during heat stress were analyzed using ShinyGO for GO molecular function. This analysis revealed CHIP dependent ubiquitination of proteins implicated in RNA binding. FIG.8D is a chart showing GO biological processes for proteins that had increased ubiquitination in a CHIP dependent manner were determined and select pathways. The different colors highlight protein classes as categorized by PANTHER Protein Class. FIG.8E is a graph showing that SCA48 mutant CHIP results in increased ubiquitination of proteins that bind RNA. ShinyGO was utilized to analyze proteins that were ubiquitinated more with CHIPA52Gcompared to CHIP. FIG.8F is a chart showing GO biological processes for proteins that had increased ubiquitination with CHIPA52Gcompared to CHIP were analyzed using ShinyGO. The different colors highlight protein classes as categorized by PANTHER Protein Class. FIG.8G is a blot showing that SCA48 mutant CHIP that is defective in HSP70 ubiquitination, ubiquitinates MAGOH. In vitro ubiquitination assays were performed with the proteins listed for the times indicated. Reactions were separated by SDS-PAGE and analyzed by Western blot with the indicated antibody. FIG.8H is a blot showing that MAGOH is ubiquitinated in a manner dependent upon SCA48 mutant CHIP. Ubiquitination reactions were carried out with the proteins indicated. Reactions were separated by SDS-PAGE and analyzed by Western blot with the indicated antibody.

[0021] FIGS.9A-N show that changes in protein levels do not correlate with alterations in the CHIP dependent ubiquitinome for RNA binding proteins. FIG.9A, FIG.9B, FIG.9C, and FIG.9D are scatter plots showing analyses of alterations of the ubiquitinome. Di-gly profiling was performed for CHIP-KO MEFs expressing mCherry or CHIP at 37°C or at 43°C or expressing CHIPA52Gat 37°C (n=3). FIG.9E, FIG.9F, and FIG.9G are scatter plots showing that whole cell proteomics reveal only subtle changes at the proteome level in CHIP-KO cells expressing wild-type CHIP at 37°C or 43°C, or SCA48 mutant CHIP at 37°C. QuantitativeMBF Ref.028193-0010-WO01 whole cell proteomics was performed for the samples indicated and fold changes were quantified and plotted (red indicates an increase, blue a decrease; n=3). FIG.9H, FIG.9I, and FIG.9J are Venn diagrams showing comparisons between proteins with altered levels and ubiquitination were made and revealed little overlap. FIG.9K, FIG.9L, and FIG.9M are Venn diagrams showing that proteins with increased ubiquitination were not due to increased total abundance. Ubiquitinome analysis of proteins with increased levels in the whole cell proteomics data herein was compared with proteins with increased ubiquitination in the ubiquitinome analysis revealing little overlap (n=3). FIG.9N is a diagram of a pathway analysis that reveals that proteins that are increased in both levels and ubiquitination in cells expressing CHIP are involved in interferon signaling. The data shown are representative for both 37°C and 43°C.

[0022] FIGS.10A-O show that CHIP is necessary for the full transcriptional response to heat stress and regulates heat stress induced splicing. FIG.10A is a plot showing that RNAseq analysis identifies transcripts with altered expression upon heat stress. RNAseq was performed on wild-type MEFs incubated at either 37°C or for one hour at 43°C. Upregulated transcripts are shown on the right and downregulated are shown on the left. Select HSF1 target genes are labeled (n=3). FIG.10B is a plot showing that CHIP-KO cells have a blunted transcriptional response to heat stress. RNAseq was performed on CHIP-KO MEFs incubated at either 37°C or for one hour at 43°C. Upregulated transcripts are shown on the right and downregulated are shown on the left. Select HSF1 target genes are labeled (n=3). FIG.10C is a Venn diagram showing that CHIP-KO cells have a deficit in the transcriptional response to cellular stress. A Venn diagram was prepared to compare the transcriptional response to the genes that were differentially regulated upon heat stress in wild-type and CHIP-KO MEFs. FIG.10D, FIG.10E, FIG.10F, FIG.10G, FIG.10H, FIG.10I, FIG.10J, and FIG.10K are graphs showing that deletion of CHIP does not disrupt HSF1 induced gene expression. Transcript levels of select HSF1 target genes were compared in wild-type and CHIP-KO MEFs at 37°C or after 1 hour at 43°C (n=3). FIG.10L is a heat map showing splicing analysis that reveals that altered splicing occurs during stress in the absence of CHIP. MAJIQ was utilized to detect local splice variations, quantified via changes in percent RNA spliced in (dPSI), between RNA isolated from wild-type or CHIP-KO MEFs at 37°C or at 43°C. FIG.10M is a plot showing that splicing changes that occur in wild-type MEFs upon heat stress have a decreased dPSI values in CHIP- KO MEFs during heat stress. The median dPSI of splicing changes that occurred in heat shocked MEFs was compared to median dPSI values of WT vs CHIP-KO during heat stress, revealing a negative correlation. FIG.10N is a diagram showing that identification of alternativeMBF Ref.028193-0010-WO01 splicing of Zfp948 during heat stress that is lost in the absence of CHIP. MAJIQ and Voila were utilized to visualize alternative splicing of Zfp948. FIG.10O is a blot showing validation of splicing changes in the presence and absence of CHIP during heat stress. mRNA was isolated from wild-type MEFs at 37°C or wild-type and CHIP-KO MEFs at 37°C and 43°C and cDNA was synthesized. The junction spanning exon 1 and exon 2 was then amplified and analyzed by gel electrophoresis to visualize alternative splicing events.

[0023] FIG.11 is a bar graph showing that splicing changes in CHIP-KO cells are not caused by changes in gene expression. The percent of genes differentially expressed (%DE) was cross referenced with genes with altered splicing patterns to determine if splicing difference caused by deletion of CHIP are due to changes in gene expression.

[0024] FIGS.12A-D show that CHIP has metamorphic properties and can revert to its normal fold upon resolution of heat stress. FIG.12A is a plot showing that structural changes to the TPR domain of CHIP induced by heat stress are reversible. 2D1H / 15N-HSQC NMR spectra of wild-type CHIP at 25°C (black), at 45°C (light grey), and again upon being returned to 25°C after being at 45°C (dark grey) were collected. Recovery of the structure of CHIP was observed upon return to 25°C. FIG.12B is a graph showing that CHIP regains the ability to bind chaperones after recovery from heat stress. FP assays were performed at 37°C (light grey line), 42°C (dark grey line) and after being returned to 37°C from 42°C (black line). A complete recovery in binding upon return to physiological temperature was observed. FIG.12C is a blot showing that CHIP regains the ability to ubiquitinate HSP70 upon return to physiological temperature. In vitro ubiquitination assays were performed at the temperatures indicated for one hour in the no recovery lane. In the recovery lane reactions were incubated at the indicated temperatures for one hour prior to being returned to 37°C for 15 minutes. Samples were separated by SDS-PAGE and analyzed by western blot with the indicated antibodies. FIG.12D is a blot showing that CHIP regains the ability to ubiquitinate HSP70 upon being returned to 37°C. Lanes from FIG.11C were cropped and placed next to each other to highlight the recovery of HSP70 ubiquitination at selected temperatures. FIG.12E is a bar graph showing that CHIP rebinds to chaperones upon resolution of cellular heat stress. Nanobit split luciferase assays were performed prior to, after heat shock, or after six hours of recovery. FIG.12F and FIG.12G are plots showing that a peptide that mimics the C-terminus of HSP70 can restore the fold of either heat stressed (FIG.12F) or SCA48 mutant CHIP (FIG.12G).MBF Ref.028193-0010-WO01

[0025] FIGS.13A-F show that FKBP51 is a prolyl-isomerase that induces structural changes to CHIP. Data herein suggest that an exchange factor that mediates the binding of - VD proteins to the C-terminus of CHIP must exist. FIG.13A is a diagram showing that SCA48 TPR mutants all exist on two helices that are separated by a turn that encodes a proline. This led to searching the proximity labeling data herein to identify prolyl isomerases that may preferentially interact with CHIP. FIG.13B is graphs showing that, from the proximity labeling experiment, FKBP51, FKBP52, and CYP40 all had increased proximity to CHIP compared to control. To determine what effect that these prolyl isomerases had on CHIP function, in vitro ubiquitination assays were performed with or without each prolyl isomerase. FIG.13C is blots showing that FKBP51 selectively inhibited CHIP activity and FIG.13D is blots showing that FKBP51 inhibits CHIP activity without inhibiting E1 and E2 enzymes. To determine if FKBP51 could function as a prolyl isomerase with the TPR domain of CHIP, protein NMR was utilized to directly monitor the isomerization state of the TPR domain of CHIP. FIG.13E is a plot showing that addition of FKBP51 led to selective loss of peaks that mimic what we have found with SCA48 mutations or heat stress. Finally, the residues that flank the proline that was hypothesized to be isomerized were analyzed. FIG.13F is a plot showing that the appearance of a new peak that would correlate with the expected position if proline isomerization had occurred.

[0026] FIG.14 shows a graphic of CHIP showing all the known CHIP mutations. A peptide or molecule described herein would restore the function of CHIP for any CHIP with a point mutation in the TPR or coiled-coil domain of CHIP.

[0027] FIGS.15A-F show that addition of a peptide (IEEVD) rescues structural defects in SCA48 mutant CHIP. FIG.15A, FIG.15B, and FIG.15C are plots of NMR spectra of wild TPR domain (Black) and SCA48 mutant CHIP TPR domain (blue or red) for the I53T mutant (FIG. 15A), Y49C mutant (FIG.15B), or A52G mutant (FIG.15C). In each case specific peaks are lost indicating a loss of structure in this region. FIG.15D, FIG.15E, and FIG.15F show that addition of a peptide that corresponds to the C-terminus of chaperones restores the fold of SCA48 mutant CHIP. Addition of four-fold molar excess -IEEVD peptide causes peaks to reappear compared to FIGS.15A-C for the I53T mutant (FIG.15D), Y49C mutant (FIG.15E), or A52G mutant (FIG.15F).

[0028] FIGS.16A-D show that CHIP is in proximity to other TPR domain-containing proteins. FIG.16A is a graph showing a proximity-labeling screen that identified proteinsMBF Ref.028193-0010-WO01 preferentially biotinylated by CHIPMTvs MT alone. Known CHIP interactors including molecular chaperones were enriched. FIG.16B is a diagram showing string analysis that identifies TPR domain-containing proteins as enriched near CHIP. FIG.16C is a graph showing the peptidyl prolyl cis-trans isomerase FKBP51 was enriched in the CHIP-MT compared to MT alone (n=3; p^0.01). FIG.16D is a graph showing validation of the CHIP / FKBP51 interaction. NanoBiT split luminescence was utilized to confirm the CHIP / FKBP51 interaction substrate. HEK 293 cells were transfected with CHIP and FKBP51 each tagged with a fragment of nanoluciferase for 24 hours prior to measuring luminescent signal (n=3; p^0.01).

[0029] FIGS.17A-D show validation of enrichment of known CHIP interactors. FIG.17A is a graph showing that normalized peptide intensity from a proximity labeling screen to identify proteins in proximity to CHIP validates that CHIP was enriched over control. FIG.17B is a graph showing that normalized peptide intensity from a proximity labeling screen to identify proteins in proximity to CHIP validates that CHIP was enriched over a known CHIP interactor, HSPA1A. FIG.17C is a graph showing that normalized peptide intensity from a proximity labeling screen to identify proteins in proximity to CHIP validates that CHIP was enriched over a known CHIP interactor, HS90AA1. FIG.17D is a graph showing that normalized peptide intensity from a proximity labeling screen to identify proteins in proximity to CHIP validates that CHIP was enriched over a known CHIP interactor, HSPAB1. n=3; ***^0.001; ****^0.0001.

[0030] FIGS.18A-C show that FKBP52 is in close proximity to CHIP. FIG.18A is a graph showing that normalized peptide intensity from a proximity labeling screen identifies FKBP52 as enriched near CHIP. FIG.18B is a graph showing that normalized peptide intensity from a proximity labeling screen identifies CYP40 as enriched near CHIP. FIG.18C is a graph showing validation of the FKBP52 / CHIP interaction by NanoBiT. NanoBiT experiments were performed with CHIP and either FKBP52 or CYP40. The CHIP / FKBP52 interaction was significant while the CHIP / CYP40 interaction trended towards an interaction but did not reach significance. n=3; **^0.01 ***^0.001; ****^0.0001.

[0031] FIGS.19A-I show that the TPR domain of CHIP is a substrate of FKBP51. For FIGS.19A-D, FKBP51 was titrated in at increasing ratios and the HSQC spectrum of CHIP TPR domain was monitored. FKBP51 induces structural changes to the CHIP TPR domain. Insets highlight the disappearance of cross peaks that correspond to residues on the first and second TPR repeat. FIG.19A is a spectra graph showing1H-15N HSQC analysis with15N labelled CHIP TPR domain. FIG.19B is a spectra graph showing1H-15N HSQC analysis with15N labelledMBF Ref.028193-0010-WO01 CHIP TPR domain and titrated in FKBP51 at a ratio of 1:0.25 CHIPTPR:FKBP51. FIG.19C is a spectra graph showing1H-15N HSQC analysis with15N labelled CHIP TPR domain and titrated in FKBP51 at a ratio of 1:0.5 CHIPTPR:FKBP51. FIG.19D is a spectra graph showing1H-15N HSQC analysis with15N labelled CHIP TPR domain and titrated in FKBP51 at a ratio of 1:1 CHIPTPR:FKBP51. FIGS.19E-I show that structural changes in the CHIP TPR domain induced by FKBP51 require the catalytic function and TPR domain of FKBP51. FIG.19E is a HSQC spectra graph showing the protein phosphatase 5 (PP5) TPR domain in unbound form. FIG. 19F is a HSQC spectra graph showing the PP5 TPR domain in the presence of FKBP51. FIG. 19G is a HSQC spectra graph showing the PP5 TPR domain in the presence of FKBP12. FIG. 19H is a HSQC spectra graph showing the PP5 TPR domain in the presence of FKBP51 preincubated with FK506. FIG.19I is a HSQC spectra graph showing the PP5 TPR domain in the presence of FKBP51ǻTPR. Arrowheads in the insets identify residues that lose intensity.

[0032] FIGS.20A-C show that FKBP52 and CY40 do not induce large changes to the spectra of the CHIP TPR domain. FIG.20A is a1H-15N HSQC spectra graph showing15N labelled CHIP TPR domain alone. FIG.20B is a1H-15N HSQC spectra graph showing15N labelled CHIP TPR domain in the presence of a 0.5 molar ratio of FKBP52. FIG.20C is a1H-15N HSQC spectra graph showing15N labelled CHIP TPR domain in the presence of a 0.5 molar ratio of CYP40.

[0033] FIGS.21A-K show that the TPR domain of CHIP is thermodynamically labile. FIG. 21A is an image of a gel showing that CHIP-mediated ubiquitination on Hsp70 is inhibited at elevated temperature. In vitro ubiquitination assays were performed at indicated temperatures for 1 hour prior to analysis by SDS-PAGE and Western blotting with the indicated antibodies. FIG.21B is an image of a gel showing that the E3 ligase function of CHIP is not inhibited at elevated temperature. In vitro lysine discharge assay was performed by charging UbcH5c with ubiquitin prior to addition of addition of 5 mM lysine and incubation at the indicated temperatures for 30 minutes. Samples were analyzed by SDS-PAGE and Coomassie staining. FIG.21C is a graph showing that CHIP has an impaired interaction with Hsp70 at elevated temperature. FP assays were performed with rhodamine-labeled Hsp70 C-terminal peptide and increasing concentrations of CHIP at indicated temperatures. FIGS.21D-F show that temperature induces a reversible structural change to the TPR domain of CHIP. FIG.21D is a HSQC spectrum of CHIP TPR domain acquired at 25°C. FIG.21E is a HSQC spectrum of CHIP TPR domain acquired at 45°C. FIG.21F is a HSQC spectrum of CHIP TPR domain acquired at 25°C after incubation at 45°C. FIG.21G is a graph showing that the CHIP / Hsp70 interaction is restoredMBF Ref.028193-0010-WO01 upon return to physiological temperature. FP was performed with Rhodamine-labeled Hsp70 peptide and increasing concentrations of CHIP at 37°C, 42°C, and returning to 37°C after 42°C as indicated. FIGS.21H-J show that thermodynamic stabilization of the TPR domain of CHIP reverses structural changes caused by heat or by FKBP51. FIG.21H is a HSQC spectra of the CHIP TPR domain that was collected in the presence of four-fold molar excess of Hsp70 peptide at 25°C. FIG.21I is a HSQC spectra of the CHIP TPR domain that was collected in the presence of four-fold molar excess of Hsp70 peptide at 45°C. FIG.21J is a HSQC spectra of the CHIP TPR domain that was collected in the presence of four-fold molar excess of Hsp70 peptide at 25°C in the presence of FKBP51. FIG.21K is a diagram showing a model of stabilization of CHIP by excess IEEVD peptide.

[0034] FIG.22 is a1H-15N HSQC spectra of the CHIP TPR domain with increasing temperature.1H-15N HSQC spectra were obtained at 25°C prior to subsequent increases in temperature. Temperature dependent shifts in spectra are highlighted in addition to the noted loss of peak intensity of N-terminal residues above 37°C.

[0035] FIGS.23A-B show that heat stressed CHIP ubiquitinates Hsp70 upon return to physiological temperature. FIG.23A is an image of a gel showing ubiquitination assays that were performed at the indicated temperatures for 45 minutes prior to remaining at the indicated temperature (no recovery) for 15 minutes or being returned to 37°C for 15 minutes (recovery). Samples were analyzed by SDS-PAGE and western blotting with indicated antibodies. FIG. 23B is cropped images from FIG.23A to highlight the increased ubiquitination upon recovery from either 42°C or 45°C.

[0036] FIGS.24A-G show that SCA48 mutations induce reversible structural changes that decrease the affinity of CHIP for chaperones. FIG.24A is a schematic of SCA48 causing mutations in the CHIP TPR domain. FIG.24B is a diagram showing that SCA48 causing mutations in the TPR domain of CHIP map to the interface between TPR1 and TPR2. The crystal structure of CHIP (pdb: 2c2l) was used to model the CHIP. The TPR (white), coiled-coil (light gray), and U-box (dark gray) domains are shown, and disease-causing mutations are show in medium gray. FIG.24C is a HSCQ spectra for wild-type CHIP. FIG.24D is a HSCQ spectra for CHIPA52Gand shows that mutations in the CHIP TPR domain that cause SCA48 induce structural changes. FIG.24E is a graph showing that mutations in the TPR domain lower their affinity for Hsp70. FP assays were performed with rhodamine-labeled Hsp70 C- terminal peptide and increasing concentrations of CHIP or SCA48 mutant CHIP. FIG.24F is aMBF Ref.028193-0010-WO01 HSCQ spectra of N-15 labeled wild-type CHIP TPR domains that were obtained in the presence of a four-fold excess of a peptide that mimics the C-termini of Hsp70. FIG.24G is a HSCQ spectra of N-15 labeled SCA48 mutant CHIP TPR domains that were obtained in the presence of a four-fold excess of a peptide that mimics the C-termini of Hsp70. It shows that structural changes induced by SCA48 mutations are reversible.

[0037] FIGS.25A-C show that additional SCA48 mutant CHIP TPR domains have a similar loss of peak intensity. FIG.25A is a1H-15N HSQC spectra that was obtained for wildtype15N labelled CHIP TPR domain. FIG.25B is a1H-15N HSQC spectra that was obtained for Y49C SCA48 mutant15N labelled CHIP TPR domain. FIG.25C is a1H-15N HSQC spectra that was obtained for I53T SCA48 mutant15N labelled CHIP TPR domain.

[0038] FIGS.26A-B show that SCA48 mutation induces structural changes to the N- terminus of CHIP. FIG.26A is an image of a gel showing that SCA48 mutant CHIP is more prone to digestion by trypsin. Wildtype or CHIPA52Gwere incubated in the presence or absence of trypsin prior to analysis by SDS-PAGE and Coomassie staining. The asterisk indicates a band that appears more intensely with SCA48 mutant CHIP. FIG.26B is a diagram representation of the region of CHIP that becomes more unstructured and accessible to proteolytic cleavage. The crystal structure of CHIP was utilized to show the region of CHIP that becomes more unstructured. Residues mutated in SCA48 are shown in dark gray and the region of CHIP that corresponds to the lost peptides from TABLE 1 is shown as partially transparent.

[0039] FIGS.27A-L show that FKBP51 induces structural changes in additional TPR domain containing co-chaperone. FIG.27A is an image of a gel showing that FKBP51 alters the stability of additional TPR domain containing co-chaperones. The recombinant TPR domains indicated were incubated at 25°C for 16 hours in the absence and presence of FKBP51 prior to centrifugation. The amount of soluble and pelleted TPR domain was then assessed by SDS-PAGE and Coomassie staining. FIG.27B is a graph showing quantification of FIG.27A. FIGS.27C-F show that FKBP51 induces structural changes to the PP5 TPR domain. FIG.27C is a HSQC spectrum of the PP5 TPR were collected in the absence of FKBP51. FIG.27D is a HSQC spectrum of the PP5 TPR was collected with the indicated ratio of FKBP51 / PP5 TPR domain (0.25:1). FIG.27E is a HSQC spectrum of the PP5 TPR was collected with the indicated ratio of FKBP51 / PP5 TPR domain (0.5:1). FIG.27F is a HSQC spectrum of the PP5 TPR was collected with the indicated ratio of FKBP51 / PP5 TPR domainMBF Ref.028193-0010-WO01 (1:1). Insets highlight regions where disappearance of cross peaks occur. FIGS.27G-K show that structural changes in the PP5 TPR domain induced by FKBP51 require the catalytic function and TPR domain of FKBP51. FIG.27G is a HSQC spectra of the PP5 TPR domain in unbound form. FIG.27H is a HSQC spectra of the PP5 TPR domain in the presence of FKBP51. FIG.27I is a HSQC spectra of the PP5 TPR domain in the presence of FKBP12. FIG. 27J is a HSQC spectra of the PP5 TPR domain in the presence of FKBP51 preincubated with FK506. FIG.27K is a HSQC spectra of the PP5 TPR domain in the presence of FKBP51ǻTPR. Arrowheads in the insets identify residues that lose intensity. FIG.27L is a diagram of a working model for the role of FKBP51 and SCA48 mutations in chaperone / co-chaperone interactions. DETAILED DESCRIPTION

[0040] Described herein are methods of treating and / or alleviating symptoms of a neurodegenerative disorder, such as spinocerebellar ataxia type 48 (SCA48). Identified herein is the discovery that heat stress induces a reversible conformational change in C-terminus of Hsc70 Interacting Protein (CHIP) that results in dissociation from chaperones. During heat stress CHIP is necessary for the proper ubiquitination of RNA binding proteins and loss of CHIP leads to a failure of full activation of the transcriptional response to stress and impairs stress induced splicing events. Further disclosed herein is the finding that mutations in the TPR domain of CHIP that cause SCA48, lock CHIP in a structure that is similar to heat-stressed wild- type CHIP and result in a gain of function.

[0041] Demonstrated herein are the metamorphic properties of the TPR domain of CHIP, where the TPR domain undergoes a reversible conformational change upon heat stress. Also, described herein are mutations in the TPR domain of CHIP that cause SCA48 also cause the TPR domain to adopt a similar, alternative conformation. In this alternate conformation CHIP disengages from chaperones yet retains E3 ligase activity. In cells, both heat stress and SCA48 mutations target CHIP to the nucleus where CHIP is in proximity to RNA binding proteins. To determine if CHIP alters the ubiquitination of proteins in these pathways, ubiquitinome analysis and in vitro ubiquitination assays were performed and showed that CHIP-dependent ubiquitination of RNA binding proteins occurs during heat stress or with SCA48 mutations. Using RNAseq, it was found herein that CHIP has functional effects on the transcriptional response to heat stress in a HSF1 independent manner. This includes a nearly 70% reduction in the upregulation of genes in response to heat shock, and the loss of stress induced splicingMBF Ref.028193-0010-WO01 events in CHIP-knockout (CHIP-KO) cells. Using pathway analysis, it was found herein that genes that are alternatively spliced during heat stress in a CHIP dependent manner are involved in preweaning lethality, abnormal survival, and mortality / aging, all phenotypes observed in CHIP knockout mice. The structural changes observed in CHIP during heat stress are reversible, and upon resolution of heat stress the TPR domain refolds, adopting its normal conformation. Herein metamorphic structural and functional properties of the E3 ubiquitin ligase CHIP are identified and provide mechanistic insight into how mutations in CHIP cause SCA48. 1. Definitions

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The meaning and scope of the terms should be clear. In case of conflict, the present document, including definitions, take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0043] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0044] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0045] The term “about” or “approximately” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptableMBF Ref.028193-0010-WO01 error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value.

[0046] The terms “active ingredient” or “active pharmaceutical ingredient” as used herein refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0047] “C terminus of HSP70-Interacting Protein” or “CHIP” as used herein may refer to wild-type CHIP or a mutant CHIP. In some instances, CHIP is specified as mutant CHIP or wild- type CHIP when needed for clarity.

[0048] The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” as used herein refers to a group of control subjects. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. Cutoff values (or predetermined cutoff values) may be determined by Adaptive Index Model (AIM) methodology. Cutoff values (or predetermined cutoff values) may be determined by a receiver operating curve (ROC) analysis from biological samples of the patient group. ROC analysis, as generally known in the biological arts, is a determination of the ability of a test to discriminate one condition from another, e.g., to determine the performance of each marker in identifying a patient having CRC. A description of ROC analysis is provided in P.J. Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of which is hereby incorporated by reference in its entirety. Alternatively, cutoff values may be determined by a quartile analysis of biological samples of a patient group. For example, a cutoff value may be determined by selecting a value that corresponds to any value in the 25th-75th percentile range, in some embodiments a value that corresponds to the 25th percentile, the 50th percentile or the 75th percentile, and inMBF Ref.028193-0010-WO01 some embodiments the 75th percentile. Such statistical analyses may be performed using any method known in the art and can be implemented through any number of commercially available software packages (e.g., from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station, TX; SAS Institute Inc., Cary, NC.). The healthy or normal levels or ranges may be defined in accordance with standard practice. A control may be a subject or cell without a drug as detailed herein. A control may be a subject, or a sample therefrom, whose disease state is known. The subject, or sample therefrom, may be healthy, diseased, diseased prior to treatment, diseased during treatment, or diseased after treatment, or a combination thereof.

[0049] The term “dose” as used herein denotes any form of the active ingredient formulation or composition that contains an amount sufficient to produce a therapeutic effect with at least a single administration.

[0050] The term “dosage” as used herein refers to the administering of a specific amount, number, and frequency of doses over a specified period of time, typically 1 day.

[0051] The term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable to one skilled in the art.

[0052] “Sample” or “test sample” as used herein can mean any sample in which the presence and / or level of a target is to be detected or determined or any sample comprising a drug as detailed herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre- treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.MBF Ref.028193-0010-WO01

[0053] “Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal that wants or is in need of the herein described compounds, compositions, or methods. The subject may be a human or a non-human. The subject may be a vertebrate. The subject may be a mammal. The mammal may be a primate or a non-primate. The mammal can be a non-primate such as, for example, cow, pig, camel, llama, hedgehog, anteater, platypus, elephant, alpaca, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse. The mammal can be a primate such as a human. The mammal can be a non-human primate such as, for example, monkey, cynomolgus monkey, rhesus monkey, chimpanzee, gorilla, orangutan, and gibbon. The subject may be of any age or stage of development, such as, for example, an adult, an adolescent, or an infant. The subject may be male. The subject may be female. In some embodiments, the subject has a neurodegenerative disorder. The subject may be undergoing other forms of treatment.

[0054] “Treatment” or “treating” when referring to protection of a subject from a disease or disorder, means suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of disease or disorder, or completely eliminating a disease or disorder. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or a disorder or symptoms associated with such disease or disorder prior to affliction with the disease or disorder. Preventing the disease or disorder involves administering a composition or compound of the present disclosure to a subject prior to onset of the disease or disorder. Suppressing the disease or disorder involves administering a composition or compound of the present disclosure to a subject after induction of the disease or disorder but before its clinical appearance. Repressing or ameliorating the disease or disorder involves administering a composition or compound of the present disclosure to a subject after clinical appearance of the disease or disorder.

[0055] “Wild-type,” “wildtype,” or “WT” as used herein refers to a phenotype, genotype, protein, or gene that predominates in a natural population of organisms or strain of organisms in contrast to that of laboratory mutant (i.e. changed) forms. It is a term used to describe a phenotype, genotype, protein, or gene when it is found in its natural, non-mutated (unchanged) form.MBF Ref.028193-0010-WO01 2. Compounds for Treatment of Neurodegenerative Diseases

[0056] Provided herein are methods for treating or reducing symptoms of a neurodegenerative disease using a small molecule or peptide. Further, provided herein are methods for treating or reducing symptoms of a neurodegenerative disease that may include administering to a subject a therapeutically effective amount of a small molecule or peptide, or a composition comprising a small molecule or peptide. In some embodiments, the small molecule or peptide or compositions comprising the same as detailed herein, or at least one component thereof, may treat or reduce the symptoms a neurodegenerative disease. 3. Pharmaceutical Compositions

[0057] Further provided herein are pharmaceutical compositions comprising small molecules or peptides or pharmaceutically acceptable salts thereof. The small molecules or peptides or pharmaceutically acceptable salts thereof may be a peptide that mimics a chaperone of CHIP or fragment thereof. The small molecules or peptides or pharmaceutically acceptable salts thereof may be a peptide that binds to the tetratricopeptide repeat (TPR) domain of wild-type or mutant CHIP. For example, the small molecules or peptides or pharmaceutically acceptable salts thereof may be a peptide that mimics the C-termini of heat shock protein 70 (HSP70) such as, but not limited to, IEEVD peptide. A peptide may be used in excess, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold molar excess. The small molecules or peptides or pharmaceutically acceptable salts thereof may bind to mutant CHIP, induce a conformational change in the mutant CHIP, increase affinity of mutant CHIP for chaperones, increase thermodynamic stability of mutant CHIP, increase thermal stability of mutant CHIP, or a combination thereof. The small molecules or peptides or pharmaceutically acceptable salts thereof may bind to wild-type CHIP, enhance the function of wild-type CHIP, induce a conformational change in the wild-type CHIP, increase affinity of wild- type CHIP for chaperones, increase thermodynamic stability of wild-type CHIP, increase thermal stability of wild-type CHIP, or a combination thereof.

[0058] Molecular chaperones are critical determinants of protein fate whose function is essential to counteract numerous age-related diseases, including neurodegenerative diseases. Co-chaperones associate with chaperones to help direct outcomes for chaperone clients. Among co-chaperones are numerous proteins that bind to the C-terminal -EEVD motif ofMBF Ref.028193-0010-WO01 chaperones via a TPR domain. A chaperone may be HSP70, HSP90, transcription factor II A (TFIIA), charged multivesicular body protein 2B (CHMP2B), or a combination thereof.

[0059] Small molecules or peptides or pharmaceutically acceptable salts thereof as detailed herein, or at least one component thereof, may be formulated into pharmaceutical compositions in accordance with standard techniques well known to those skilled in the pharmaceutical art. The pharmaceutical compositions can be formulated according to the mode of administration to be used. In cases where pharmaceutical compositions are injectable pharmaceutical compositions, they are sterile, pyrogen free, and particulate free. An isotonic formulation is in some embodiments used. Generally, additives for isotonicity may include sodium chloride, dextrose, mannitol, sorbitol and lactose. In some cases, isotonic solutions such as phosphate buffered saline can be used. Stabilizers include gelatin and albumin. In some embodiments, a secondary treatment for a neurodegenerative disease is added to the composition. In some embodiments, anti-inflammatories, antibiotics, analgesics, or combinations thereof are added to the composition. Anti-inflammatories may include one or more of the following: corticosteroids,5-aminosalicylates, mesalamine (Asacol® HD, Delzicol®, etc.), balsalazide (Colazal®), olsalazine(Dipentum®). Antibiotics may include metronidazole or ciprofloxacin. Analgesics may include one or more of the following: opioids (narcotics), such as Avinza®, Kadian®, MS Contin®(morphine), OxyContin®(oxycodone), Dolophine®or Methadose®(methadone), Dilaudid®(hydromorphone), codeine, Demerol®(meperidine), Duragesic®or Actiq®(fentanyl); Tylenol®(acetaminophen); non-steroidal anti-inflammatory drugs (NSAIDs), such as Advil®(ibuprofen), Aleve®(naproxen), Celebrex®(celecoxib), Aspirin (acetylsalicylic acid).

[0060] The composition may further comprise a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be functional molecules as vehicles, adjuvants, carriers, or diluents. The term “pharmaceutically acceptable carrier,” may be a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Pharmaceutically acceptable carriers include, for example, diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, emollients, propellants, humectants, powders, pH adjusting agents, and combinations thereof. The pharmaceutically acceptable excipient may be a transfection facilitating agent, which may include surface active agents, such as immune- stimulating complexes (ISCOMS), Freunds incomplete adjuvant, LPS analog including monophosphoryl lipid A, muramyl peptides, quinone analogs, vesicles such as squalene and squalene, hyaluronic acid, lipids, liposomes, calcium ions, viral proteins, polyanions,MBF Ref.028193-0010-WO01 polycations, or nanoparticles, or other known transfection facilitating agents. The transfection facilitating agent may be a polyanion, polycation, including poly-L-glutamate (LGS), or lipid.

[0061] In another aspect, the present disclosure provides a pharmaceutical composition comprising a small molecule or peptide as described herein, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In a further embodiment, the composition comprises at least two pharmaceutically acceptable carriers, such as those described herein. For purposes of the present disclosure, unless designated otherwise, solvates and hydrates are generally considered compositions. Preferably, pharmaceutically acceptable carriers are sterile. The pharmaceutical composition can be formulated for particular routes of administration such as oral administration, parenteral administration, and rectal administration, etc. In addition, the pharmaceutical compositions of the present disclosure can be made up in a solid form (including without limitation capsules, tablets, pills, granules, powders or suppositories), or in a liquid form (including without limitation solutions, suspensions or emulsions). The pharmaceutical compositions can be subjected to conventional pharmaceutical operations such as sterilization and / or can contain conventional inert diluents, lubricating agents, or buffering agents, as well as adjuvants, such as preservatives, stabilizers, wetting agents, emulsifiers and buffers, etc. In some embodiments, the pharmaceutical compositions are tablets or gelatin capsules comprising the active ingredient together with one or more of: diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and / or glycine; lubricants, e.g., silica, talcum, stearic acid, its magnesium or calcium salt and / or polyethyleneglycol; binders, e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidone; disintegrants, e.g., starches, agar, alginic acid or its sodium salt, or effervescent mixtures; and absorbents, colorants, flavors and sweeteners.

[0062] Certain injectable compositions are aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions. Said compositions may be sterilized and / or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and / or buffers. In addition, they may also contain other therapeutically valuable substances. Said compositions may be prepared according to conventional mixing, granulating or coating methods, respectively, and contain about 0.1-100%, or about 0.1-75%, or contain about 1-50%, of the active ingredient such as a small molecule or peptide as described herein, or a pharmaceutically acceptable salt thereof.MBF Ref.028193-0010-WO01 4. Administration

[0063] The small molecules or peptides or pharmaceutically acceptable salts thereof as detailed herein, or at least one component thereof, may be administered or delivered to a subject or a cell of the subject. Small molecules or peptides or pharmaceutically acceptable salts thereof as detailed herein, or at least one component thereof, may be administered or delivered to a subject or a cell of the subject in a therapeutically effective amount. For example, the peptide may be used in excess, such as 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, or 10-fold molar excess. Such compositions can be administered in dosages and by techniques well known to those skilled in the medical arts taking into consideration such factors as the age, sex, weight, and condition of the particular subject, and the route of administration. The presently disclosed small molecules or peptides or pharmaceutically acceptable salts thereof, or compositions comprising the same, may be administered to a subject by different routes including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, intranasal, intravaginal, via inhalation, via buccal administration, intrapleurally, intravenous, intraarterial, intraperitoneal, subcutaneous, intradermally, epidermally, intramuscular, intranasal, intrathecal, intracranial, and intraarticular or combinations thereof.

[0064] The term “a therapeutically effective amount” of a compound as described herein refers to an amount of the compound as described herein that will elicit the biological or medical response of a subject, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc. In one non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound as described herein that, when administered to a subject, is effective to (1) at least partially alleviate, inhibit, prevent and / or ameliorate a condition, or a disorder or a disease (i) mediated by a CHIP mutant, or (ii) associated with mutant or wild-type CHIP activity, or (iii) characterized by activity (normal or abnormal) of mutant or wild-type CHIP; or (2) reduce or inhibit the activity of mutant or wild-type CHIP. In another non-limiting embodiment, the term “a therapeutically effective amount” refers to the amount of the compound of the present disclosure that, when administered to a cell, or a tissue, or a non-cellular biological material, or a medium, is effective to at least partially reducing or inhibiting the activity of a mutant CHIP or enhancing the activity of wild-type CHIP. a. Cell TypesMBF Ref.028193-0010-WO01

[0065] Any of the delivery methods and / or routes of administration detailed herein can be utilized with a myriad of cell types, for example, those cell types currently under investigation for neurodegenerative disorders, including, but not limited to, sensory neurons, neurons of the central nervous system, neurons of the autonomic nervous system, neurons of the enteric nervous system, motor neurons, glial cells, immune cells, fibroblasts, epithelial cells, or cell lines such as, but not limited to, HEK293T cells and mouse embryonic fibroblasts (MEFs). Cells can be modified in vivo or ex vivo. 5. Methods a. Methods of Restoring the Structure of Mutant CHIP

[0066] Provided herein are methods of restoring the tertiary structure of a mutant C terminus of HSP70-Interacting Protein (CHIP) to the tertiary structure of wild-type CHIP. The method may include contacting cells with a small molecule or a peptide. The mutant CHIP may comprise a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. The mutation may be a mutation in the TPR domain of CHIP, coiled-coil domain of CHIP, the U-box domain of CHIP, or a combination thereof. The mutation may be a mutation in the TPR domain of CHIP, such as N34S, G33S, A45V, A46P, Y49C, A52G, I53T, P57L, L58P, C69Y, A67T / V, R66P, N65S, A79P, Q74P, A86T, G101R, A113D / V, or a combination thereof. The mutation may be a mutation in the coiled-coil domain of CHIP, such as D134*, D133V, F131L, W147*, R146C, K143WDfs*4, K143E, K143*, Q157*, R154C, Q196*, R182*, R175E,176ins33 / 63, R175fs*93, L168F, R225*, R222K, or a combination thereof. The mutation may be a mutation in the U-box domain of CHIP, such as C232G, Y230Cfs*9, P228S, G249V, C244Yfs*24, P243L, R241W, G265D, V26Gfs*4, E278Nfs*9, L275Rfs*12 / 16, L275*, P274Afs*3, V289G, E288*, N283K, or a combination thereof.

[0067] The TPR domain may be partially unfolded. The small molecule or the peptide may bind to the mutant CHIP. The cells may be in a subject or in vitro. The mutant CHIP may be in subject that has a neurodegenerative disorder. The neurodegenerative disorder may be Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s disease (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). b. Methods of Increasing Ubiquitination of a Protein by Mutant CHIPMBF Ref.028193-0010-WO01

[0068] Provided herein are methods of increasing ubiquitination of a protein by a mutant C terminus of HSP70-Interacting Protein (CHIP). The method may include contacting cells with a small molecule or a peptide. The small molecule or the peptide may induce a conformational change in the mutant CHIP. The mutant CHIP may comprise a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. The mutation may be a mutation in the TPR domain of CHIP, coiled-coil domain of CHIP, the U-box domain of CHIP, or a combination thereof. The mutation may be a mutation in the TPR domain of CHIP, such as N34S, G33S, A45V, A46P, Y49C, A52G, I53T, P57L, L58P, C69Y, A67T / V, R66P, N65S, A79P, Q74P, A86T, G101R, A113D / V, or a combination thereof. The mutation may be a mutation in the coiled-coil domain of CHIP, such as D134*, D133V, F131L, W147*, R146C, K143WDfs*4, K143E, K143*, Q157*, R154C, Q196*, R182*, R175E,176ins33 / 63, R175fs*93, L168F, R225*, R222K, or a combination thereof. The mutation may be a mutation in the U-box domain of CHIP, such as C232G, Y230Cfs*9, P228S, G249V, C244Yfs*24, P243L, R241W, G265D, V26Gfs*4, E278Nfs*9, L275Rfs*12 / 16, L275*, P274Afs*3, V289G, E288*, N283K, or a combination thereof.

[0069] The cells may be in a subject or in vitro. The subject may have a neurodegenerative disorder. The neurodegenerative disorder may be Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s disease (PD), Machado- Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). The small molecule or the peptide may bind to the mutant CHIP. Affinity between the protein and the mutant CHIP may be increased. The protein may be a binding partner of the TPR domain of CHIP. The protein may be heat shock protein 70 (HSP70), transcription factor II A (TFIIA), charged multivesicular body protein 2B (CHMP2B), or a combination thereof. c. Methods of Treating a Neurodegenerative Disorder

[0070] Provided herein are methods of treating a neurodegenerative disorder. The method may include administering a therapeutically effective amount of a small molecule or a peptide to a subject in need thereof. The subject may have a mutant C terminus of HSP70-Interacting Protein (CHIP) that may comprise a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. The subject may have a wild-type CHIP. The neurodegenerative disorder may be Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’sMBF Ref.028193-0010-WO01 disease (AD), Parkinson’s disease (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). The small molecule or the peptide may bind to the mutant CHIP and / or to the wild-type CHIP.

[0071] The mutation may be a mutation in the TPR domain of CHIP, coiled-coil domain of CHIP, the U-box domain of CHIP, or a combination thereof. The mutation may be a mutation in the TPR domain of CHIP, such as N34S, G33S, A45V, A46P, Y49C, A52G, I53T, P57L, L58P, C69Y, A67T / V, R66P, N65S, A79P, Q74P, A86T, G101R, A113D / V, or a combination thereof. The mutation may be a mutation in the coiled-coil domain of CHIP, such as D134*, D133V, F131L, W147*, R146C, K143WDfs*4, K143E, K143*, Q157*, R154C, Q196*, R182*, R175E,176ins33 / 63, R175fs*93, L168F, R225*, R222K, or a combination thereof. The mutation may be a mutation in the U-box domain of CHIP, such as C232G, Y230Cfs*9, P228S, G249V, C244Yfs*24, P243L, R241W, G265D, V26Gfs*4, E278Nfs*9, L275Rfs*12 / 16, L275*, P274Afs*3, V289G, E288*, N283K, or a combination thereof. d. Methods of Enhancing the Function of Wild-Type CHIP

[0072] Provided herein are methods of enhancing the function of a wild-type C terminus of HSP70-Interacting Protein (CHIP). The method may include contacting cells with a small molecule or a peptide. The small molecule or the peptide may bind to the wild-type CHIP. The cells may be in a subject or in vitro. The wild-type CHIP may be in subject that has a neurodegenerative disorder. The neurodegenerative disorder may be Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s disease (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). e. Methods of Increasing Ubiquitination of a Protein by Wild-Type CHIP

[0073] Provided herein are methods of increasing ubiquitination of a protein by a wild-type C terminus of HSP70-Interacting Protein (CHIP). The method may include contacting cells with a small molecule or a peptide. The small molecule or the peptide may induce a conformational change in the wild-type CHIP. The cells may be in a subject or in vitro. The subject may have a neurodegenerative disorder. The neurodegenerative disorder may be Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s diseaseMBF Ref.028193-0010-WO01 (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). The small molecule or the peptide may bind to the wild-type CHIP. The function of wild-type CHIP may be enhanced. Affinity between the protein and the wild-type CHIP may be increased. The protein may be a binding partner of the TPR domain of CHIP. The protein may be heat shock protein 70 (HSP70), transcription factor II A (TFIIA), charged multivesicular body protein 2B (CHMP2B), or a combination thereof. 6. Examples

[0074] The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention. The present disclosure has multiple aspects and embodiments, illustrated by the appended non-limiting examples. Example 1 Materials and Methods

[0075] The materials and methods described within Example 1 were used for Examples 2- 10 below.

[0076] Cell lines. HEK293T cells and mouse embryonic fibroblasts (MEFs) were grown in DMEM media (11995063, GibcoTM, Thermo Fisher, Waltham, MA) supplemented with 10% (v / v) FBS, 1% (v / v) Penicillin / Streptomycin, and 1% (v / v) Non-Essential Amino Acids (NEAA). Cells were grown in adherent cultures in 10 cm tissue-culture treated dishes (Bio-LiteTM, Thermo Fisher, Waltham, MA) at 37°C with five percent CO2. HEK293T cells were purchased from Duke University Cell Culture Facility. MEF WT and MEF CHIP-KO cells were a gift from Schisler lab.

[0077] SDS-PAGE and Western blot analysis. Samples were combined with Laemmli buffer supplemented with ȕ-ME and boiled at 100°C for 4 minutes unless otherwise indicated. Samples were run on 4-20% Tris-glycine gradient gels in SDS-PAGE running buffer (25 mM Tris, 192 mM glycine, 0.1% (w / v) SDS) at 175 V for 1 hour. For Coomassie stain analysis, gels were fixed with fixation buffer (40% ethanol, 10% acetic acid) at room temperature with gentle rocking for 15 minutes, then stained with Coomassie stain (Bio-RadTM, Thermo Fisher, Waltham, MA) at room temperature (e.g., from about 20°C to about 22°C) overnight with gentleMBF Ref.028193-0010-WO01 rocking. Gels were destained with water by gentle rocking at room temperature. For Western blot analysis, gels were transferred onto PVDF membrane (Bio-RadTM, Thermo Fisher, Waltham, MA) using Criterion transfer apparatus (Bio-RadTM, Thermo Fisher, Waltham, MA) in transfer buffer (25 mM Tris, 192 mM glycine, 20% (v / v) methanol) either at 30 V overnight or at 75 V for 1 hour. Western blots were blocked in 5% milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween-20, pH 7.6) for at least 30 minutes, then incubated with primary antibody diluted in 5% milk in TBST solution either 3 hours at room temperature or overnight at 4°C. For streptavidin blots, Western blots were blocked in 5% BSA in TBST for 1 hour at room temperature and incubated with streptavidin antibody dissolved in 5% BSA in TBST solution at 4°C overnight. Western blots were then washed for 10 minutes with TBST three times and incubated with respective secondary antibodies 1-3 hours at room temperature. Western blots were then washed for 10 minutes with TBST three times and developed by incubating with ECL buffer (50 mM Na2HPO4, 50 mM Na2CO3, 10 mM NaBO3·4H2O, 250 mM luminol, 90 mM coumaric acid, pH 9.5, and 0.025% hydrogen peroxide (v / v)) for 2-5 minutes prior to imaging (ChemiDocTM, Bio-RadTM, Thermo Fisher, Waltham, MA).

[0078] Antibodies were used at the noted concentrations (v / v): Anti-HSP70 (InvitrogenTM, Thermo Fisher, Waltham, MA), anti-CHIP (Cell Signaling Technology, Danvers, MA), anti-Ub (Cell Signaling Technology, Danvers, MA), anti-Myc (Cell Signaling Technology, Danvers, MA) 1:1000; anti-tubulin 1:100; anti-streptavidin (Cytiva, Marlborough, MA) 1:2500. Goat-anti- mouse-HRP (Jackson ImmunoResearch, West Grove, PA) and goat-anti-rabbit-HRP (Jackson ImmunoResearch, West Grove, PA) secondary antibodies were diluted 1:5000 (v / v) in TBST.

[0079] Recombinant protein expression and purification. Recombinant proteins were purified from BL21 (DE3) or Rosetta cells as previously described (Scaglione et al., Mol Cell, 2011.43(4): p.599-612; Umano et al., J Biol Chem, 2022.298(5): p.101899; Madrigal et al., J Biol Chem, 2019.294(50): p.19236-19245; Kanack et al., J Biol Chem, 2018.293(8): p.2735- 2743; Kanack et al., Biochemistry, 2020.59(22): p.2078-2088; Kanack et al., Chembiochem, 2022.23(6): p. e202100633; Smith et al., Biochemistry, 2013.52(32): p.5354-64). For purification of GST tagged proteins, bacteria were lysed in NETN buffer (50 mM Tris, 150 mM NaCl, 0.5% IGEPAL, pH 7.5) supplemented with 1 mM DTT, 5 μg / mL aprotinin, 1 μg / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson Ultrasonics Corporation, Brookfield, CT) at power output of 5 for 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to addition of 500 μL glutathione agarose beads (GoldBio, St. Louis, MO) and tumbling for 3 hours.MBF Ref.028193-0010-WO01 The glutathione beads were then washed three times with NETN and three more times with kinase buffer (50 mM Tris pH 7.5, 50 mM KCl, 0.2 mM DTT) prior to addition of H3C protease. Cleaved proteins were further purified using size-exclusion chromatography (ENrichTMSEC 650, Bio-RadTM, Thermo Fisher, Waltham, MA).

[0080] For purification of UbcH5, bacteria were lysed in MES lysis buffer (30 mM MES, pH 6.0), supplemented with 1 mM DTT, 5 ^g / mL aprotinin, 1 ^g / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson Ultrasonics Corporation, Brookfield, CT) at power output 5 for 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to application onto SP-sepharose column equilibrated with 3 column volumes of MES lysis buffer. Unbound proteins were washed with 5 column volumes of MES lysis buffer. Then bound proteins were eluted using AKTA Start Protein Purification System (Cytiva, Marlborough, MA) at salt gradient from 0% to 100% MES elution buffer (30 mM MES, 500 mM NaCl, pH 6.0) supplemented with 2 mM DTT and 1:1000 (v / v) supersaturated PMSF. Each fraction was analyzed by SDS-PAGE gel and Coomassie stain. Fractions with protein of interest were further purified using size-exclusion chromatography (ENrichTMSEC 75, Bio-RadTM, Thermo Fisher, Waltham, MA). Each fraction was analyzed using SDS-PAGE gel and Coomassie stain. And protein fractions were combined and concentrated using concentrator (Sartorius, Göttingen, Germany) by spinning at 4000 g.

[0081] For purification of His tagged proteins, bacteria were lysed in buffer A (50 mM Tris, 1M NaCl, 0.5% (v / v) Triton-X100), supplemented with 1 mM DTT, 5 ^g / mL aprotinin, 1 ^g / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson Ultrasonics Corporation, Brookfield, CT) at power output 5 for 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to addition of 500 μL of Ni-HTC beads (GoldBio, St. Louis, MO) and tumbling for 1 hour. The Ni-HTC beads were then washed three times with buffer A, three times with buffer B (50 mM Tris, 1 M NaCl, 20 mM imidazole, pH 7.5), and three more times with buffer C (50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.5) prior to elution 3 times each time with 500 μL of elution buffer (50 mM Tris, 150 mM NaCl, 400 mM imidazole, pH 7.5) by tumbling at 4°C for 15 minutes. Proteins were further purified using size-exclusion chromatography (ENrichTMSEC 650, Bio-RadTM, Thermo Fisher, Waltham, MA).

[0082] For purification of ubiquitin, bacteria were lysed in ubiquitin lysis buffer (50 mM Tris, 1 mM EDTA, 0.05% (v / v) Tween-20, pH 7.6) supplemented with 1 mM DTT, 5 ^g / mL aprotinin,MBF Ref.028193-0010-WO01 1 ^g / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson Ultrasonics Corporation, Brookfield, CT) at power output 6 with 80% interval 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to dialyzing in 50 mM sodium acetate (pH 4). Samples were then applied to SP-Sepharose column and eluted with a linear gradient of NaCl prior to size-exclusion chromatography (ENrichTMSEC 75, Bio-RadTM, Thermo Fisher, Waltham, MA).

[0083] Partial proteolysis assays and mass spectrometry. Partial proteolysis assays were modified based on previously published protocols (Fontana et al., Methods Mol Biol, 2012.896: p.297-318). CHIP proteins were diluted to 1 μg / μL in a trypsin digestion buffer (50 mM Tris, 50 mM KCl, pH 8.0). 5 μg of CHIP proteins were prepared in each reaction, mixed with 1:3000 trypsin:CHIP w / w ratio of trypsin (unless mentioned otherwise) in trypsin digestion buffer, and incubated at respective temperatures for 5 minutes. For partial proteolysis assays comparing CHIP and CHIPA52G, trypsin digestions were performed at 37°C for 5 minutes. Samples were then stopped with the addition of 4X Laemmli buffer and separated by SDS-PAGE and visualized with Coomassie. For partial proteolysis mass spectrometry analysis, the band corresponding to the undigested samples and a band indicated by an asterisk were stored in 100 μL water, and immediately subjected to mass spectrometry analysis for each sample.

[0084] For gel band analysis, SDS-PAGE gel bands were subjected to reduction, alkylation, and in-gel tryptic digestion as described here: genome.duke.edu / sites / genome.duke.edu / files / In- gelDigestionProtocolrevised_0.pdf. Digested peptides were lyophilized to dryness and resuspended in 12 μL of 0.2% formic acid / 2% acetonitrile. Each sample was subjected to chromatographic separation on a Waters MClass UPLC equipped with a 1.7 μm HSS T3 C1875 μm I.D. X 250 mm reversed-phase column (NanoFlow data). The mobile phase consisted of (A) 0.1% formic acid in water and (B) 0.1% formic acid in acetonitrile. 3 μL was injected and peptides were trapped for 3 minutes on a 5 μm Symmetry C18180 μm I.D. X 20 mm column at 5 μl / minute in 99.9% A. The analytical column was then switched in-line and a linear elution gradient of 5% B to 40% B was performed over 60 minutes at 400 nL / minute. The analytical column was connected to a Fusion Lumos mass spectrometer (Thermo Fisher, Waltham, MA) through an electrospray interface operating in a data-dependent mode of acquisition. The instrument was set to acquire a precursor MS scan from m / z 375-1500 at R=120,000 (target AGC 2×105, max IT 50 milliseconds) with MS / MS spectra acquired in the ion trap (target AGC 1×104, max IT 100 milliseconds). For all experiments, HCD energy settings were 30 V and a 20-second dynamic exclusion was employed for previously fragmented precursor ions.MBF Ref.028193-0010-WO01

[0085] Raw LC-MS / MS data files were processed in Proteome Discoverer 2.5 (Thermo Fisher, Waltham, MA) and then submitted to independent Sequest database searches against a Human protein (containing the custom CHIP sequence) database containing both forward (20260 entries) and reverse entries of each protein. Search tolerances were 2 ppm for precursor ions and 0.8 Da for product ions using trypsin specificity with up to two missed cleavages. Carbamidomethylation (+57.0214 Da on C) was set as a fixed modification, whereas oxidation (+15.9949 Da on M) was considered a dynamic mass modifications. Following percolator scoring and adjustment to a maximum 1% false discovery rate, peptide identifications were imported into Skyline (UWashington, MacCoss Laboratory) and extracted ion chromatograms were extracted for confidently identified peptides. Note that total CHIP expression levels were normalized using peptide sequences found in the middle of the sequence.

[0086] Nuclear magnetic resonance spectroscopy. The TPR domain of CHIP was expressed in Rosetta cells and purified based on previously published protocols (Zhang et al., Biomol NMR Assign, 2017.11(1): p.5-9; Marley et al., J Biomol NMR, 2001.20(1): p.71-5). CHIP-TPR domain constructs were transformed into Rosetta cells and inoculated in 10 mL overnight cultures to incubate at 37°C overnight. The next day, each overnight culture (4X) was used to inoculate a 1 L culture to incubate at 37°C. The optical density (OD600) of each 1 L culture was determined. When the OD was between 0.6 to 0.8, cells were collected and washed twice with 100 mL PBS. The harvested cells from four liters of culture were then transferred to 1 L of minimal media (50 mM Na2HPO4, 50 mM KH2PO4, 5 mM Na2SO4, 3 g / L15NH4Cl, 5 g / L NaCl) supplemented with 0.8% glucose, 2 mM MgSO4, 30 μM CaCl2, vitamin solution and antibiotics, and induced with 1 mM of IPTG at 16°C overnight. Cells were then collected and lysed in CHIP TPR lysis buffer (180 mM Tris, 450 mM NaCl, 10% glycerol, 5 mM ȕ-ME, pH 7.8), and tumbled with glutathione agarose beads for 3 hours. Proteins bound on beads were collected and washed with CHIP TPR lysis buffer and CHIP TPR cleavage buffer (20 mM HEPES, 50 mM NaCl, 1 mM EDTA, 10% glycerol, 5 mM ȕ-mercaptoethanol, pH 7.5). Proteins were cleaved by H3C protease in CHIP TPR cleavage buffer at 4°C with overnight tumbling. The next day proteins were collected and concentrated to final concentrations 200 μM for CHIP-TPR and 100 μM for CHIPA52G-TPR.

[0087] 1H / 15N-HSQC NMR spectra were acquired using a Varian / Agilent 800MHz DirectDrive2 spectrometer. Experiments were performed with concentrations at 200 μM for CHIPWT-TPR and 100 μM for CHIPA52G-TPR in buffer supplemented with 10% D2O. 8MBF Ref.028193-0010-WO01 acquisitions per FID for CHIP-TPR samples and 16 acquisitions per FID for CHIPA52G-TPR samples were collected with 256 increments each in the indirect15N dimensions for a total acquisition time of 57 and 114 minutes respectively at 25°C. For collection of data for CHIPWT- TPR at 45°C, the samples were equilibrated at 45°C in spectrometer for at least 15 minutes prior to data collection. For data collection of CHIPWT-TPR at 45°C, 16 acquisitions per FID were required because some precipitation was observed at elevated temperatures. All spectra were analyzed using NMRPipe and NMRViewJ. Peaks were identified and labeled based on previous assignments (Zhang et al., Biomol NMR Assign, 2017.11(1): p.5-9).

[0088] In vitro ubiquitination assay. In vitro ubiquitination assays were performed as previously described (Umano et al., J Biol Chem, 2022.298(5): p.101899; Kanack et al., J Biol Chem, 2018.293(8): p.2735-2743). For substrate ubiquitination, reaction mixtures with 1 μM CHIP or mutant CHIP, 100 nM E1, 1 μM UbcH5c, 1 μM Hsp70, 5 mM ATP, 5 mM MgCl2, and 500 μM Ub were prepared in kinase buffer. Reactions were incubated at 37°C unless otherwise indicated for 1 hour prior to the addition of 4X Laemmli buffer and boiling. Proteins were separated using SDS-PAGE and visualized using Western blotting with the indicated antibodies.

[0089] Fluorescence polarization assays. Fluorescence polarization assays were performed as previously described (Umano et al., J Biol Chem, 2022.298(5): p.101899; Kanack et al., J Biol Chem, 2018.293(8): p.2735-2743; Ricci and Williams, Curr Chem Genomics, 2008.2: p.90-5). Hsc70 peptide labeled with Rhodamine B were diluted in FP buffer (50 mM HEPES, 75 mM NaCl, and 0.01% Triton X-100, pH 7.4) to 20 nM and added to 384-well black flat-bottom assay plates. CHIP was then added at the indicated concentrations, to each sample. Samples were incubated at the indicated temperatures for 30 minutes in the dark prior to taking measurements. Data were analyzed by GraphPad Prism and exhibited as average with SD display.

[0090] Transfection of cells. HEK293T cells were transfected with lipofectamine 3000 per the manufacturer protocol. HEK293T cells were grown on 12-well or 10-cm tissue-culture treated plates until 60-80% confluency. For 12-well transfection, 1 μg of plasmid and 2 μL of P3000 reagent were mixed with 50 μL Opti-MEM media (GibcoTM, Thermo Fisher, Waltham, MA) while 1 μL of lipofectamine was mixed with 50 μL Opti-MEM media prior to incubation at room temperature for 5 minutes. Lipofectamine in Opti-MEM was then combined with plasmid DNA and P3000 reagent and allowed to incubate for 15 minutes at room temperature. Media was then changed on the cells and the DNA and lipofectamine mixture were applied.MBF Ref.028193-0010-WO01

[0091] NanoBiT assays. NanoBiT assays were performed following published protocols (Umano et al., J Biol Chem, 2022.298(5): p.101899). In short, HEK293T cells were grown in 12-well plates and transfected using lipofectamine P3000 (InvitrogenTM, Thermo Fisher, Waltham, MA). Twenty-four hours post transfection cells were trypsinized, collected, and resuspended in 2 mL OptiMEM (GibcoTM, Thermo Fisher, Waltham, MA). Next, 100 μL of cell suspension was plated on 96-well white flat-bottom tissue-culture treated plate (cite plate) and incubated at 37°C for 45 minutes. Nano-Glo Live Cell Reagent was prepared by mixing 1:20 (v / v) ratio of live cell substrate and Nano-Glo Live Cell buffer. 25 μL of Nano-Glo Live Cell Reagent (Promega, Madison, WI) was added each well. Samples were shaken for 15 seconds and read for luminescence counts. For NanoBiT assays with heat shock conditions, after cells were plated on white flat-bottom tissue-culture treated 96-well plate, cells were incubated at 43°C for 45 minutes. Data were analyzed using GraphPad Prism.

[0092] Thioester formation and lysine discharge assays. Thioester formation and lysine discharge assays were performed as previously described (Umano et al., J Biol Chem, 2022. 298(5): p.101899; Kanack et al., J Biol Chem, 2018.293(8): p.2735-2743). For thioester formation assays, a mixture of 20 μM CHIP, 1 μM E1, 20 μM UbcH5c, 5 mM ATP, 5 mM MgCl2, and 20 mM Ub were prepared in kinase buffer and incubated at indicated temperatures for 30 minutes. Reactions were stopped with 4X Laemmli buffer and boiling at 100°C 4 minutes or with 4X Laemmli buffer without ȕ-ME or boiling. For lysine discharge assays the reactions were stopped by the addition of 50 mM EDTA prior to addition of 5 mM lysine for the times indicated.

[0093] Electroporation of MEFs. MEFs were plated in 6-well plates at the density of 2.5×105cells per well 24 hours prior to electroporation. Each well was trypsinized, collected by centrifuging at 1200 rpm for 3 minutes, resuspended in 250 μL GlutaMAX (GibcoTM, Thermo Fisher, Waltham, MA), and mixed with 10 μg of plasmid. The mixtures were then transferred to 4 mm cuvettes (Bulldog Biosciences, Portsmouth, NH), and electroporated with square-wave protocol at 250 V, 2 pulses with each 10 milliseconds pulse and a 10 seconds interval prior to being re-plated.

[0094] Confocal microscopy. MEFs were electroporated with GFP-tagged CHIP constructs as previously described herein. Twenty-four hours post electroporation, cells were trypsinized, resuspended in 4 mL of media, and re-plated to one well of 4-well Lab-Tek chamber slides (Thermo Fisher, Waltham, MA). Cells were allowed to adhere and grow at 37°C 5% CO2for 24 hours prior to confocal microscopy. Live cells were incubated with Hoechst stainingMBF Ref.028193-0010-WO01 (InvitrogenTM, Thermo Fisher, Waltham, MA) for 10 minutes, then imaged using Zeiss LSM 880 Inverted Laser Scanning Confocal Microscope following manufacturer instructions under 37°C and 5% CO2incubation with 63X magnification. For confocal microscopy of CHIP localization under heat shock and recovery conditions, cells were heat shocked at 43°C for 30 minutes, then imaged immediately using Zeiss LSM 880 for heat shock images. Cells were incubated at 37°C 5% CO2for time indicated for recovery after heat shock images. Confocal microcopy images were analyzed using Fiji.

[0095] Cytosolic and nuclear extraction to verify CHIP localization in cells. Cytosolic and nuclear extraction was performed as previously described (Nabbi and Riabowol, Cold Spring Harb Protoc, 2015.2015(8): p.769-72). Myc / His tagged CHIP (MHCHIP) or mutant CHIP (MHCHIPA52G) were electroporated into CHIP-KO MEFs in 6-well plates as previously described herein. 24 hours after electroporation, fresh media was added to cells. Twenty-four hours later cells were washed twice with PBS and collected in 450 μL PBS. Samples were briefly centrifuged, and supernatant was removed. Cells were then resuspended in 450 μL PBS supplemented with 0.1% Triton, and 150 μL of suspension was saved and mixed with 4X Laemmli buffer as the whole cell lysate. The remainder suspension was briefly centrifuged, and 150 μL supernatant was saved and mixed with 4X Laemmli buffer as the cytosolic fraction. The remainder of supernatant was removed. Pellet was washed with 500 μL PBS with 0.1% Triton, then resuspended in 90 μL 1X Laemmli buffer as the nuclear fraction. Samples were next briefly sonicated and 20 μL of each sample was loaded to an SDS-PAGE gel for Western blot analysis with the indicated antibodies.

[0096] Thermal shift assay. Ube1, UbcH5c, and CHIP proteins were diluted into PBS to 5 μM in 4°C. 45 μL of proteins were combined with 5 μL of 200X SYPRO Orange protein stain diluted in PBS in each well of 96-well plates. The plates were briefly centrifuged prior to analysis for fluorescence intensity at excitation wavelength 490 nm and emission wavelength 580 nm. Temperature was increased starting from 25°C to 95°C with accelerating temperature increase at 1°C / minute each cycle. Data was analyzed using GraphPad Prism and thermal melting curves were generated with Gaussian curve regression to fit points between 25°C to 95°C for each sample (Kanack et al., J Biol Chem, 2018.293(8): p.2735-2743, 87).

[0097] Bio-ID Sample Preparation. Expression plasmids for miniTurbo was obtained from Addgene, Watertown, MA (plasmid #107170) and modified to remove the nuclear export signal. CHIP was cloned into the vector using the HindIII and NheI restriction sites. The A52G mutationMBF Ref.028193-0010-WO01 was added by site-directed mutagenesis (Agilent Technologies, Santa Clara, CA, QuikChange Lightning). MiniTurbo tagged CHIP (MTCHIP), mutant CHIP (MTCHIPA52G), or miniTurbo vector were transfected into HEK293 cells at 60% confluency in 10 cm dishes according to manufacturer’s directions (Lipofectamine 3000, InvitrogenTM, Thermo Fisher, Waltham, MA). 24 hours after transfection the media was changed and 16 hours later 500 μM of biotin was added. The cells were incubated for 30 minutes prior to termination of the reaction by placing the cells on ice and washing 5 times with ice-cold PBS. Cells were then harvested in PBS and spun down at 1500 RPM for 3 minutes at 4°C. The cell pellets were resuspended in 500 μL of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% IGEPAL, pH 8.0) with PMSF and protease inhibitor cocktail. The cells were then allowed to lyse on ice for 5 minutes prior to sonication and centrifugation at 4°C for 10 minutes at 10,000 rpm. The supernatant was removed and saved and a BCA assay (PierceTM, Thermo Fisher, Waltham, MA) was performed to determine total protein content.

[0098] In a 1.7 mL tube, 30 μL of Neutravidin beads (Thermo Fisher, Waltham, MA) was washed 3 times with 500 μL of RIPA buffer. Lysate was added to the beads (total of 1.6 mg of protein) and allowed to tumble overnight at 4°C. The next morning, the beads were spun at 600 g at 4°C and the lysate removed. The beads were transferred to low-binding Epitubes (Eppendorf, Hamburg, Germany) and a series of room temperature washes were done with a 21-gauge needle and 1 mL syringe to remove any unbound or nonspecific proteins. 500 μL of each wash solution was applied and allowed to rock for 10 minutes before removal and application of the next wash. The washes consisted of two washes with 2% SDS, one wash with 1% TritonX100, 1% sodium deoxycholate, 25 mM lithium chloride, two wash with 1 M sodium chloride, and five washes with 50 mM ammonium bicarbonate. After the final wash, bound proteins were eluted from the bead by adding 70 μL elution buffer (125 mM Tris pH 6.8, 4% SDS, 10 mM DTT, 5 mM biotin), boiling at 95°C for 5 minutes and vortexing 3X during this time. The beads were then spun briefly at 600 g and the supernatant transferred to a new low- binding Epitube and stored at -80°C until delivery to the proteomics core. The experiment was repeated in triplicate.

[0099] For miniTurbo ID, samples were spiked with undigested bovine casein at a total of either 1 or 2 pmol as an internal quality control standard. Next, samples were reduced with 10 mM dithiolthreitol for 30 minutes at 80°C, alkylated with 20 mM iodoacetamide for 30 minutes at room temperature, then supplemented with a final concentration of 1.2% phosphoric acid and 623 ^L of S-Trap (ProtiFi, Fairport, NY) binding buffer (90% MeOH / 100 mM TEAB). ProteinsMBF Ref.028193-0010-WO01 were trapped on the S-Trap microcartridge, digested using 20 ng / ^L sequencing grade trypsin (Promega, Madison, WI) for 1 hour at 47°C, and eluted using 50 mM TEAB, followed by 0.2% FA, and lastly using 50% ACN / 0.2% FA. All samples were then lyophilized to dryness. Samples were resolubilized using 12 ^L of 1% TFA / 2% ACN with 25 fmol / ^L yeast ADH. [000100] Whole cell proteomics and diGLY-profiling sample preparation. Protein lysates were collected from WT or CHIP-KO MEFs with or without heat shock at 43°C for 1 hour. Fresh media was supplied to 2 plates of each MEF WT and MEF CHIP- / - each cell line at 80% confluency prior to temperature treatment. Prior to collection cells were washed and cell lysates were immediately collected in urea lysis buffer (8 M urea, 20 mM HEPES, pH 8.0). Cell lysates were stored at -80°C. [000101] For whole cell proteomics, samples were normalized to 50 ^g using 8 M urea and spiked with undigested bovine casein at a total of either 200 fmol / ^g or 400 fmol / ^g as an internal quality control standard. Next, samples were reduced with 10 mM dithiolthreitol for 45 minutes at 32°C and alkylated with 20 mM iodoacetamide for 30 minutes at room temperature. Proteins were digested using sequencing grade trypsin (Promega, Madison, WI) at an enzyme:protein ratio of 1:50 overnight at 37°C. All samples were acidified using TFA, subjected to SPE clean-up (Waters SEP-PAK, 50 mg), then lyophilized to dryness. Samples were resolubilized using 100 ^L of 1% TFA / 2% ACN with 12.5 fmol / ^L yeast ADH. [000102] For di-GLY profiling, samples were normalized to 1.5 mg (except 2 samples which did not have enough material) using 8 M urea and spiked with BSA-GG at either 20 ng or 40 ng as an internal quality control standard. Next, they were reduced with 10 mM dithiolthreitol for 30 minutes at 32°C and alkylated with 20 mM iodoacetamide for 30 minutes at room temperature. Proteins were digested using 5 ^g / ^L sequencing grade trypsin (Promega, Madison, WI) at an enzyme:protein ratio of 1:50 overnight at 32°C. Samples were then acidified with TFA and subjected to C18 clean-up (Sep-Pak, 50 mg bed). All samples were then lyophilized to dryness. Peptides were enriched using the PTMScan®HS Ubiquitin / SUMO Remnant Motif (K-İ-GG) Kit (Cell Signaling Technology, Danvers, MA) according to manufacturer protocol. After elution, samples were lyophilized to dryness (Kim et al., Mol Cell, 2011.44(2): p.325-40). [000103] LC-MS / MS Analysis. For miniTurbo ID, quantitative LC / MS / MS was performed on 4 ^L using a nanoAcquity UPLC system (Waters Corp., Milford, MA) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (ThermoMBF Ref.028193-0010-WO01 Fisher, Waltham, MA) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C1820 mm × 180 ^m trapping column (5 ^L / minute at 99.9 / 0.1 v / v water / acetonitrile), after which the analytical separation was performed using a 1.8 ^m Acquity HSS T3 C1875 ^m × 250 mm column (Waters Corp., Milford, MA) with a 90-minute linear gradient of 5% to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nL / minute with a column temperature of 55°C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40 V, -60 V, -80 V). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (at m / z 200) full MS scan from m / z 375–1500 with a target AGC value of 4×105ions was performed. MS / MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 104and max fill time of 35 millisecond. The total cycle time for each CV was 0.66 second, with total cycle times of 2 seconds between like full MS scans. A 20-second dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hours. [000104] For whole cell proteomics, quantitative LC / MS / MS was performed on 2 ^L using an MClass UPLC system (Waters Corp., Milford, MA) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo Fisher, Waltham, MA) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C1820 mm × 180 ^m trapping column (5 ^L / minute at 99.9 / 0.1 v / v water / acetonitrile), after which the analytical separation was performed using a 1.8 ^m Acquity HSS T3 C1875 ^m × 250 mm column (Waters Corp., Milford, MA) with a 90-minute linear gradient of 5% to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nL / minute with a column temperature of 55°C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (-40 V, -60 V, -80 V). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (at m / z 200) full MS scan from m / z 375–1500 with a target AGC value of 4×105ions was performed. MS / MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 104and max fill time of 35 milliseconds. The total cycle time for each CV was 0.66 seconds, with total cycle times of 2 seconds between like full MS scans. A 20-second dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hours. [000105] For diGLY profiling, quantitative LC / MS / MS was performed on 3 ^L of each sample, using an MClass UPLC system (Waters Corp., Milford, MA) coupled to a Thermo OrbitrapMBF Ref.028193-0010-WO01 Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo Fisher, Waltham, MA) via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C1820 mm × 180 ^m trapping column (5 ^L / minute at 99.9 / 0.1 v / v water / acetonitrile), after which the analytical separation was performed using a 1.8 ^m Acquity HSS T3 C1875 ^m × 250 mm column (Waters Corp., Milford, MA) with a 90-minute linear gradient of 3% to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nL / minute with a column temperature of 55°C. Data collection on the Fusion Lumos mass spectrometer was in a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (at m / z 200) full MS scan from m / z 375–1500 with a target AGC value of 4×105ions was performed. MS / MS scans were acquired in the linear ion trap in “rapid” mode with a target AGC value of 104and max fill time of 50 milliseconds. There was a total cycle times of 2 seconds between like full MS scans. A 20-second dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each sample injection was approximately 2 hours. [000106] Quantitative Data Analysis. For miniTurbo ID, following UPLC-MS / MS analyses, data were imported into Proteome Discoverer 2.5 (Thermo Fisher, Waltham, MA) and individual LCMS data files were aligned based on the accurate mass and retention time of detected precursor ions (“features”) using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was measured based on peak intensities of selected ion chromatograms of the aligned features across all runs. The MS / MS data was searched against the SwissProt H. sapiens database (downloaded in Nov 2019), a common contaminant / spiked protein database (bovine albumin, bovine casein, yeast ADH, etc.), and an equal number of reversed-sequence “decoys” for false discovery rate determination. Sequest with Infernys enabled (v 2.5, Thermo PD) were utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and a variable modification on Met (oxidation). Precursor mass tolerances were 2.0 ppm and product ion mass tolerances were 0.8Da with full trypsin enzyme rules required. Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate based on q-value calculations. Note that peptide homology was addressed using razor rules in which a peptide matched to multiple different proteins was exclusively assigned to the protein has more identified peptides. Protein homology was addressed by grouping proteins that had the same set of peptides to account for their identification. A master protein within a group was assigned based on percent coverage.MBF Ref.028193-0010-WO01 [000107] For whole cell proteomics and diGLY profiling, following UPLC-MS / MS analyses, data were imported into Proteome Discoverer 3.0 (Thermo Fisher, Waltham, MA). In addition to quantitative signal extraction, the MS / MS data was searched against the SwissProt M. musculus database (downloaded in Nov 2019) and a common contaminant / spiked protein database (bovine albumin, bovine casein, yeast ADH, human keratin, etc.), and an equal number of reversed-sequence “decoys” for false discovery rate determination. Sequest with Infernys enabled (v 3.0, Thermo PD) was utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and variable modification on Met (oxidation) and Lys (-GG, ubiquitin remnant). Search tolerances were 2 ppm precursor and 0.8 Da product ion with full trypsin enzyme rules. Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate based on q-value calculations. Note that peptide homology was addressed by only using unique peptides for quantitation. Protein homology was addressed by grouping proteins that had the same set of peptides to account for their identification. A master protein within a group was assigned based on percent coverage. [000108] Prior to normalization, a filter was applied such that a peptide was removed if it was not measured in at least 50% of the samples in a single group. Next, the following imputation strategy was applied to missing values. If less than half of the values were missing in a biological group, values were imputed with an intensity derived from a normal distribution of all values defined by measured values within the same intensity range (20 bins). If greater than half values were missing for a peptide in a group and a peptide intensity was > 5×106, then it was concluded that peptide was misaligned and its measured intensity is set to 0. All remaining missing values were imputed with the lowest 2% of all detected values. Lastly, the peptide intensities were then subjected to a trimmed-mean normalization in which the top and bottom 10 percent of the signals were excluded and the average of the remaining values was used to normalize across all samples. For –GG modified peptides, only these modified peptides were considered in the normalization procedure. For the BioID and whole cell proteomics, all peptides belonging to the same protein were then summed into a single intensity. These normalized level intensities were what was used for the remained of the analysis. [000109] Isolation of RNA samples for RNA-seq. RNA samples were isolated from WT and CHIP-KO MEFs with or without heat shock. Fresh media was supplied to 2 plates of each cell line at 80% confluency prior to temperature treatment. Next, one plate of each WT and CHIP-MBF Ref.028193-0010-WO01 KO MEFs were incubated at 37°C and the other plate of each cell line were incubated at 43°C for 1 hour. After temperature treatment, RNA was collected from each treatment following manufacturer instructions (Qiagen, Hilden, Germany). For all samples, total amounts of RNA were above 400 ng, A260 / 280 values were between 1.8 and 2.2, and A260 / 230 values were above 1.8. Samples were stored at -80°C prior to sequencing (Novogene, China). The experiment was performed in triplicate. [000110] Splicing analysis. Spicing analyses was performed using MAJIQ (v2.4) as described previously (Vaquero-Garcia et al., Elife, 2016.5: p. e11752). Initial analyses of local splice variants (LSVs) in biological triplicate datasets: WT MEF, CHIP KO MEF, WT:HS MEF, and CHIP KO MEF:HS were performed in parallel using aligned .bam files (see RNA-seq methods herein) and MAJIQ builder with default settings. Mus_musculus.GRCm38.102.chr.gff3 from Esembl v102 was used as the transcript feature reference for defining LSVs. ǻPSI values were quantified between pairwise comparisons of datasets using the MAJIQ deltapsi package (v2.4) at a threshold of ǻPSI ^ 20. Splicing changes in pairwise datasets were compared using the MAJIQ package Voila. These specific LSVs were then compared against calculated expression changes (see RNA-seq methods herein) and hierarchically clustered using the python package seaborn.clustermap. GO enrichment analyses of calculated clusters was performed using STRING v11.5 (Szklarczyk et al., Nucleic Acids Res, 2021.49(D1): p. D605-D612). [000111] Reverse transcription cDNA synthesis and PCR to verify splicing differences in RNA- seq. Wild-type and CHIP-KO MEFs were heat shocked, and RNA samples were collected as in sample preparation for the RNA-seq. cDNA samples were synthesized from collected RNA samples using SuperScript III First -Strand Synthesis System (InvitrogenTM, Thermo Fisher, Waltham, MA). 5 μg RNA was used for each sample and first pre-treated by incubating with 5n g / μL random hexamers and 1 mM dNTPs in 10 μL volume at 65°C for 5 minutes prior to being chilled on ice for 1 minute. Pre-treated samples were incubated with 10 μL of RT buffer, 10 mM MgCl2, 20 mM DTT, 4 U / μL RNAseOUT (InvitrogenTM, Thermo Fisher, Waltham, MA), and 20 U / μL SuperScript III reverse transcriptase (InvitrogenTM, Thermo Fisher, Waltham, MA) at 25°C for 10 minutes, 50°C for 50 minutes, followed by chilling on ice for 1 minute. RNA templates were then digested by RNAse H (InvitrogenTM, Thermo Fisher, Waltham, MA) at 37°C for 20 minutes. Synthesized cDNA samples were stored at -20°C until further analysis or immediately proceeded to the next steps. cDNA samples were used as templates in PCR reactions. 100 ng of cDNA was used in each reaction, 1 μL of 10 μM primer forward (5’- GACACAGAGGACTTCGTGGG-3’), 1 μL of 10μM primer reverse (5’-MBF Ref.028193-0010-WO01 ACAGATAAGCAGGTCTGGGTG-3’) and 12.5 μL 2X Red Taq master mix (Azura Genomics, Raynham, MA) were used in 25 μL reaction. PCR reactions were performed with the following thermal cycles: 95°C for 1 minute, 35 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 2 minutes, then hold at 4°C. PCR products were run on 3.5% agarose gel for 1 hour before imaging using gel imager (ChemiDoc, Bio-RadTM, Thermo Fisher, Waltham, MA). Example 2 Both heat stress and SCA48 mutations induce a conformational change in the TPR domain of CHIP [000112] Utilization of circular dichroism (CD) spectroscopy coupled with thermal melt analysis demonstrated that recombinant CHIP has an unusual unfolding profile with two unfolding transitions. Interestingly, the first unfolding event occurs slightly above physiological temperature and a mutation in the TPR domain of CHIP (CHIPA52G) that causes SCA48 results in decreased helical content prior to this first unfolding event. This suggests that mutations in the TPR domain of CHIP that cause SCA48 may result in CHIP adapting an abnormal conformation that is similar to one induced by heat stress. In addition to the CHIPA52Gmutation, seven additional mutations have been identified in the TPR domain of CHIP and cause SCA48. These mutations occur in highly conserved residues, and map to a single helical interface in CHIP (FIGS.1A-B). Thus, this region of the TPR domain has important unknown biological functions. [000113] To begin experimentally assessing how SCA48 mutations and heat stress alter the structure of CHIP, partial proteolysis experiments were performed that compared the digestion pattern of CHIP with heat shock (42°C) or at 37°C with SCA48 mutations (FIGS.2A-B). With both SCA48 mutant CHIP and with heat stress increased proteolysis was observed, consistent with a decrease in secondary structure (FIGS.1C-D). To determine what regions of CHIP become more disordered with SCA48 mutations or heat stress, mass spectrometry was performed on undigested CHIP and on a partially digested form of either wild-type or SCA48 mutant CHIP. Consistent with both heat stress and SCA48 mutations inducing partial unfolding of the TPR domain of CHIP, a loss of peptides that mapped to the most N-terminal TPR repeat in the partially digested CHIP samples was observed (FIG.1E and TABLE 1). TABLE 1 shows that the first TPR repeat of CHIP is susceptible to trypsin cleavage. Either uncut or cleaved CHIP (full-length CHIP and the band at the position of the asterisk in FIG.1C) were analyzed byMBF Ref.028193-0010-WO01 mass spectrometry. Peptides that are decreased in the cleaved version of CHIP were quantitated and are listed (n=3). Also, one cleavage site was missed in the TPR domain of SCA48 mutant CHIP more frequently than in wildtype CHIP (TABLE 2). In addition to increased proteolysis in the TPR domain of CHIP, one peptide that had increased cleavage in the linker domain of SCA48 mutant CHIP was also identified (TABLE 2). TABLE 2 shows that one peptide in the TPR domain of CHIP is preferentially lost in SCA48 mutant CHIP compared to wild-type CHIP. The band corresponding the asterisk in FIG.1C for both CHIP and CHIPA52Gwere analyzed by mass spectrometry. The peptide in the TPR domain of CHIP that was decreased in CHIPA52Gis shown. One peptide in the linker domain of CHIP is preferentially cleaved in SCA48 mutant CHIP. The band corresponding to the asterisk in FIG.1C for both CHIP and CHIPA52Gwere analyzed by mass spectrometry. The peptide in the linker domain of CHIP that was decreased in CHIPA52Gis shown in TABLE 2. TABLE 1. First TPR repeat of CHIP is susceptible to trypsin cleavage.MBF Ref.028193-0010-WO01 TABLE 2. One peptide in the TPR domain of CHIP is preferentially lost or cleaved in SCA48 mutant CHIP.[000114] While the partial proteolysis results herein are consistent with both heat stress and SCA48 mutations inducing structural changes to CHIP, they do not directly identify the exact regions where structural alterations occur. Therefore, to directly observe structural changes that occur with SCA48 mutations and heat stress, 2D1H / 15N-HSQC NMR spectra on the TPR domain of CHIP were collected. The spectra of wild-type CHIP herein closely matched previously published 2D1H / 15N-HSQC NMR spectra for the TPR domain of CHIP. Consistent with the partial proteolysis results herein, a loss in signal was observed for amino acids that correspond to the first three helices of the TPR domain of CHIP with both the introduction of SCA48 mutations or with increased temperature (FIGS.1F-G). Of note, an even more pronounced change in spectra for heat stressed TPR domain was observed (FIG.1H). Together these data are consistent with both SCA48 mutations and heat stress inducing partial unfolding of the TPR domain of CHIP. Example 3 Heat stress and SCA48 mutations decrease the affinity of CHIP for chaperones but do not disrupt E3 ligase activity [000115] The finding that both the induction of heat stress or the presence of SCA48 mutations results in partial unfolding of the TPR domain suggests that in both conditions CHIP may have defects in its ability to bind chaperones and ubiquitinate chaperone clients. To test this, ubiquitination assays were performed with either wild-type CHIP or one of three SCA48 CHIP mutants (CHIPG33S, CHIPA52G, or CHIPA67T). While wild-type CHIP efficiently polyubiquitinated HSP70, defects in HSP70 ubiquitination with each of the SCA48 mutants tested were observed (FIG.3A). To compare this to heat stressed CHIP, ubiquitination assays at either 25°C, 37°C, 42°C, 45°C, or 50°C were performed. Interestingly, similar to SCA48 mutant CHIP, heat stressed CHIP also had decreased ability to ubiquitinate HSP70 (FIG.3B).MBF Ref.028193-0010-WO01 To validate that Ube1, UbcH5, and ubiquitin were still functional under increased temperatures, their thermostability and function were analyzed, and no defects were found (FIGS.5A-C). Together these data are consistent with CHIP being unable to efficiently ubiquitinate chaperones or their clients during heat stress or with SCA48 mutations. [000116] In the ubiquitination assays herein, the loss of HSP70 ubiquitination may be due to a loss of E3 ligase activity or could be due to decreased affinity between CHIP and chaperones. To begin differentiating between these two possibilities, fluorescence polarization (FP) was utilized to monitor the interaction between a fluorescently labeled peptide that mimics the C- terminus of chaperones and CHIP. FP was performed with wild-type CHIP at 25°C, 37°C, 42°C, and 45°C and heat stressed CHIP had decreased interactions with chaperones in vitro (FIG. 3C). To confirm that these results recapitulate what is occurring in cells, the interaction between CHIP and HSP70 was measured using the NanoBiT platform. Similar to the in vitro results herein, a decreased interaction between CHIP and HSP70 in cells under conditions of heat stress was observed (FIG.3D). Finally, to validate that SCA48 mutant CHIP also had decreased interaction with HSP70 in cells, the NanoBiT assay was performed with SCA48 mutant CHIP variants. Like with heat stress, a decrease in the interaction between SCA48 mutant CHIP and HSP70 in cells was observed (FIG.3E). Together these data indicate that both SCA48 mutations and heat stress disrupt the CHIP / chaperone interaction. [000117] Because a decrease in the CHIP / chaperone interaction with both SCA48 mutations and heat stress was observed herein, the E3 ligase activity of both SCA48 mutant and heat stressed CHIP was tested. To begin assessing E3 ligase activity of SCA48 mutant CHIP, autoubiquitination of CHIP was assessed in an in vitro ubiquitination assay and SCA48 mutant CHIP retained the ability to autoubiquitinate (FIG.3F). To further validate that SCA48 mutant CHIP retained E3 ligase activity, thioester discharge assays were performed, and these mutants retained the ability to discharge ubiquitin (FIG.3G). Similarly, wild-type CHIP retained E3 ligase activity up to 50°C (FIG.3H). Together these results indicate that both SCA48 mutations and heat stress disrupt chaperone binding by CHIP but do not inhibit E3 ligase activity. Example 4 SCA48 mutations and heat stress result in nuclear localization of CHIP [000118] Next, the aim was to begin to understand the physiological consequences of heat stress and SCA48 mutations on CHIP function. Previous work demonstrated that uponMBF Ref.028193-0010-WO01 induction of heat stress, CHIP localizes to the nucleus and this heat-induced translocation of CHIP to the nucleus was also detected herein (FIG.6A). Because both heat stress and SCA48 mutations induce similar structural and biochemical defects in CHIP, it was hypothesized that, like heat stress, TPR mutations that cause SCA48 would also result in nuclear localization of CHIP. To test this, GFP-tagged CHIP constructs were generated for each SCA48 TPR mutation and they were expressed in cells. As with heat stress, each of these eight mutations resulted in nuclear localization of CHIP at 37°C (FIGS.6B-C). To validate that nuclear localization of SCA48 mutant CHIP was not due to an artifact generated by GFP fusion, cellular fractionation was performed on cells expressing either wildtype or SCA48 mutant CHIP. Similar to GFP tagged protein, CHIP was predominantly cytosolic while SCA48 mutant CHIP was present in the nuclear fraction (FIG.6D). Together these data indicate that both heat stress and SCA48 mutations result in partial localization of CHIP to the nucleus. Example 5 SCA48 mutant CHIP is in proximity to RNA binding proteins implicated in pre-mRNA processing and splicing [000119] Because both SCA48 mutations and heat stress induced nuclear localization of CHIP, the aim was to determine what biological processes CHIP regulates in the nucleus. To accomplish this, proximity labeling was utilized to identify proteins that were near wild-type CHIP and SCA48 mutant CHIP. The analysis identified 319 proteins that had decreased proximity to SCA48 mutant CHIP compared to wildtype CHIP, and 260 proteins that had increased labeling with SCA48 mutant CHIP compared to wild-type CHIP (FIG.7A). Next, pathway analysis was performed to identify physiological processes that CHIP and SCA48 mutant CHIP differentially regulate. Consistent with previous results, compared to wild-type CHIP, SCA48 mutant CHIP had decreased proximity to heat shock binding proteins including molecular chaperones (FIGS. 7B-C). Interestingly, SCA48 mutations resulted in a decrease in proximity to BAG2 and HSPBP1, two CHIP inhibitors (FIG.7C). In addition to finding proteins with decreased labeling with SCA48 mutant CHIP, a subset of proteins that had increased proximity to SCA48 mutant CHIP were identified. Pathway analysis was performed on these proteins and a strong association with RNA binding proteins involved in regulating mRNA production was found, including proteins involved in pre-mRNA splicing (FIGS.7D-F). Together these data are consistent with CHIP playing a role in regulating mRNA production and splicing.MBF Ref.028193-0010-WO01 Example 6 CHIP dependent ubiquitination of RNA binding proteins is increased upon heat stress or with SCA48 mutation [000120] The proximity labeling results suggest that SCA48 mutant CHIP is in proximity to RNA binding proteins. Because CHIP is an E3 ubiquitin ligase, the aim was to test the hypothesis that the presence of CHIP results in ubiquitination of proteins involved in regulating RNA. To test this, the ubiquitinome of CHIP-KO MEFs expressing either CHIP or mCherry as a negative control was analyzed at 37°C, or incubated for one hour at 43°C. In addition, the ubiquitinome of SCA48 mutant CHIP at 37°C was also analyzed (FIG.8A). The analysis identified nearly 17,000 ubiquitinated peptides including a significant amount that had increased ubiquitination (FIGS.9A-D). Proteins that had significant increases in ubiquitination were subjected to bioinformatic analysis to identify cellular pathways with significant increases in ubiquitinated proteins. Consistent with previous work, proteins involved in processes that regulate mRNA production were found to be among the top hits. [000121] To determine if CHIP was important for the induction of ubiquitination of proteins involved in regulation of mRNA production, the ubiquitinomes of unstressed and heat stressed CHIP-KO MEFs were compared. Like in the presence of CHIP, a robust induction of ubiquitination upon heat stress was observed (FIG.9B). Proteins that have increased ubiquitination in MEFs expressing CHIP were compared to CHIP-KO MEFs to identify proteins that are ubiquitinated in a CHIP dependent manner. From this analysis a number of cellular pathways involved in RNA binding were identified, including many pathways associated with regulating RNA transport and ribonucleoprotein (RNP) complex biogenesis (FIGS.8B-C). Because SCA48 mutant CHIP phenocopies heat stressed CHIP, it was hypothesized that the presence of SCA48 mutant CHIP would also lead to an increase in the ubiquitination of RNA binding proteins at 37°C. To test this, the ubiquitinomes of CHIP-KO cells expressing either wildtype or SCA48 mutant CHIP were compared and proteins with increased ubiquitination in the presence of SCA48 mutant CHIP were identified. Similar to the heat shock data, RNA binding proteins were identified, including those that are implicated in RNP complex biogenesis to have increased ubiquitination in the presence of SCA48 mutant CHIP (FIGS.8D-E). Together these data demonstrate that factors involved in RNA binding are ubiquitinated during heat stress and that SCA48 mutant CHIP can phenocopy heat stress resulting in increases in ubiquitination at physiological temperature.MBF Ref.028193-0010-WO01 [000122] Ubiquitination commonly leads to substrate degradation. To determine if CHIP- mediated ubiquitination of RNA binding proteins targets these proteins for degradation, whole cell proteomics was performed on CHIP-KO cells expressing either mCherry (negative control) or CHIP at either 37°C or 43°C for one-hour, or expressing SCA48 mutant CHIP at 37°C. The presence of CHIP did not lead to the degradation of many proteins at either 37°C or at 43°C (FIGS.9E-F). Similarly, expression of SCA48 mutant CHIP did not lead to large changes in the proteome when compared to expression of wild-type CHIP (FIG.9G). Very little overlap between ubiquitinated proteins and proteins that had decreased steady-state levels was observed in the presence of CHIP, indicating that CHIP-dependent ubiquitination is not targeting substrates for degradation during heat stress or with SCA48 mutations (FIGS.9H-J). Importantly, increases in the steady state levels of ubiquitinated proteins involved in mRNA binding were also not seen, indicating that increased ubiquitination is not an artifact of increased protein levels (FIGS.9K-M). However, an increase in both the levels and ubiquitination of pathways associated with interferon signaling was seen, a pathway that CHIP is implicated in regulating (FIG.9N). [000123] The ubiquitination of RNA binding proteins in the presence of CHIP could be due to their ubiquitination by CHIP or could be indirect. To discriminate between these two possibilities, spliceosomal proteins were focused on as the spliceosome was enriched in the proximity labeling experiment (FIG.7E). Spliceosomal proteins were rank ordered that had significantly increased ubiquitination in the presence of CHIP (FIG.8F). The protein with the largest fold increase was Protein mago nashi homolog (MAGOH), a member of the exon junction complex and a regulator of mRNA processing (FIG.8F). To determine if MAGOH is ubiquitinated by CHIP, in vitro ubiquitination assays were performed with MAGOH and SCA48 mutant CHIP that were previously found to be deficient in ubiquitinating HSP70 (FIG.3A). In vitro, CHIPA52Gwas able to directly facilitate the ubiquitination of MAGOH (FIG.8G and TABLE 3). TABLE 3 shows a ranking of peptides associated with mRNA splicing with increased ubiquitination with heat shock in the presence of CHIP. Proteins implicated in mRNA splicing were analyzed to identify peptides with increased ubiquitination in the presence of CHIP with heat shock compared to heat shock in the absence of CHIP. Peptides were ordered by rank of fold increase in ubiquitination. Together these data are consistent with CHIP dependent ubiquitination of RNA binding proteins in a manner that does not induce their degradation during heat stress or with SCA48 mutant CHIP.MBF Ref.028193-0010-WO01 TABLE 3. Ranking of peptides associated with mRNA splicing with increased ubiquitination with heat shock in the presence of CHIP.Example 7 CHIP is necessary for the full induction of stress induced alterations to transcription and splicingMBF Ref.028193-0010-WO01 [000124] The results herein suggest that CHIP ubiquitinates proteins involved in regulating RNA biology including mRNA production during heat stress. This led to the hypothesis that CHIP plays an important role in controlling the transcriptional and splicing response to heat stress. To test this hypothesis, the transcriptomes of unstressed and heat stressed wild-type or CHIP-KO MEFs were compared. Consistent with published literature, a robust induction of genes upon heat stress was observed, including HSF1 target genes in wild-type MEFs (FIG. 10A). In CHIP-KO MEFs, however, a blunted transcriptional stress response with a loss of over two-thirds of differentially expressed genes in CHIP-KO cells was observed (FIGS.10B-C). Because CHIP has been implicated in the translocation of HSF1 to the nucleus to induce the transcription of heat shock genes, the transcript levels of HSF1 target genes in CHIP-KO cells were also analyzed. Unexpectedly, HSF1 target genes were still robustly induced by heat shock in the absence of CHIP to a level similar to wild-type cells (FIGS.10A-B and 10D-K). Together these data suggest that CHIP is important for the full transcriptional response to heat stress and are inconsistent with a role for CHIP in regulating HSF1 activation and induction of HSF1 target genes. [000125] The proximity labeling screen and ubiquitinome analysis identified splicing as a pathway that is regulated by CHIP (FIG.7E). To determine if CHIP is important for regulating stress induced splicing events, splicing analysis of the RNA-seq data sets herein was performed. Consistent with previous literature, stress induced splicing changes upon heat stress in both wild-type and CHIP-KO MEFs that are largely independent of expression changes (FIG.10L, FIG.11). Interestingly, in CHIP-KO cells a subset of stress-induced splicing changes were smaller or non-changing, suggesting CHIP-dependent splicing regulation during heat stress (FIG.10L). Indeed, direct comparison of heat-stressed WT and CHIP-KO data revealed a set of misregulated splicing events that were inversely correlated to stress-induced splicing in WT cells (FIGS.10L and 10M). These data suggest CHIP is required for a complete alternative splicing response to stress, similar to its role in completing the stress-induced transcriptional response. In addition, splicing changes were observed in CHIP-KO, non-stressed cells that are not seen in heat-stressed cells, suggesting CHIP regulates splicing of specific genes independent of heat stress (FIG.10L). To validate the splicing analysis, the splicing of Zfp948 was analyzed, a gene that is alternatively spliced in a CHIP dependent manner during heat stress (FIG.10N). Consistent with the RNAseq splicing data herein, heat stress induced splicing changes to Zfp948 in wild-type cells and this change was absent in CHIP-KO cells (FIG. 10O). To gain more insight into the physiological roles of CHIP-dependent splicing events,MBF Ref.028193-0010-WO01 STRING analysis was performed on genes with altered splicing patterns. While STRING analysis did not detect any significant enrichment in alternatively spliced genes between wild- type and knockout cells, a strong enrichment was identified when comparing wild-type and knockout cells during heat stress. Strikingly the phenotypes that were enriched for were preweaning lethality, abnormal survival, and mortality and ageing, all phenotypes that are associated with CHIP knockout mice (TABLE 4). TABLE 4 shows STRING analysis performed on genes that were alternatively spliced during heat shock in wild-type but not CHIP-KO cells. The analysis revealed significant enrichment of interactions that correlate with phenotypes of preweaning lethality, abnormal survival, and mortality / aging. Splicing events with a dPSI ^0.20 and a probability of changing ^ 0.95 were used for STRING analysis. Together the data herein indicate that CHIP plays an important role in the transcriptional and splicing response to heat stress. TABLE 4. Mammalian phenotype ontology.Example 8 CHIP has metamorphic properties [000126] The results so far indicate that both SCA48 mutations and heat stress induce a conformational change in the structure of CHIP that disrupts the CHIP / chaperone interaction and rewires CHIP to regulate mRNA production. Because heat stress is typically a transient stress, CHIP may be able to interconvert between these two conformations in a reversible manner. To test this, NMR was utilized to monitor the structural changes that occur in heat stressed CHIP upon return to room temperature. Strikingly, the TPR domain of CHIP refolded upon return to 25°C after being incubated at 45°C (FIG.12A). To validate that CHIP regained the ability to bind chaperones upon being returned to physiological temperature, FP was performed to assess binding of CHIP to chaperones. While CHIP has decreased interactionMBF Ref.028193-0010-WO01 with chaperones at 42°C, this interaction could be restored by incubating the samples at 37°C (FIG.12B). Similarly, restoration of HSP70 ubiquitination was observed after incubation at 42°C or 45°C, consistent with restoration of the CHIP / HSP70 interaction (FIGS.12C-D). Together these data indicate that CHIP has metamorphic properties and undergoes a reversible conformational change upon heat stress. Example 9 Discussion I [000127] Previous work has demonstrated that CHIP is essential for viability upon heat stress. This was thought to be due to the ability of CHIP to bind to chaperones and ubiquitinate misfolded proteins, targeting them for degradation. Conversely, it was found herein that CHIP disengages from chaperones during heat stress due to a conformational change that occurs in the TPR domain of CHIP resulting in CHIP adopting an alternative conformation (FIGS.1C-K; FIGS.3A-E; FIGS.7A-C). In this alternate conformation CHIP can bind to and ubiquitinate RNA binding proteins and regulate the production and processing of mRNAs (FIG.7, FIG.8, and FIG.10). Upon resolution of heat stress, the TPR domain of CHIP can refold and re- engage chaperones (FIG.12). Importantly, mutations in the TPR domain of CHIP that cause SCA48 result in CHIP adopting this alternate conformation and phenocopying CHIP function during heat stress. Together the findings herein define CHIP as a stress dependent sensor that alters its mechanism of substrate recruitment via its metamorphic properties. [000128] During cellular stress CHIP plays a critical role in suppressing cell death in the presence of stressors including heat stress. CHIP is proposed to function primarily as an E3 ligase that ubiquitinates chaperone bound clients mediating their proteasomal degradation. However recent data are beginning to challenge this notion. For example, recent work suggests that CHIP has low E3 ligase activity when bound to chaperones. Moreover, CHIP has also recently been shown to function as a monomer that ubiquitinates the insulin receptor in a chaperone independent manner. Herein, it was found that during heat stress CHIP dissociates from chaperones and its inhibitors BAG2 and HSPBP1, suggesting that CHIP has increased activity in its chaperone free form (FIGS.7A-C). In addition, despite numerous reports of proteins being CHIP substrates both under physiological temperature and during cellular stress, the presence of CHIP was found to only lead to the decrease in the steady state level of about 30-40 proteins, however during cell stress 360 proteins were identified that are ubiquitinated in aMBF Ref.028193-0010-WO01 CHIP dependent manner (FIGS.8F-G). Together this suggests that the major cellular functions of CHIP may be occurring in a chaperone independent manner during cellular stress and may not be dependent upon proteasomal degradation. [000129] In addition to structural changes, it was found herein that SCA48 mutant CHIP, like heat stressed CHIP, partially translocates to the nucleus. While some stressors have previously been shown to target CHIP to the nucleus, the function of nuclear CHIP was unknown. Utilizing a proximity labelling approach, CHIP was found in proximity to proteins that regulate pre-mRNA processing and splicing. This is important as recent work has identified ubiquitin dependent changes to RNP complexes during heat stress, however the enzymes involved in this process are unknown. The results herein indicate that CHIP is one E3 ligase that plays an important role in this process. To examine the role of CHIP in regulating RNA biology, RNAseq and splicing analysis were performed. These analyses revealed that over two-thirds of transcripts that are induced during heat stress are induced in a manner dependent upon CHIP (FIGS.10A- C). Moreover, a subset of heat stress dependent splicing events were almost entirely dependent upon CHIP (FIGS.10L-O). Interestingly, upon performing STRING analysis on transcripts that had altered stress induced splicing, there was significant enrichment and the top hits in mammalian phenotype ontology were mortality / aging, preweaning lethality, and abnormal survival (TABLE 4). This is important because around 20% of CHIP knockout mice die prior to weaning and the major phenotype of the mice that survive is a decrease in longevity and accelerated aging. This suggests that an impaired stress response, including stress dependent RNA processing and splicing, may be a major contributor to phenotypes observed in CHIP knockout mice. [000130] This disclosure also provides important information about potential routes to develop therapeutics for the treatment of SCA48. This is highly important as little progress has been made in developing therapies for neurodegenerative diseases. One major reason for the foregoing is that mechanistic details of how mutations disrupt function for most proteins that cause neurodegenerative diseases are unknown. Herein it has been identified that clear structural changes in SCA48 mutant CHIP induced by mutations cause SCA48 (FIG.1C and FIGS.1E-I). This raises the possibility that small molecules that restore this structural defect may be useful for treating SCA48. Finally, in addition to SCA48 it is also possible that therapeutics that target CHIP may also be useful for treating more common neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, and the polyglutamine diseases as CHIP is neuroprotective in these diseases.MBF Ref.028193-0010-WO01 Example 10 Compound Identification [000131] Small molecules will be identified by high throughput screening. For this a fluorescence polarization assay will be used to identify small molecules that restore the interaction between SCA48 mutant CHIP and a peptide that mimics the C-terminus of HSP70. Compounds that restore CHIP function will be validated using a cellular based assay that monitors the interaction between CHIP and HSP70 via luciferase complementation. Compounds that are validated will undergo rounds of structure activity relationship studies where higher efficacy lead compounds will be developed. Next, the efficacy of these lead molecules will be tested in a mouse model of SCA48 that has been developed in-house. If protective, a series of experiments will occur to define the toxicity of the compound, how it is metabolized, and other pharmacodynamic properties. Optimal lead compounds will then be identified and move to clinical trials in humans. [000132] For dosage, a compound identified as protective will be administered orally or by intrathecal delivery. If the compound would be delivered orally, it may be taken daily. If intrathecal delivery is required, dosing may be every 3 to 6 months. Example 11 Materials and Methods [000133] The materials and methods described within Example 11 were used for Examples 12-17 below. [000134] Plasmid construction. Full-length FKBP51, FKBP52, and cyclophilin 40 (CYP40) were amplified from plasmids pCMV-FKBP51-Myc-DDK (Origene, Rockville, MD: RC210608), pCMV-FKBP52-Myc-DDK (Origene, Rockville, MD: RC200713), and pCMV-CYP40-Myc-DDK (Origene, Rockville, MD: RC206039), and cloned into pET28a(+) with NotI and XhoI (FKBP51 and FKBP52) or EcoRI and HindIII (CYP40) to yield N-terminally taggedHisFKBP51,HisFKBP52, andHisCYP40 constructs.HisFKBP12 expression construct was a gift from Duke Magnetic Resonance Spectroscopy Center. To construct FKBP51, FKBP52, and CYP40 expressing constructs for NanoBiT®assay, FKBP51, FKBP52, and CYP40 were amplified and cloned into pBit1.1N, pBit1.1C, pBit2.1N, and pBit2.1C with XhoI and NheI (FKBP51 and FKBP52) or EcoRI and NheI (CYP40). Correct sequences were confirmed by whole plasmid sequencing.MBF Ref.028193-0010-WO01 [000135] Protein expression and purification. Recombinant proteins were purified from BL21 (DE3) or Rosetta E. coli bacterial cells as previously described (Kanack et al., J Biol Chem. 2018; 293 (8): 2735-43; Umano et al., J Biol Chem.2022; 298(5): 101899; Kanack et al., Biochemistry.2020; 59(22): 2078-88; Kanack et al., Chembiochem.2022; 23(6): e202100633; Madrigal et al., J Biol Chem.2019; 294(50): 19236-45; Scaglione et al., Mol Cell.2011; 43(4): 599-612; Smith et al., Biochemistry.2013; 52(32): 5354-64; Zhang et al., Biomol NMR Assign. 2017; 11(1): 5-9; Zheng et al., Mol Biol Rep.2010; 37(3): 1413-9). Overnight cultures were inoculated in 10 mL lauria broth (LB) supplemented with 100 μg / mL carbenicillin or 50 μg / mL kanamycin. 25 μg / mL chloramphenicol was supplemented with Rosetta cells. Cultures were then incubated at 37°C with 200 rpm orbital shaking overnight. The next day the overnight cultures were added to 1L LB supplemented with respective antibiotics and grown to an OD600 of 0.6-0.8 unless otherwise stated. The 1L LB culture was induced with 1 mM IPTG at 16°C with 200 rmp orbital shaking overnight. [000136] Purification of CHIP-TPR domain and variants. CHIP-TPR domain and variants were purified as previously described (Zhang et al., Biomol NMR Assign.2017; 11(1): 5-9). Cells were lysed in CHIP TPR lysis buffer (180 mM Tris, 450 mM NaCl, 10% v / v glycerol, 5 mM ȕ- mercaptoethanol (ȕ-ME), pH 7.8), and tumbled with glutathione agarose beads for 3 hours. Proteins bound on beads were collected and washed with CHIP TPR lysis buffer and CHIP TPR cleavage buffer (20 mM HEPES, 50 mM NaCl, 1 mM EDTA, 10% v / v glycerol, 5 mM ȕ-ME, pH 7.5). Proteins were cleaved by H3C protease in CHIP TPR cleavage buffer at 4ºC with overnight tumbling. The next day proteins were collected and concentrated to desired concentrations. [000137] Purification of His-tagged proteins. For the purification ofHisFKBP51 and variants,HisFKBP52,HisCyp40,HisFKBP12,HisHIP-TPR domain,HisHOP-TPR domains, andHisPP5-TPR domain, bacteria were lysed in buffer A (50 mM Tris, 1 M NaCl, 0.5% v / v Triton X-100, pH 7.5) supplemented with 1 mM DTT, 5 μg / mL aprotinin, 1 μg / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson, Brookfield, CT) at power output of 5 for 3 times with 30 seconds duration each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to addition of 500 μL Ni-HTC agarose beads (GoldBio, St. Louis, MO) and tumbling for 3 hours. The proteins bound to glutathione beads were then washed three times with buffer A, three times with buffer B (50 mM Tris, 1 M NaCl, 20 mM imidazole, pH 7.5), and three times with buffer C (50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.5). Proteins were eluted by tumbling in 500 μL elution buffer (50 mM Tris,MBF Ref.028193-0010-WO01 150 mM NaCl, 300 mM imidazole, pH 7.5) for 3 times 15 minutes each time. Eluted protein fractions were analyzed using SDS-PAGE and Coomassie staining. Fractions containing proteins of interest were combined and concentrated (Sartorius, Göttingen, Germany) by spinning at 4000 g until desired concentrations were reached (Scaglione et al., Mol Cell.2011; 43(4): 599-612). [000138] Purification of CHIP and variants. Bacterial cells were lysed in NETN buffer (50 mM Tris, 150 mM NaCl, 0.5% IGEPAL, pH 7.5) supplemented with 1 mM DTT, 5 μg / mL aprotinin, 1 μg / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson®) at power output of 5 for 3 times with 30 seconds duration each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to addition of 500 μL glutathione agarose beads (GoldBio, St. Louis, MO) and tumbling for 3 hours. The proteins bound to glutathione beads were then washed three times with NETN and three more times with Kinase Buffer (50 mM Tris pH 7.5, 50 mM KCl, 0.2 mM DTT) prior to addition of H3C protease. Cleaved proteins were further purified using size-exclusion chromatography (ENrich SEC 650, Bio-Rad, Hercules, CA). Each fraction was analyzed using SDS-PAGE gel and Coomassie stain. And protein fractions were combined and concentrated (Sartorius, Göttingen, Germany) by spinning at 4000 g until desired concentrations were obtained (Umano et al., J Biol Chem. 2022; 298(5): 101899; Kanack et al., Chembiochem.2022; 23(6): e202100633; Madrigal et al., J Biol Chem.2019; 294(50): 19236-45; Scaglione et al., Mol Cell.2011; 43(4): 599-612; Smith et al., Biochemistry.2013; 52(32): 5354-64). [000139] Purification of UbcH5c. Cells were lysed in MES lysis buffer (30 mM MES, pH 6.0), supplemented with 1 mM DTT, 5 μg / mL aprotinin, 1 μg / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson, Brookfield, CT) at power output 5, 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to application onto SP-Sepharose column equilibrated with 3 column volumes of MES lysis buffer. Unbound proteins were washed with 5 column volumes of MES lysis buffer. Then bound proteins were eluted using AKTA Start Protein Purification System (Cytiva, Marlborough, MA) at salt gradient from 0% to 100% MES elution buffer (30 mM MES, 500 mM NaCl, pH 6.0) supplemented with 2 mM DTT and 1:1000 (v / v) supersaturated PMSF. Each fraction was analyzed by SDS-PAGE gel and Coomassie stain. Fractions with protein of interest were further purified using size-exclusion chromatography (ENrich SEC 75, Bio-Rad, Hercules, CA). Each fraction was analyzed using SDS-PAGE gel and Coomassie stain. And protein fractions were combined and concentrated using concentrator (Sartorius,MBF Ref.028193-0010-WO01 Göttingen, Germany) by spinning at 4000 g until desired concentrations were reached (Kanack et al., J Biol Chem.2018; 293 (8): 2735-43; Umano et al., J Biol Chem.2022; 298(5): 101899; Kanack et al., Biochemistry.2020; 59(22): 2078-88; Scaglione et al., Mol Cell.2011; 43(4): 599- 612). [000140] Purification of humanHisUbe1. Bacteria transformed with His-tagged Ube1 construct were grown and induced at optical density (OD) 0.3. Bacteria were lysed in buffer A (50 mM Tris, 1 M NaCl, 0.5% (v / v) Triton-X100), supplemented with 1 mM DTT, 5 μg / mL aprotinin, 1 μg / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson, Brookfield, CT) at power output 5, 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to addition of 500 μL of Ni-HTC beads (GoldBio, St. Louis, MO) and tumbling for 1 hour. The Ni-HTC beads were then washed three times with buffer A, three times with buffer B (50 mM Tris, 1 M NaCl, 20 mM imidazole, pH 7.5), and three more times with buffer C (50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.5) prior to elution 3 times each time with 500 μL of elution buffer (50 mM Tris, 150 mM NaCl, 300 mM imidazole, pH 7.5) and tumbling at 4°C for 15 minutes. Each elution fraction was analyzed using SDS-PAGE and Coomassie stain. Proteins were dialyzed into buffer with 20 mM Tris, 50 mM NaCl, pH 7.5 prior to loading onto a mono Q column (BioRad, Hercules, CA) and were eluted with 0-400 mM NaCl gradient. Fractions with proteins were collected and further purified using size-exclusion chromatography (Enrich SEC 650, Bio-Rad, Hercules, CA). Each fraction was analyzed using SDS-PAGE and Coomassie stain. Protein fractions were combined and concentrated (Sartorius, Göttingen, Germany) by spinning at 4000 g to a final concentration at 10 μM (Zheng et al., Mol Biol Rep.2010; 37(3): 1413-9). [000141] Purification of ubiquitin. Bacteria were lysed in ubiquitin lysis buffer (50 mM Tris, 1 mM EDTA, 0.05% v / v Tween-20, pH 7.6) supplemented with 1 mM DTT, 5 μg / mL aprotinin, 1 μg / mL leupeptin, and 1:1000 supersaturated PMSF. Cell lysates were then sonicated (Branson®) at power output 6 with 80% interval 3 times for 30 seconds each time. Sonicated lysates were then centrifuged at 12,000 rpm for 30 minutes at 4°C prior to dialyzing using dialysis tubing (Thermo Fisher,Waltham, MA) to 3.5 L ubiquitin wash buffer (50 mM sodium acetate, pH 4.5) twice at least 4 hours each time. Dialyzed samples were then centrifuged at 12,000 rpm for 30 minutes to remove precipitates. Centrifuged samples were then applied to SP-sepharose column equilibrated with 4 column volumes of ubiquitin wash buffer. Proteins were eluted with ubiquitin wash buffer supplemented with 50 mM, 100 mM, 200 mM, 300 mM, 400 mM, and 500 mM NaCl 3 times at each salt concentration. Fractions were analyzed usingMBF Ref.028193-0010-WO01 SDS-PAGE and Coomassie stain. Fractions with ubiquitin were concentrated using concentrator (Sartorius, Göttingen, Germany) by spinning at 4000 g for to final a concentration of 20 mg / mL. Concentrated proteins were further purified using size-exclusion chromatography (Enrich SEC 75, Bio-Rad, Hercules, CA). Each fraction was analyzed using SDS-PAGE and Coomassie stain. And protein fractions were combined and concentrated using concentrator (Sartorius, Göttingen, Germany) by spinning at 4000 g to a final concentration of 10 mM (Kanack et al., J Biol Chem.2018; 293 (8): 2735-43; Umano et al., J Biol Chem.2022; 298(5): 101899; Kanack et al., Biochemistry.2020; 59(22): 2078-88; Scaglione et al., Mol Cell.2011; 43(4): 599-612). [000142] Cell lines and reagents. HEK293T cells (ATCC CRL-3216) were grown in DMEM media (11995063, Gibco®, Thermo Fisher, Waltham, MA) supplemented with 10% v / v FBS (Cytiva, Marlborough, MA), 1% v / v Penicillin / Streptomycin (Gibco®, Thermo Fisher, Waltham, MA), and 1% v / v Non-Essential Amino Acids (NEAA, Gibco®, Thermo Fisher, Waltham, MA). Cells were grown in adherent cultures in 10 cm tissue-culture treated dishes (Bio-Lite®, Thermo Fisher, Waltham, MA) at 37°C with 5% CO2. [000143] HEK293T cells were transfected with lipofectamine 3000 (InvitrogenTM, Thermo Fisher, Waltham, MA) per manufacturer protocol. HEK293T cells were grown on 12-well or 10- cm tissue-culture treated plates until 60-80% confluency. For 12-well transfection, 1 μg of plasmid and 2 μL of P3000 reagent were mixed with 50 μL Opti-MEM media (Gibco®, Thermo Fisher, Waltham, MA) while 1 μL of lipofectamine was mixed with 50 μL Opti-MEMTMmedia prior to incubation at room temperature for 5 minutes. Lipofectamine in Opti-MEMTMwas then combined with plasmid DNA and P3000 reagent, and allowed for incubation for 15 minutes at room temperature (i.e., from about 20°C to about 22°C). Media was then changed on the cells and the DNA and lipofectamine mixture were applied. Transfection protocol was the same for 10 cm dish transfection except 5 μg plasmid DNA and 10 μL P3000 reagent were mixed with 300 μL Opti-MEMTMmedia, while 5 μL lipofectamine was incubated with 300 μL Opti-MEMTM. [000144] Proximity labeling screen. Expression plasmids for miniTurbo was obtained from Addgene, Watertown, MA (plasmid #107170) and modified to remove the nuclear export signal (Branon et al., Nat Biotechnol.2018; 36(9): 880-7). CHIP was cloned into the vector using the HindIII and NheI restriction sites. The A52G mutation was added by site-directed mutagenesis (Agilent, Santa Clara, CA, QuikChange®Lightning). MiniTurbo tagged CHIP (MTCHIP), mutant CHIP (MTCHIPA52G) or miniTurbo vector were transfected into HEK293 cells at 60% confluency inMBF Ref.028193-0010-WO01 10 cm dishes according to manufacturer’s directions (Lipofectamine 3000, InvitrogenTM, Thermo Fisher, Waltham, MA). 24 hours after transfection the media was changed and 16 hours later 500 μM of biotin was added. The cells were incubated for 30 minutes prior to termination of the reaction by placing the cells on ice and washing 5 times with ice-cold PBS. Cells were then harvested in PBS and spun down at 1500 RPM for 3 minutes at 4°C. The cell pellets were resuspended in 500 μL of RIPA buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% IGEPAL, pH 8.0) with PMSF and protease inhibitor cocktail. The cells were then allowed to lyse on ice for 5 minutes prior to sonication and centrifugation at 4°C for 10 minutes at 10,000 rpm. The supernatant was removed and saved, and a BCA assay (PierceTM, Thermo Fisher, Waltham, MA) was performed to determine total protein content. [000145] In a 1.7 mL tube, 30 μL of Neutravidin beads (Thermo Fisher, Waltham, MA) was washed 3 times with 500 μL of RIPA buffer. Lysate was added to the beads (total of 1.6 mg of protein) and allowed to tumble overnight at 4°C. The next morning, the beads were spun at 600 g at 4°C and the lysate removed. The beads were transferred to low-binding Epitubes (Eppendorf, Hamburg, Germany) and a series of room temperature washes were done with a 21-gauge needle and 1 mL syringe to remove any unbound or nonspecific proteins. 500 μL of each wash solution was applied and allowed to rock for 10 minutes before removal and application of the next wash. The washes consisted of two washes with 2% SDS, one wash with 1% TritonX100, 1% sodium deoxycholate, 25 mM lithium chloride, two wash with 1 M sodium chloride, and five washes with 50 mM ammonium bicarbonate. After the final wash, bound proteins were eluted from the bead by adding 70 μL elution buffer (125 mM Tris, 4% SDS, 10 mM DTT, 5 mM biotin, pH 6.8), boiling at 95°C for 5 minutes and vertexing 3X during this time. The beads were then spun briefly at 600 g and the supernatant transferred to a new low-binding Epitube and stored at -80°C until delivery to the proteomics core. The experiment was repeated in triplicate. [000146] Mass spectrometry and data analysis. Proximity labeling samples were spiked with undigested bovine casein at a total of either 1 or 2 pmol as an internal quality control standard. Next, samples were reduced with 10 mM dithiolthreitol for 30 minutes at 80ºC, alkylated with 20 mM iodoacetamide for 30 minutes at room temperature, then supplemented with a final concentration of 1.2% phosphoric acid and 623 ^L of S-Trap (Protifi, Fairport, NY) binding buffer (90% MeOH / 100 mM TEAB). Proteins were trapped on the S-Trap microcartridge, digested using 20 ng / ^L sequencing grade trypsin (Promega, Madison, WI) for 1 hour at 47ºC, and eluted using 50 mM TEAB, followed by 0.2% FA, and lastly using 50% ACN / 0.2% FA. All samplesMBF Ref.028193-0010-WO01 were then lyophilized to dryness. Samples were resolubilized using 12 ^L of 1% TFA / 2% ACN with 25 fmol / ^L yeast ADH. [000147] Quantitative LC / MS / MS was performed on 4 ^L using a nanoAcquity UPLC®system (Waters Corp., Milford, MA) coupled to an OrbitrapTMFusionTMLumosTMhigh resolution accurate mass tandem mass spectrometer (Thermo Fisher, Waltham, MA) equipped with a FAIMSProTMdevice via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C1820 mm × 180 ^m trapping column (5 ^L / minute at 99.9 / 0.1 v / v water / acetonitrile), after which the analytical separation was performed using a 1.8 ^m Acquity®HSS T3 C1875 ^m × 250 mm column (Waters Corp., Milford, MA) with a 90-minute linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nL / minute with a column temperature of 55ºC. Data collection on the FusionTMLumosTMmass spectrometer was performed for three difference compensation voltages (-40 v, -60 v, and -80 v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m / z 200) full MS scan from m / z 375 – 1500 with a target AGC value of 4×105ions was performed. MS / MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 104and max fill time of 35 millisecond. The total cycle time for each CV was 0.66 second, with total cycle times of 2 seconds between like full MS scans. A 20-second dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hours. [000148] Following UPLC-MS / MS analyses, data were imported into Proteome Discoverer 2.5 (Thermo Fisher, Waltham, MA) and individual LCMS data files were aligned based on the accurate mass and retention time of detected precursor ions (“features”) using Minora Feature Detector algorithm in Proteome Discoverer. Relative peptide abundance was measured based on peak intensities of selected ion chromatograms of the aligned features across all runs. The MS / MS data was searched against the Swiss-Prot H. sapiens database (downloaded in November 2019), a common contaminant / spiked protein database (bovine albumin, bovine casein, yeast ADH, etc.), and an equal number of reversed-sequence “decoys” for false discovery rate determination. Sequest with Infernys enabled (v 2.5, Thermo PD, Thermo Fisher, Waltham, MA) were utilized to produce fragment ion spectra and to perform the database searches. Database search parameters included fixed modification on Cys (carbamidomethyl) and a variable modification on Met (oxidation). Precursor mass tolerances were 2.0 ppm and product ion mass tolerances were 0.8 Da with full trypsin enzyme rules required. Peptide Validator and Protein FDR Validator nodes in Proteome Discoverer were used to annotate the data at a maximum 1% protein false discovery rate based on q-valueMBF Ref.028193-0010-WO01 calculations. Note that peptide homology was addressed using razor rules in which a peptide matched to multiple different proteins was exclusively assigned to the protein has more identified peptides. Protein homology was addressed by grouping proteins that had the same set of peptides to account for their identification. A master protein within a group was assigned based on % coverage. [000149] Prior to imputation, a filter was applied such that a peptide was removed if it was not measured in at least 2 unique samples (50% of a single group). After that filter, any missing data missing values were imputed using the following rules: 1) if only a single signal was missing within the group of three, an average of the other two values was used or 2) if two out of three signals were missing within the group of three, a randomized intensity within the bottom 2% of the detectable signals was used. A normalization was then applied to the data by excluding the highest and lowest 10% of the signals and then making the average of the remaining signals be the same across all samples. To summarize to the protein level, all peptides belonging to the same protein were summed into a single intensity. These normalized protein level intensities were what was used for the remained of the analysis. [000150] NanoBiT®split luminescence assay. NanoBiT®assays were performed following published protocols (Umano et al., J Biol Chem.2022; 298(5): 101899). In short, HEK293T cells were grown in 12-well plates and transfected using lipofectamine P3000 (InvitrogenTM, Thermo Fisher, Waltham, MA). Twenty-four hours post transfection cells were trypsinized, collected, and resuspended in 2 mL Opti-MEM media (GibcoTM, Thermo Fisher, Waltham, MA). 100μL of cell suspension was plated on 96-well white flat-bottom tissue-culture treated plate (cite plate) and incubated at 37ºC for 45 minutes. Nano-Glo®Live Cell Reagent was prepared by mixing 1:20 v / v ratio of live cell substrate (Promega, Madison, WI) and Nano-Glo®Live Cell buffer (Promega, Madison, WI). 25 μL of Nano-Glo®Live Cell Reagent was added each well. Samples were shaken for 15 seconds and read for luminescence counts. Data were analyzed using GraphPad Prism. [000151] Nuclear magnetic resonance (NMR). Proteins subjected to NMR analysis were expressed and purified as preciously described (Zhang et al., Biomol NMR Assign.2017; 11(1): 5-9; Marley et al., J Biomol NMR.2001; 20(1): 71-5). Rosetta cells transformed with protein expression constructs were inoculated in 10 mL overnight cultures to incubate at 37ºC overnight. The next day, each overnight culture was used to inoculate a 200 mL culture to incubate at 37ºC. The optical density (OD) of each 200 mL culture was determined. When theMBF Ref.028193-0010-WO01 OD was between 0.6 to 0.8, cells were collected and washed twice with 100 mL PBS prior to being transferred to 1L of minimal media (50 mM Na2HPO4, 50 mM KH2PO4, 5 mM Na2SO4, 3 g / L15NH4Cl, 5 g / L NaCl) supplemented with 0.8% glucose, 2 mM MgSO4, 30 μM CaCl2, vitamin solution and antibiotics. When the OD of the minimal media cultured reached 0.6-0.8, cells were induced with 1 mM of IPTG at 16ºC overnight. [000152]1H / 15N-HSQC NMR analysis was performed using Burker 600MHz Avance IIITMspectrometer equipped with cryogenic probes.15N labeled proteins were concentrated to 100- 200 μM and supplemented with 10% deuterated water prior to analysis. The samples were locked, tuned, and shimmed as instructed by manufacturer. NMR analysis was performed at 25ºC unless otherwise indicated. For collection of data for CHIP-TPR at 45ºC, spectrometer was warmed to 45ºC, samples were equilibrated in spectrometer for at least 15 minutes prior to data collection. The optimal number of acquisitions per FID were determined prior to data collection. 8-32 acquisitions per FID were performed except for 0.5:1 and 1:1 FKBP51 titration where 128 acquisitions were used per FID. All spectra were analyzed using NMRPipe and NMRViewJ. Cross peaks for CHIP-TPR spectra were identified and labeled based on previous assignments. [000153] To determine the conformational changes on the TPR domains of co-chaperones induced by PPIase and Hsp70 peptide, NMR titration analysis was performed on15N-labeled CHIP-TPR or PP5-TPR (200 μM) with increasing amounts of PPIase (FKBP51 and variants, FKBP52, CYP40, and FKBP52) or Hsp70 peptide. The cross peaks were recorded using heteronuclear single quantum coherence (HSQC) spectra. All spectra were analyzed using NMRPipe and NMRViewJ. [000154] In vitro ubiquitination assay. In vitro ubiquitination assays were performed as previously documented (Kanack et al., J Biol Chem.2018; 293 (8): 2735-43; Umano et al., J Biol Chem.2022; 298(5): 101899). For substrate ubiquitination, reaction mixtures with 1 μM CHIP or mutant CHIP, 100 nM E1, 1μM UbcH5c, 1μM Hsp70, 5 mM ATP, 5 mM MgCl2, and 500 μM Ub were prepared in a kinase buffer (50 mM Tris, 50 mM KCl, 0.2 mM DTT, pH 7.5). Reactions were incubated at 37ºC for comparison between CHIP and mutants, or indicated temperatures for temperature comparison for 1 hour, prior to be stopped with the addition of 4x Laemmli buffer with ȕ-ME and boiling. Proteins were separated using SDS-PAGE and analyzed using Western blotting with the indicated antibodies.MBF Ref.028193-0010-WO01 [000155] Lysine discharge assay. Lysine discharge assay was performed as previously described (Kanack et al., J Biol Chem.2018; 293 (8): 2735-43; Umano et al., J Biol Chem. 2022; 298(5): 101899). For thioester formation assays, a mixture of 20 μM CHIP, 1 μM E1, 20 μM UbcH5c, 5 mM ATP, 5 mM MgCl2, and 20 mM Ub were prepared in kinase buffer (50 mM Tris, 50 mM KCl, 0.2 mM DTT, pH 7.5) and incubated at 37°C for 30 minutes. Reactions were stopped with the addition of 50 mM EDTA. 5 mM lysine was then added to each sample and incubated at respective temperatures. For lysine discharge assays with CHIP, lysine discharge reactions were incubated at 37ºC for 30 minutes for comparison between CHIPWTand mutants, or at indicated temperatures for 30 minutes for temperature comparison. For lysine discharge assays without CHIP, lysine discharge reactions were incubated at indicated temperatures for 2 hours. [000156] Fluorescence polarization assay. Fluorescence polarization assay was performed as previously described (Kanack et al., J Biol Chem.2018; 293 (8): 2735-43; Umano et al., J Biol Chem.2022; 298(5): 101899; Ricci et al., Curr Chem Genomics.2008; 2: 90-5). Hsc70 peptide labeled with Rhodamine B was diluted in FP buffer (50 mM HEPES, 75 mM NaCl, and 0.01% Triton X-100, pH 7.4) to 20 nM and added to 384-well black flat-bottom assay plates. CHIP was then added to the indicated concentrations, to a reaction volume 50 μL each sample. Samples were incubated at the indicated temperatures for 30 minutes in the dark prior to taking measurements. Data were analyzed by GraphPad Prism and exhibited as average with SD display. [000157] Co-chaperone TPR domain stability analysis in the absence or presence of FKBP51. To assess the stability change on the TPR domains of co-chaperones in the presence of FKBP51, a precipitation analysis was performed. Co-chaperone TPR domains were diluted to 200 μM in 50 μL of buffer C (50 mM Tris, 150 mM NaCl, 20 mM imidazole, pH 7.5) supplemented with 1 mM DTT. 200 μM of FKBP51 (stock concentration was 1 mM) or same volume of buffer C were added to TPR domain proteins for stability analysis. 10 μL of pre- incubation samples were collected, mixed with 4x Laemmli buffer, boiled for 4 minutes and frozen at -20°C until ready for analysis. The rest samples were incubated at 25°C for 16 hours. After incubation, soluble and insoluble fractions were separated by centrifuging at 12,000 rpm for 10 minutes. Soluble fractions were collected and mixed with 4x Laemmli buffer. Insoluble fractions were washed twice with 50 μL buffer C, and resuspended in 50 μL 1x Laemmli buffer. All samples were boiled for 4 minutes prior to separation on SDS-PAGE gel and visualization with Coomassie stain.MBF Ref.028193-0010-WO01 Example 12 CHIP Interacts with TPR Domain-Containing Proline Isomerases [000158] The co-chaperone CHIP plays a critical role in facilitating the degradation of chaperone clients, and therefore understanding factors that regulate CHIP may provide insight into pathways that suppress neurodegeneration. To identify co-factors that regulate CHIP function a proximity labeling screen was performed. It was confirmed that known CHIP interacting proteins including molecular chaperones were enriched with miniturbo (MT) tagged CHIP (CHIPMT) compared to MT alone (FIG.16A; FIGS.17A-D). Among enriched proteins it was found that proteins that contain a TPR domain were highly enriched (FIG.16B and TABLE 5). TPR domains are known to self-associate suggesting that they may interact with and modulate CHIP function (FIG.16B and TABLE 5). Among the TPR domain-containing proteins enriched in the CHIPMTsample, it was found that all known TPR domain co-chaperone PPIases were also enriched (FIGS.16B-C: FIGS.17A-D). To validate that CHIP is in proximity to FKBP51, FKBP52, and CYP40 in cells, NanoBiT analysis was performed, and it was found that in each case there was an increased signal with CHIP compared to a negative control (FIG. 16D; FIGS.18A-C). Together these data suggest that TPR domain containing PPIases interact with CHIP. TABLE 5. String analysis identifies TPR domain-containing proteins as enriched near CHIP; protein domains and features (InterPro).Example 13 CHIP is a Substrate of FKBP51 [000159] Next, it was determined if any of the PPIases identified induced structural changes to the TPR domain of CHIP.1H-15N HSQC analysis was performed with15N labelled CHIP TPR domain and titrated in increasing amounts of FKBP51. A loss of intensity of N-terminal peaks upon addition of FKBP51 was observed (FIGS.19A-D), with a more global loss of signalMBF Ref.028193-0010-WO01 occurring at a 1:1 ratio (FIG.19D). Interestingly, neither FKBP52 or CYP40 induced a similar change in spectra, however broadening of some peaks and chemical shift perturbations (CSPs) of other residues was observed, suggesting that FKBP52 and CYP40 could bind to the TPR domain of CHIP, but do not induce large structural changes at the ratios tested (FIGS.20A-C). To validate that this structural change is selective for FKBP51, FKBP12 was also titrated in. FKBP12 is a PPIase that contains the highly conserved FK domain but lacks the second inactive FK2 domain and TPR domain present in FKBP51. Titrating in FKBP12 resulted in no observable changes in the spectra of the TPR domain of CHIP (FIGS.19E-G). To confirm that the changes in spectra that occurred in the presence of FKBP51 were due to FKBP51 PPIase activity, FKBP51 was preincubated with the PPIase inhibitor FK506 prior to addition. Consistent with FKBP51 enzymatic function being necessary, the dramatic change in spectra as observed in the absence of FK506 was not observed (FIGS.19E-F, H). Finally, to determine if the TPR domain of FKBP51 was necessary for FKBP51 to induce structural changes to CHIP, a TPR- less FKBP51 (FKBP51ǻTPR) was generated. Consistent with the TPR domain of FKBP51 being necessary to induce structural changes to CHIP, the robust structural changes in the presence of FKBP51ǻTPRas compared with wild-type FKPB51 was not observed (FIGS.19E-F, I). Together these data identify FKBP51 as a PPIase that induces structural changes to the TPR domain of CHIP in a manner dependent upon its catalytic activity and the presence of its TPR domain. Example 14 Thermodynamic Lability is a Feature of the CHIP TPR Domain [000160] Previously, Applicant found that mutations that cause SCAR16 lower the thermal stability of CHIP to below physiological temperature. Applicant also found that while these mutants had impaired ability to ubiquitinated Hsp70 at 37°C they were active at 25°C. By performing CD thermal melt analysis of wild-type and SCA48 mutant CHIP, it was found that CHIP unfolds with two distinct transitions, with the first transition occurring slightly above physiological temperature. Because PPIases are ATP-independent enzymes, it was hypothesized that being thermodynamically labile may be a feature that allows for PPIases to bypass energy barriers that may be associated with proline isomerization. To test this, in vitro ubiquitination assays were performed at temperatures ranging from 25°C to 45°C. It was found that at slightly elevated temperatures ubiquitination of Hsp70 was impaired (FIG.21A). This loss of ubiquitination was not due to impaired E3-ligase activity, but instead was caused by aMBF Ref.028193-0010-WO01 decrease in the interaction between CHIP and Hsp70 (FIGS.21B-C). To determine if thermal challenge resulted in changes to the structure of the TPR domain of CHIP, NMR spectroscopy was utilized to monitor the fold of the TPR domain of CHIP with increasing temperature. It was found that at elevated temperatures there was a loss of peaks that map to the N-terminal helices of the CHIP TPR domain similar to those changes induced at a 1:0.5 ration of CHIPTPRto FKBP51 (FIGS.21D-E; FIG.19A and FIG.19C; FIG.22). To determine if this structural change was reversible, the temperature was lowered in the NMR from 45°C back to 25°C and it was found that that the peaks lost at the elevated temperature returned indicating that the structural changes caused by increased temperature were reversible (FIGS.21D-F). Consistent with this, it was also found that the interaction between CHIP and Hsp70 could be restored upon reduction of temperature (FIGS.21G-J, FIGS.23A-B). Finally, it was reasoned that stabilization of the CHIP TPR domain should prevent structural changes induced by heat or by FKBP51. To test this, NMR spectroscopy was performed with the TPR domain of CHIP at either 25°C or 45°C, or with FKBP51 at 25°C and a peptide that mimics the C-terminus of Hsp70 and increases the thermal stability of CHIP was added. Consistent with increased thermal stability preventing structural changes with heat stress or with FKPB51, large differences in spectra in any condition in the presence of the peptide was not observed (FIGS. 21H-J). Together, these data identify the TPR domain of CHIP as being thermodynamically labile and sensitive to conformational change. These data also suggest that this property is important for the induction of structural changes by FKBP51. Example 15 SCA48 Mutations Disrupt the Structure of the TPR Domain of CHIP in a Reversible Manner [000161] In addition to being generally neuroprotective, when mutated, CHIP causes the dominant neurodegenerative disease SCA48. When mapping mutations in the TPR domain of CHIP that cause SCA48, it was observed that nearly all cluster to the interface that stabilizes the interaction between the first and second CHIP TPR repeat (FIGS.24A-B). Because this region stabilizes the CHIP TPR domain and is thermodynamically labile (FIGS.21A-K) it was reasoned that SCA48 mutations may induce similar structural changes as observed with heat stress and with FKBP51. To test this, the15N-1H HSCQ spectra of wildtype and SCA48 mutant CHIP TPR domain was analyzed. Similar to in the presence of a substoichiometric concentrations of FKBP51 a loss of signal from peaks in the first and second TPR repeat ofMBF Ref.028193-0010-WO01 CHIP in the presence of SCA48 mutations was observed, consistent with structural alterations to this region (FIGS.24C-D; FIGS.25A-C). To confirm this result with full length CHIP partial trypsin digestion coupled with quantitative mass spectrometry was performed to assess the structurally stability of the N-terminus of CHIP. It was found that the N-terminal region of SCA48 CHIP was more susceptible to cleavage, consistent with decreased structural stability (FIGS. 26A-B and TABLE 1). The first TPR repeat of CHIP is susceptible to trypsin cleavage. Either uncut or cleaved CHIP (full-length CHIP and the band at the position of the asterisk in FIG.26A) were analyzed by mass spectrometry. Peptides that are decreased in the cleaved version of CHIP were quantitated and are listed in TABLE 1 (n=3). [000162] The ability of SCA48 mutant CHIP to bind to chaperones and to transfer ubiquitin was also assessed. It was found that SCA48 mutations in the CHIP TPR domain resulted in decreased affinity for chaperones but did not impair E3 ligase activity (FIG.24E). Finally, to determine if the structural defects associated with SCA48 mutants could be restored excess peptide that corresponds to the C-terminus of Hsp70 was added and its fold was monitored via HSCQ. Similar to with heat stress or in the presence of FKBP51, it was found that addition of peptide resulted in a restoration of peak intensity of N-terminal TPR residues (FIGS. 24F-G). Together these data are consistent with mutations that cause SCA48 inducing a reversible structural change to the CHIP TPR domain that resembles what occurs in the presence of FKBP51 and results in decreased affinity for chaperones. Example 16 TPR Domaining Co-Chaperones are Substrates of FKBP51 [000163] Because Applicant found that FKBP51 can induce structural changes to the TPR domain of CHIP, it was decided to determine if this was a more common phenomena shared among TPR containing co-chaperones. To test this, each TPR domain of the co-chaperones Hsc70 Interacting Protein (HIP), Hsp70-Hsp90 Organizing Protein (HOP), and protein phosphatase 5 (PP5) was purified to identify additional TPR domains that may be FK506 binding protein 51 (FKBP51) substrates. To initially assess if FKBP51 induced unfolding of additional TPR domains, the solubility of each TPR domain with or without incubation with FKBP51 was measured. It was observed that TPR1 and TPR2A of HOP remained soluble, however TPR2B of HOP and the TPR domains of HIP and PP5 all had reduced solubility after incubation with FKBP51 (FIGS.27A-B). To confirm that this decrease in solubility correlatedMBF Ref.028193-0010-WO01 with structural changes of additional TPR domains, the spectra of each of the TPR domains that were purified were analyzed and it was found that only PP5 was amenable to analysis by15N-1H HSCQ. Similar to the TPR domain of CHIP, it was found that addition of FKBP51 resulted in a loss of peaks and decreased solubility in a dose dependent manner (FIGS.27C-F). Similar to the CHIP TPR domain, changes in the spectra were specific for FKBP51 as FKBP12 did not result in changes to the PP5 spectra (FIGS.27G-I). It was also found that the changes in PP5 spectra were also dependent upon the catalytic activity of FKBP51, as preincubation of FKBP51 with FK506 prevented changes to the PP5 TPR spectra (FIGS.27G-H, J). Finally, similar to the CHIP TPR domain the spectral changes were dependent upon the presence of the FKBP51 TPR domain (FIGS.27G-H, K). Together these data are consistent with the TPR domains of additional co-chaperones being substrates for FKBP51 and suggests that FKBP51 may influence the formation and dynamics of chaperone / co-chaperone complexes (FIG.27L). Example 17 Discussion II [000164] PPIases comprise a large class of chaperones that play important biological functions. However, precise substrates for PPIases have remained largely elusive. This is true for FKBP51 which is known to play a role in numerous biological pathways, however direct evidence of the induction of changes to the structure of substrates has been lacking. Herein the TPR-containing co-chaperones have been identified as FKBP51 substrates (FIGS.16A-D, FIGS.19A-I, FIGS.27A-L). The data herein support a role for FKBP51 in altering the fold of the TPR domains of these co-chaperones in a manner that depends both upon the catalytic activity of FKBP51 and the presence of its TPR domain (FIGS.19E-I; FIGS.27G-K). These findings also suggest that FKBP51 alters the affinity of TPR-containing co-chaperones for chaperones and thus influences chaperone complex assembly (FIG.27L). [000165] FKBP51 is thought to play diverse roles by promoting the folding of substrates and reorganizing protein complexes. Herein it was found that FKPB51 can promote the unfolding of co-chaperone TPR domains. One key feature of FKBP51 substrates that appears to be necessary for unfolding is low thermodynamic stability. Previous work has identified the CHIP and PP5 TPR domains as being thermodynamically labile with a TM also only slightly above physiological temperatures. PPIases isomerize proline residues in an ATP-independent manner and the low thermodynamic stability of the TPR domains of CHIP and PP5 is likely necessaryMBF Ref.028193-0010-WO01 for FKPB51 for being able to induce this structural change. Consistent with this, the addition of a peptide that increases the stability of the TPR domain of CHIP is sufficient to overcome structural changes induced by FKBP51 (FIGS.24F-G). This suggests that low thermodynamic stability may be a feature of FKBP51 and potentially other PPIase substrates. [000166] In addition to FKBP51 and heat stress inducing changes to the structure of CHIP TPR domain, it was also found that mutations that cause SCA48 also results in a structural change (FIGS.24C-D). It was observed that the intensity of similar peaks are lost in the presence of FKBP51, with increased temperature, or with SCA48 mutations (FIGS.19E-F; FIGS.21D-E; FIGS.24C-D). This suggests that mutations that cause SCA48 may promote CHIP to have similar structural changes as is normally induced by FKBP51. Because the presence of the peptide that mimics the C-terminus of Hsp70 increases the thermal stability of CHIP, the affinity of isomerized CHIP for Hsp70 cannot be directly measured. However, SCA48 mutant CHIP appears to be prone to more stably adopt a structure similar to the isomerized state, a state at which it has decreased affinity for chaperones (FIGS.24C-E). Strikingly, this altered structural state was reversible and addition of a 4-fold molar excess of peptide that mimics the C-termini of Hsp70 restored the structure of SCA48 mutant CHIP (FIGS.24F-G). This suggests that development of small molecules that thermodynamically stabilize the CHIP TPR domain may be therapeutic. [000167] In addition to SCA48, CHIP is generally neuroprotective and deletion of CHIP results in accelerated aging phenotypes. Aging is the top risk factor associated with neurodegeneration and FKBP51 levels increase with age. Additionally, in a meta-analysis of genome wide DNA methylation changes in people with Alzheimer’s disease, Parkinson’s disease, or Amyotrophic Lateral Sclerosis (ALS), demethylation of FKBP5, the gene that encodes FKBP51, was the top gene association with disease. In addition, decreases in proteostasis are known to occur with aging. Given the findings herein, one possibility is that increased levels of FKBP51 results in changes to chaperone complexes that leads to decreased proteostatic capacity and neurodegeneration. In the future, more work is warranted to understand how FKBP51 influences chaperone complexes with aging and neurodegeneration. * * * [000168] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readilyMBF Ref.028193-0010-WO01 modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [000169] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents. [000170] All publications, patents, patent applications, and / or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and / or other document were individually indicated to be incorporated by reference for all purposes. [000171] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses: [000172] Clause 1. A method of restoring the tertiary structure of a mutant C terminus of HSP70-Interacting Protein (CHIP) to the tertiary structure of wild-type CHIP in cells, the method comprising contacting the cells with a small molecule or a peptide. [000173] Clause 2. The method of clause 1, wherein the mutant CHIP comprises a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. [000174] Clause 3. The method of clause 2, wherein the TPR domain is partially unfolded. [000175] Clause 4. The method of any one of clauses 1-3, wherein the small molecule or the peptide bind to the mutant CHIP. [000176] Clause 5. The method of any one of clauses 1-4, wherein the cells are in a subject or in vitro. [000177] Clause 6. The method of clause 5, wherein the subject has a neurodegenerative disorder.MBF Ref.028193-0010-WO01 [000178] Clause 7. A method of increasing ubiquitination of a protein by a mutant C terminus of HSP70-Interacting Protein (CHIP) or a wild-type CHIP in cells, the method comprising contacting the cells with a small molecule or a peptide. [000179] Clause 8. The method of clause 7, wherein the small molecule or the peptide induces a conformational change in the mutant CHIP or the wild-type CHIP. [000180] Clause 9. The method of clause 7 or clause 8, wherein the mutant CHIP comprises a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain. [000181] Clause 10. The method of any one of clauses 7-9, wherein the cells are in a subject or in vitro. [000182] Clause 11. The method of clause 10, wherein the subject has a neurodegenerative disorder. [000183] Clause 12. The method of any one of clauses 7-11, wherein the small molecule or the peptide bind to the mutant CHIP or the wild-type CHIP. [000184] Clause 13. The method of any one of clauses 7-12, wherein affinity between the protein and the mutant CHIP or the wild-type CHIP is increased. [000185] Clause 14. The method of any one of clauses 7-13, wherein the protein is a binding partner of the TPR domain of the mutant CHIP or the wild-type CHIP. [000186] Clause 15. The method of any one of clauses 7-14, wherein the protein is heat shock protein 70 (HSP70), transcription factor II A (TFIIA), charged multivesicular body protein 2B (CHMP2B), or a combination thereof. [000187] Clause 16. A method of treating a neurodegenerative disorder, the method comprising administering a therapeutically effective amount of a small molecule or a peptide to a subject in need thereof. [000188] Clause 17. The method of clause 16, wherein the subject comprises a mutant C terminus of HSP70-Interacting Protein (CHIP) comprising a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain or a wild-type CHIP.MBF Ref.028193-0010-WO01 [000189] Clause 18. The method of clause 16 or clause 17, wherein the neurodegenerative disorder is Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s disease (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA). [000190] Clause 19. The method of any one of clauses 16-18, wherein the small molecule or the peptide bind to the mutant CHIP or the wild-type CHIP. [000191] Clause 20. The method of any one of clauses 17-19, wherein the mutation is a mutation in the TPR domain of CHIP, coiled-coil domain of CHIP, the U-box domain of CHIP, or a combination thereof. SEQUENCES SEQ ID NO: 1 Amino Acids 30-70 of Human TPR Domain of CHIP Amino Acid Sequence KEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCY SEQ ID NO: 2 Amino Acids 31-71 of Mouse TPR Domain of CHIP Amino Acid Sequence KEQGNRLFVGRKYPEAAACYGRAITRNPLVAVYYTNRALCY SEQ ID NO: 3 Amino Acids 19-59 of Fish TPR Domain of CHIP Amino Acid Sequence KEQGNRLFLSRKYQEAVTCYSKAINRNPSVAVYYTNRALCY SEQ ID NO: 4 Amino Acids 18-58 of Fly TPR Domain of CHIP Amino Acid Sequence KEQGNCLFAARKYDDAINCYSKAIIKNPTNATYFTNRALCN SEQ ID NO: 5 Amino Acids 14-54 of Plant TPR Domain of CHIP Amino Acid Sequence KEDGNNCFKKERFGAAIDAYTEAIALSPNVPAYWTNRALCMMBF Ref.028193-0010-WO01 SEQ ID NO: 6 Amino Acids 3-12 of Human TPR Domain of CHIP Amino Acid Sequence GKEEKEGGAR SEQ ID NO: 7 Amino Acids 8-22 of Human TPR Domain of CHIP Amino Acid Sequence EGGARLGAGGGSPEK SEQ ID NO: 8 Amino Acids 13-30 of Human TPR Domain of CHIP Amino Acid Sequence LGAGGGSPEKSPSAQELK SEQ ID NO: 9 Amino Acids 23-30 of Human TPR Domain of CHIP Amino Acid Sequence SPSAQELK SEQ ID NO: 10 Amino Acids 23-35 of Human TPR Domain of CHIP Amino Acid Sequence SPSAQELKEQGNR SEQ ID NO: 11 Amino Acids 41-51 of Human TPR Domain of CHIP Amino Acid Sequence KYPEAAACYGR SEQ ID NO: 12 Amino Acids 42-51 of Human TPR Domain of CHIP Amino Acid Sequence YPEAAACYGR SEQ ID NO: 13 Amino Acids 155-173 of Human SCA48 mutant TPR Domain of CHIP Amino Acid Sequence IHQESELHSYLSRLIAAER SEQ ID NO: 14 Amino Acids 36-51 of Human SCA48 mutant TPR Domain of CHIP Amino Acid SequenceMBF Ref.028193-0010-WO01 LFVGRKYPEAACYGR SEQ ID NO: 15 Amino Acids 49-62 of Human Magoh Amino Acid Sequence EAYVHKSVMEELKR SEQ ID NO: 16 Amino Acids 89-111 of Human Ddx5 Amino Acid Sequence GHNCPKPVLNFYEANFPANVMDVIAR SEQ ID NO: 17 Amino Acids 44-53 of Human Ddx5 Amino Acid Sequence KKWNLDELPK SEQ ID NO: 18 Amino Acids 77-97 of Human Prpf38a Amino Acid Sequence MLQIQPEKDIIVEFIKNEDFK SEQ ID NO: 19 Amino Acids 179-203 of Human Prpf6 Amino Acid Sequence LTPVPDSFFAKHLQTGENHTSVDPR SEQ ID NO: 20 Amino Acids 667-684 of Human Hnmpm Amino Acid Sequence MLKDKFNECGHVLYADIK SEQ ID NO: 21 Amino Acids 114-129 of Human Hnrnpa1 Amino Acid Sequence IFVGGIKEDTEEHHLR SEQ ID NO: 22 Amino Acids 36-57 of Human Hnrnpa3 Amino Acid Sequence KLFIGGSFETTDDSLREHFEKMBF Ref.028193-0010-WO01 SEQ ID NO: 23 Amino Acids 1589-1603 of Human Snrnp200 Amino Acid Sequence FLHCTEKDLIPYLEK SEQ ID NO: 24 Amino Acids 32-53 of Human Hnrnpa1 Amino Acid Sequence SHFEQWGTLTDCVVMRDPNTKR SEQ ID NO: 25 Amino Acids 1032-1057 of Human Sf3b3 Amino Acid Sequence WVTTASLLDYDTVAGADKFGNICVVR SEQ ID NO: 26 Amino Acids 93-106 of Human Hnrnpa1 Amino Acid Sequence EDSQRPGAHLTVKK SEQ ID NO: 27 Amino Acids 38-48 of Human Hnmpm Amino Acid Sequence GEERPTQNEKR SEQ ID NO: 28 Amino Acids 180-195 of Human Eif4a3 Amino Acid Sequence AIKMLVLDEADEMLNK SEQ ID NO: 29 Amino Acids 239-252 of Human Eif4a3 Amino Acid Sequence ILVKRDELTEGIK SEQ ID NO: 30 Amino Acids 144-151 of Human Snrpa Amino Acid Sequence VLDFQKVK

Claims

MBF Ref.028193-0010-WO01 CLAIMS What is claimed is:

1. A method of restoring the tertiary structure of a mutant C terminus of HSP70-Interacting Protein (CHIP) to the tertiary structure of wild-type CHIP in cells, the method comprising contacting the cells with a small molecule or a peptide.

2. The method of claim 1, wherein the mutant CHIP comprises a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain.

3. The method of claim 2, wherein the TPR domain is partially unfolded.

4. The method of claim 1, wherein the small molecule or the peptide bind to the mutant CHIP.

5. The method of claim 1, wherein the cells are in a subject or in vitro.

6. The method of claim 5, wherein the subject has a neurodegenerative disorder.

7. A method of increasing ubiquitination of a protein by a mutant C terminus of HSP70- Interacting Protein (CHIP) or a wild-type CHIP in cells, the method comprising contacting the cells with a small molecule or a peptide.

8. The method of claim 7, wherein the small molecule or the peptide induces a conformational change in the mutant CHIP or the wild-type CHIP.

9. The method of claim 7, wherein the mutant CHIP comprises a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain.

10. The method of claim 7, wherein the cells are in a subject or in vitro.

11. The method of claim 10, wherein the subject has a neurodegenerative disorder.MBF Ref.028193-0010-WO01 12. The method of claim 7, wherein the small molecule or the peptide bind to the mutant CHIP or the wild-type CHIP.

13. The method of claim 7, wherein affinity between the protein and the mutant CHIP or the wild-type CHIP is increased.

14. The method of claim 7, wherein the protein is a binding partner of the TPR domain of the mutant CHIP or the wild-type CHIP.

15. The method of claim 7, wherein the protein is heat shock protein 70 (HSP70), transcription factor II A (TFIIA), charged multivesicular body protein 2B (CHMP2B), or a combination thereof.

16. A method of treating a neurodegenerative disorder, the method comprising administering a therapeutically effective amount of a small molecule or a peptide to a subject in need thereof.

17. The method of claim 16, wherein the subject comprises a mutant C terminus of HSP70- Interacting Protein (CHIP) comprising a mutation in its tetratricopeptide repeat (TPR) domain or coiled-coil domain or a wild-type CHIP.

18. The method of claim 16, wherein the neurodegenerative disorder is Spinocerebellar ataxia (SCA) 48, SCA1, SCA2, SCA6, SCA7, SCA17, Alzheimer’s disease (AD), Parkinson’s disease (PD), Machado-Joseph disease (MJD / SCA3), Huntington's disease (HD), dentatorubral pallidoluysian atrophy (DRPLA), amyotrophic lateral sclerosis (ALS), or spinal and bulbar muscular atrophy, X-linked 1 (SMAX1 / SBMA).

19. The method of claim 16, wherein the small molecule or the peptide bind to the mutant CHIP or the wild-type CHIP.

20. The method of claim 17, wherein the mutation is a mutation in the TPR domain of CHIP, coiled-coil domain of CHIP, the U-box domain of CHIP, or a combination thereof.

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