Method for screening lysophagy activator and composition containing lysophagy activator

Abstract

The present invention relates to a method for screening a lysophagy activator by promoting ubiquitination, and a pharmaceutical composition that contains the lysophagy activator selected by the screening method, and thus can treat or prevent lysosomal regulatory disorders, such as lysosomal storage disorders, neurodegenerative diseases, diabetes, or cancers.

Description

Method for screening lysophagy activators and composition containing lysophagy activators

[0001] The present invention relates to a method for screening lysophagi activators by promoting ubiquitination, and to a pharmaceutical composition or functional food composition containing a lysophagi activator selected by such screening method, which can treat or prevent lysosomal dysregulation, such as lysosomal storage disorder, neurodegenerative disease, diabetes, or cancer.

[0002] Lysosomes are single-membrane organelles that play a pivotal role in cell degradation and nutrient recycling. Mature lysosomes have an acidic interior (pH 4.5 to 5.0), which is generated and maintained by proton-pumping v-type ATPases. 1 These acidic conditions optimally activate and stabilize various lysosomal hydrolases. 2 These unique characteristics regulate lysosomal degradation function. 3 The lysosomal membrane is composed of numerous membrane proteins, which highlights the important role lysosomes play in various cellular functions such as nutrient sensing, ion signaling, and lipid homeostasis. 4,5 Lysosomal membrane proteins undergo extensive glycosylation to protect from the acidic lumen environment and maintain lysosomal integrity. 5,6,7 .

[0003] Lysosomal dysregulation is associated with various diseases, including lysosomal storage disorders, neurodegenerative diseases, diabetes, and cancer. 8 Therefore, lysosome integrity is essential for cellular health and overall homeostasis. Lysosomotropic agents, such as L-leucyl-L-leucine methyl ester (LLOMe), induce lysosomal membrane permeability9,10,11,12 and release lysosome contents into the cytoplasm, thereby increasing cytoplasmic acidity and inducing apoptotic pathways through released lysosomal proteases. 13In response to partial damage, the endosome sorting complex (ESCRT-III) apparatus required for transport is recruited to the damaged lysosome membrane and repaired. 14 Then, the transcription factor EB (TFEB) is activated through a decrease in phosphorylation, moves from the cytoplasm to the nucleus, and increases lysosomal gene expression to replace damaged lysosomes. 15,16 If this mechanism fails, the damaged lysosome undergoes lysosome-selective autophagy (i.e., lysophage) for removal.

[0004] In selective autophagy, cargo is typically labeled with ubiquitin via E3 ligase. Ubiquitin is a small protein recognized by autophagy adapter proteins such as Sequestosome 1 (SQSTM1 / p62). 17 Lysosomes ruptured during lysophagy are selectively labeled with galectin and ubiquitin. 9 Galectin, a conserved family of cytoplasmic lectins with carbohydrate recognition domains, - Shows high affinity for galactoside glycoconjugates 18 Since most lysosomal membrane proteins are glycosylated, β-galactosides are exposed in the cytoplasm after lysosomal rupture and are recognized by galectins19,20,21,22,23. Among galectins, galectin 3 (Gal3) is used as a widely used marker for lysosomal injury. 9,24 Damaged lysosomes are also marked with ubiquitin. Some lysosomal membrane proteins have been reported to be ubiquitinated as substrates for lysosomal damage, including lysosome-associated membrane protein 1 (LAMP1), LAMP2, and transmembrane protein 192 (TMEM192). 25Several enzymes contribute to the ubiquitination of lysosomal membrane proteins during lysophage. For example, LAMP2 is part of the SCF E3 ligase complex, which consists of S-phase-kinase-associated protein 1 (SKP1), Cullin-1 (CUL1), F-box protein 27 (FBXO27), and the CUL4A-DDB1-WDFY1 complex. FBXO27 It is ubiquitinated as a substrate through 25,26 In addition, the ubiquitin-conjugated E2 enzyme (UBE2QL1) participates in the ubiquitination of LAMP1 in cooperation with lysophage effectors. 27 Autophagy adapter proteins such as SQSTM1, tax1-binding protein 1 (TAX1BP1), and calcium-binding and coiled-coil domain 2 (CALCOCO2) recognize ubiquitin at the lysosome membrane and recruit autophagy machinery to facilitate lysophagi9,27,28,29. Thus, ruptured lysosomes are eventually degraded by lysophagi.

[0005] The most well-established ubiquitin-dependent mechanism in selective autophagy is mitochondrial selective autophagy. As a regulatory mechanism of mitophagy, PTEN-induced kinase 1 (PINK1) and the E3 ligase Parkin participate in the ubiquitination of damaged mitochondrial membrane proteins. When mitochondria are damaged, PINK1 stabilizes and accumulates on the mitochondrial membrane, recruiting cytoplasmic Parkin to mediate the ubiquitination of mitochondrial outer membrane proteins such as voltage-dependent anion channel 1 (VDAC1)31. 30 Then, adapter proteins (e.g., SQSTM1, optinurin (OPTN), and CALCOCO2) recognize ubiquitination and recruit autophagy machinery to degrade mitochondria. 32 Therefore, in mitophagy, PINK1 and Parkin act as a mitochondrial damage sensor kinase and a downstream E3 ligase, respectively. Thus, the PINK1-Parkin-VDAC1-SQSTM1 axis is a well-known scenario for mitophagy.

[0006] Although the importance of lysophages in maintaining cell homeostasis and cell fate is well established, the precise regulatory mechanisms that orchestrate the sensor kinase-E3 ligase-membrane ubiquitination target-adapter recruitment scenario remain unclear.

[0007] This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HP24C1234).

[0008] The inventors screened a library of ubiquitination-related compounds to identify lysophagy activators and discovered that a novel lysophagy pathway, the TBK1-FBXO3-TMEM192-TAX1BP1 axis, promotes ubiquitination and regulates lysophagy, thereby completing the present invention.

[0009] Accordingly, the present invention aims to provide a method for screening lysophagy activators using the TBK1-FBXO3-TMEM192-TAX1BP1 axis.

[0010] In addition, the present invention aims to provide a composition capable of treating or preventing lysosomal dysregulation by containing a lysophagi activator.

[0011] To achieve the above objective, the present invention provides a method for screening lysophagy activators by controlling the ubiquitination of the TBK1-FBXO3-TMEM192-TAX1BP1 axis.

[0012] In addition, the present invention provides a pharmaceutical composition for treating or preventing lysosomal dysregulation by containing a lysophagi activator screened through the above method.

[0013] The present invention can easily determine the efficacy and utility of substances having lysophage-regulating activity based on the newly identified TBK1-FBXO3-TMEM192-TAX1BP1 axis, a lysophage regulatory pathway. The present invention can effectively screen substances with excellent effects for the prevention, alleviation, improvement, or treatment of lysosomal dysregulation. By providing an in-depth understanding of lysosomal dysregulation, the present invention can present new approaches to therapies, including innovative therapeutic agents.

[0014] Figure 1 shows screening data for lysophage inhibitors, representing the relative grades of lysophage inhibitors through ubiquitination compound library screening in HepG2 / GFP-Gal3 cells. Bafilomycin A1 (10 nM) was treated as a positive control for lysophage inhibitors.

[0015] Figure 2 shows that FBXO3 inhibition reduces the clearance of Gal3 puncta and lysophage flux in HepG2 cells. A: HepG2 / GFP-Gal3 cells were co-treated with LLOMe (750 μM) for 2 hours with 10 nM bafilomycin A1 (Baf) or 50 μM BC-1215. After 2 hours of treatment, cells were washed with fresh medium and cultured for an additional 24 hours in standard culture medium containing Baf or BC-1215. Then, the number of puncta structures expressing GFP-Gal3 per cell was quantified. b, c: HepG2 / GFP-Gal3 cells were transfected for 48 hours with scrambled siRNA (Sc), siRNA targeting FBXO3 (siFBXO3), or siRNA targeting ATG5 (siATG5). After transfection, cells were treated with LLOMe for 2 hours. After 2 hours of treatment, cells were washed and incubated in normal cell culture medium for 24 hours. The number of GFP-Gal3 spots per cell was counted, and cells were further analyzed by Western blot using the anti-FBXO3 antibody. d: HepG2 / CH-Gal3 cells were co-treated with LLOMe and Baf or BC-1215 for 2 hours. After 2 hours of treatment, LLOMe was removed, and cells were cultured in normal cell medium containing Baf or BC-1215 for 24 hours. mCherry and SEpHluorin double-positive Gal3 spots per cell (mCherry + / SEpHluorin + ) or mCherry-positive / SEpHluorin-negative Gal3 spots (mCherry + / SEpHluorin -The number of ) was counted. e: HepG2 cells were treated with LLOMe containing Baf or BC-1215. After 2 hours of treatment, the LLOMe was washed with normal cell culture medium, and the cells were incubated with Baf or BC-1215 for another 24 hours. The cells were then analyzed by Western blot to evaluate LC3 conversion. Data for all panels are expressed as mean ± SEM (n = 300, *p < 0.0001). Scale bar: 10 μm.

[0016] Figure 3 shows the effect of BC-1215 on GFP-Gal3 spot formation in HepG2 cells. a, b: HepG2 / GFP-Gal3 cells were treated with the indicated concentrations of BC-1215 for 24 hours. The number of GFP-Gal3 spots was then counted. Data are expressed as mean ± SEM (n = 300, *p < 0.0001). Scale bar: 10 μm.

[0017] Figure 4 shows the effects of lysophage regulators on HepG2 cells treated with LLOMe. a, b: HepG2 / GFP-Gal3 cells were transfected with labeled siRNA, which is known as a ubiquitination regulator in lysophages. The number of GFP-Gal3 spots per cell was counted. Data are expressed as mean ± SEM (n = 300, *p < 0.0001). Scale bar: 10 μm.

[0018] Figure 5 shows the verification results of ubiquitination targets regulated by FBXO3. a: HepG2 cells overexpressing both HA-FBXO3 and GFP-LAMP1 were treated with LLOMe (750 μM) for 2 hours and immunoprecipitated with an HA antibody conjugated to agarose beads. b: HepG2 cells transiently expressing both HA-FBXO3 and GFP-VAMP3 were treated with LLOMe (750 μM) for 2 hours and immunoprecipitated with an anti-HA antibody conjugated to agarose beads. c: HepG2 cells overexpressing both HA-ubiquitin and GFP-VAMP3 were treated with LLOMe (750 μM) for 2 hours and immunoprecipitated with an anti-GFP antibody conjugated to agarose beads. d, e: HepG2 cells transiently expressing both HA-FBXO3 and TMEM192-FLAG were treated with LLOMe (750 μM) for 2 hours and immunoprecipitated with an agarose bead-conjugated anti-HA antibody or an anti-FLAG antibody. The immunoprecipitate was analyzed by Western blotting with the labeled antibody. f: HepG2 cells overexpressing both HA-ubiquitin and TMEM192-FLAG were treated with LLOMe (750 μM) for 2 hours and down-conjugated with an agarose bead-conjugated anti-FLAG antibody. The immunoprecipitate was further analyzed by Western blotting with the labeled antibody.

[0019] Figure 6 indicates that FBXO3 interacts with TMEM192 and mediates the ubiquitination of TMEM192 in LLOMe-treated HepG2 cells. a, b: HepG2 cell lysates were treated with LLOMe (750 μM) for 2 hours, and immunoprecipitation was performed using anti-FBXO3 or anti-TMEM192 antibodies. Immunocomplexes were then analyzed by Western blot using the indicated antibodies. c: Schematic diagram of FBXO3 WT and deletion mutations. d: HepG2 cells were transfected with HA-tagged FBXO3 wild-type (WT), FBXO3 ΔF-box domain, FBXO3 ΔSUKH domain, and FBXO3 ΔApaG domain along with TMEM192-FLAG. After transfection, cells were treated with LLOMe, and then immunoprecipitation was performed using anti-HA antibodies conjugated to agarose beads. Immunoprecipitates were analyzed by Western blot using the labeled antibodies. e: HepG2 cells were transfected with a plasmid expressing TMEM192-FLAG wild-type (WT) or mutant (TMEM192 K201,211,237,246,254R) along with HA-ubiquitin. After transfection, cells were treated with LLOMe for 2 hours, followed by immunoprecipitation using an agarose-conjugated anti-FLAG antibody. Immunoprecipitates were analyzed by Western blot using the labeled antibodies. f: HepG2 cells overexpressing both HA-ubiquitin and TMEM192-FLAG were pretreated with BC-1215 and then co-treated with LLOMe for an additional 2 hours. Subsequently, cells were immunoprecipitated using an anti-FLAG antibody conjugated to agarose beads. Immunoprecipitates were further analyzed by Western blot using the labeled antibodies. g: HepG2 cells were transfected for 72 hours with scrambled siRNA or siRNA targeting FBXO3siRNA together with HA-ubiquitin and TMEM192-FLAG.After 72 hours, cells were treated with LLOMe (750 μM) for 2 hours and immunoprecipitated using an agarose-conjugated anti-FLAG antibody. Immunocomplexes were analyzed by Western blot using the labeled antibody.

[0020] Figure 7 indicates that downregulation of TMEM192 reduces the clearance of Gal3 spots. a: HepG2 / GFP-Gal3 cells were transiently transfected with scrambled siRNA (Sc) or TMEM192-targeted siRNA (siTMEM192) for 48 hours. Then, the cells were treated with LLOMe (750 μM) for 2 hours. After 2 hours, the cells were washed and incubated in normal cell culture medium for 24 hours. Then, the number of GFP-Gal3 spots was counted. Data are expressed as mean ± SEM (n = 300, *p < 0.0001). Scale bar: 10 μm. b: Cells were analyzed by Western blot using an anti-TMEM192 antibody.

[0021] Figure 8 shows the screening results of adapter proteins that recognize TMEM192 ubiquitination. a, c: HepG2 cells expressing GFP-TMEM192 and mCherry-NBR1 were treated with LLOMe (750 μM) for 2 hours. After 2 hours, the cells were fixed and imaged using a confocal microscope. b: HepG2 cells expressing TMEM192-FLAG and OPTN-GFP were treated with LLOMe (750 μM) for 2 hours. After 2 hours, the cells were stained with an anti-FLAG antibody and imaged using a confocal microscope. The degree of co-localization between TMEM192 and NBR1 or OPTN was analyzed using Pearson correlation coefficients. Data are expressed as mean ± SEM (n = 12). Scale bar: 10 μm. c, d: HepG2 cells transfected with mCherry-NBR1 or OPTN-GFP with TMEM192-FLAG were treated with LLOMe (750 μM) for 2 hours. Then, the cells were immunoprecipitated with agarose-conjugated anti-FLAG and analyzed by Western blot with the labeled antibodies.

[0022] Figure 9 shows that TMEM192 interacts with SQSTM1 and TAX1BP1 as an autophagy adapter in response to lysosomal injury. a, b, c: HepG2 cells were transiently transfected with mCherry-SQSTM1 or TAX1BP1-FLAG along with GFP-TMEM192 and then treated with LLOMe (750 μM, 2 hours). The cells were then stained with anti-FLAG antibodies and imaged using a confocal microscope. The degree of co-localization of TMEM192 with SQSTM1 or TAX1BP1 was analyzed using Pearson correlation coefficients. Data are expressed as mean ± SEM (n = 12, ***p = 0.0001, **p = 0.0020). d: HepG2 cells expressing both TMEM192-FLAG and GFP-SQSTM1 were treated with LLOMe. Then, cells were immunoprecipitated with anti-FLAG antibodies conjugated with agarose beads and analyzed by Western blot using the labeled antibodies. e: HepG2 cells transiently expressing both GFP-TMEM192 and TAX1BP1-FLAG were treated with LLOMe. After 2 hours, cells were immunoprecipitated with anti-GFP antibodies conjugated with agarose beads and analyzed by Western blot using the labeled antibodies. f, g, h: HepG2 / GFP-Gal3 cells were transiently transfected for 48 hours with scrambled siRNA (Sc), SQSTM1-targeted siRNA (siSQSTM1), or TAX1BP1-targeted siRNA (siTAX1BP1). Then, cells were treated with LLOMe, washed, and incubated for an additional 24 hours in standard cell culture medium. Afterward, the number of GFP-Gal3 spots was counted, and the cells were analyzed by Western blot using the labeled antibodies. Data were expressed as mean ± SEM (n = 300, *p< 0.0001).

[0023] Figure 10 shows that TBK1 is activated to mediate the phosphorylation of FBXO3 in HepG2 cells treated with LLOMe. a, b: HepG2 / GFP-Gal3 cells were treated with MRT67307 (2 μM) or GSK8612 (10 μM) along with LLOMe (750 μM). After 2 hours of treatment, the LLOMe was washed away with normal cell culture medium, and the cells were incubated with MRT67307 or GSK8612 for another 24 hours. The number of GFP-Gal3 spots was quantified, and the cells were further analyzed by Western blot using the indicated antibodies. Data are expressed as mean ± SEM (n = 300, *p < 0.0001). Scale bar: 10 μm. c: HepG2 cells transiently expressing both GFP-TBK1 and HA-FBXO3 were treated with LLOMe. Then, cells were immunoprecipitated using anti-HA antibodies conjugated with agarose beads. The immunoprecipitates were further analyzed by Western blot using the labeled antibodies. d: HepG2 cells overexpressing HA or HA-FBXO3 were pretreated with MRT67307 (2 μM) for 2 hours and then co-treated with LLOMe. The cells were then analyzed by Phos-tag SDS-PAGE and Western blotted using the labeled antibodies.

[0024] Figure 11 indicates that Gal3 spot clearance decreases when TBK1 is depleted. a: HepG2 / GFP-Gal3 cells were transiently transfected with scrambled siRNA (Sc) or TBK1-targeted siRNA (siTBK1) for 48 hours. Then, the cells were treated with LLOMe (750 μM) for 2 hours. After 2 hours, the cells were washed and incubated in normal cell culture medium for 24 hours. Then, the number of GFP-Gal3 spots was counted. Data are expressed as mean ± SEM (n = 300, *p < 0.0001). Scale bar: 10 μm. b: HepG2 / GFP-Gal3 cells transfected with siRNA against TBK1 were harvested and analyzed by Western blotting using an anti-TBK1 antibody.

[0025] Figure 12 indicates that FBXO3 or TBK1 inhibitors reduce the FBXO3-TMEM192 interaction. a, b: HepG2 cells were pretreated with BC-1215 (50 μM) or GSK8612 (10 μM) for 22 hours. Then, they were co-treated with LLOMe for an additional 2 hours. The cells were then immunoprecipitated with an anti-FBXO3 antibody or an anti-TMEM192 antibody. The immune complexes were then analyzed by Western blot using the indicated antibodies.

[0026] Figure 13 shows the effect of the TBK1-FBXO3-TMEM192-TAX1BP1 axis on LLOMe-induced lysophages. a: HepG2 cells were transfected with both HA-ubiquitin and TMEM192-FLAG and pretreated with MRT67307 (2 μM) or GSK8612 (10 μM). They were co-treated with LLOMe for an additional 2 hours. Then, the cells were immunoprecipitated with an agarose bead-conjugated anti-FLAG antibody and further analyzed by Western blot using the labeled antibody. b: HepG2 cells overexpressing both GFP-TMEM192 and TAX1BP1-FLAG were pretreated with BC-1215 or GSK8612. They were co-treated with LLOMe for an additional 2 hours. Then, the cells were pulled up with an agarose bead-conjugated anti-GFP antibody and further analyzed by Western blot using the labeled antibody. c: HepG2 cells transiently expressing GFP-LC3 and TMEM192-FLAG were pretreated with BC-1215 or GSK8612 and co-treated with LLOMe. The cells were then stained with anti-FLAG antibodies and images were taken using a confocal microscope. The degree of co-localization of TMEM192 and LC3 was analyzed using Pearson correlation coefficients. Data were expressed as mean ± SEM (n = 15, * p = 0.0031, ** p < 0.0001).

[0027] Figure 14 shows the association between FBXO3 and LLOMe-induced lysophages in FBXO3 and FBXO3 knockout HepG2 cells. a: FBXO3 wild-type (WT) and knockout (KO) HepG2 cells were analyzed using Western blot with FBXO3 antibodies. B: FBXO3 WT and KO HepG2 cells transiently expressing GFP-Gal3 were treated with 2 mM LLOMe for 3 hours. After treatment, cells were washed and incubated with fresh medium. The number of GFP-Gal3 spots per cell was counted. Data are expressed as mean ± SEM (n = 470, *p < 0.0001). Scale bar: 10 μm. c: FBXO3 WT and KO HepG2 cells expressing HA-ubiquitin and TMEM192-FLAG were treated with 2 mM LLOMe for 3 hours. Then, cells were immunoprecipitated with FLAG antibodies conjugated to agarose beads and analyzed by Western blot using the labeled antibodies. d: FBXO3 WT and KO HepG2 cells expressing GFP-TMEM192 and TAX1BP1-FLAG were treated with 2 mM LLOMe for 3 hours. After 3 hours, cells were immunoprecipitated with anti-GFP antibodies conjugated to agarose beads and analyzed by Western blot using the labeled antibodies. e: FBXO3 WT and KO HepG2 cells transiently expressing mCherry-Gal3 were reconstituted with empty GFP vector, GFP-FBXO3 WT, or GFP-FBXO3 V221I. Then, cells were treated with LLOMe. After treatment, cells were washed and incubated in fresh culture medium for 24 hours. The number of mCherry-Gal3 spots per cell was counted. Data are expressed as mean ± SEM (n = 200, *p< 0.0001). Scale bar: 10 μm. f: KO HepG2 cells expressing HA-ubiquitin with empty GFP, GFP-FBXO3 WT, or V221I were treated with LLOMe.Then, cells were immunoprecipitated with anti-TMEM192 antibody and analyzed by Western blot using the labeled antibody.

[0028] Figure 15 shows a schematic diagram of a novel lysophagy regulatory mechanism by the TBK1-FBXO3-TMEM192-TAX1BP1 axis. Lysosomal agonists such as LLOMe induce lysosome damage. When severe damage occurs, the ruptured lysosome is degraded by lysophagi, the selective autophagy of lysosomes. In response to lysosome rupture, TBK1 is activated via phosphorylation at S172 and mediates the phosphorylation of FBXO3, a component of the SCF E3 ligase complex. Subsequently, FBXO3 mediates the ubiquitination of the lysosome membrane protein TMEM192. This ubiquitination leads to the recruitment of autophagy adapter proteins SQSTM1 or TAX1BP1 and other autophagy machinery, resulting in the degradation of the damaged lysosome.

[0029] Lysophages remove damaged lysosomes and are essential for cellular homeostasis. However, their underlying mechanism is not currently fully understood.

[0030] Accordingly, the inventors screened a library of ubiquitination-related compounds and found an SCF type E3 ubiquitin ligase complex, SCF FBXO3 We confirmed that the substrate recognition component of (FBXO3) is a novel and important lysophage regulator. Inhibition of FBXO3 reduced lysophage and lysophage flux in response to L-leucyl-L-leucine methyl ester (LLOMe). Furthermore, FBXO3 interacted with TMEM192, leading to ubiquitination in LLOMe-treated cells.

[0031] In addition, the inventors confirmed that TAX1BP1 is an important autophagy adapter that recognizes ubiquitinated TMEM192 during lysophagy and discovered that TBK1 activation is essential for lysophagy because it phosphorylates FBXO3 in response to lysosome damage.

[0032] Knockout of FBXO3 significantly impaired lysophages, and reconstitution with a loss-of-function mutant (V221I) further confirmed that it plays an essential role in lysophage regulation. Overall, the inventors' findings highlight the importance of the TBK1-FBXO3-TMEM192-TAX1BP1 axis in lysophages and the important role of FBXO3 in lysosome integrity.

[0033] The present invention provides a method for screening lysophagi activators by controlling the ubiquitination of the TBK1-FBXO3-TMEM192-TAX1BP1 axis.

[0034] The present invention relates to a method for screening lysophagy activators, comprising:

[0035] 1) A step of preparing a HepG2 cell line expressing the TBK1, FBXO3, TMEM192, TAX1BP1 genes and Enhanced Green Fluorescent Protein (EGFP);

[0036] 2) A step of inducing lysophage activation by treating the prepared HepG2 cell line with a lysosomotropic agent without or with the test substance;

[0037] 3) a step of capturing a fluorescence image of the cells and measuring the number of spots exhibiting the green fluorescence of EGFP; and

[0038] 4) A step of determining a test substance as a lysophagy activator by reducing the number of spots exhibiting green fluorescence compared to the case where a lysosomal homing agent is administered without the test substance,

[0039] Includes

[0040] In the present invention, the lysosome-oriented agent comprises, but is not limited to, L-leucil-L-leucine methyl ester (LLOMe).

[0041] In step 2) above, lysophage activation is induced through the TBK1-FBXO3-TMEM192-TAX1BP1 signaling cascade, which consists of i) activation of TBK1, ii) phosphorylation of FBXO3, iii) ubiquitination of TMEM192, and iv) activation of TAX1BP1.

[0042] The method of the present invention may further include the step of determining whether a test substance determined to be a regulator activating lysophagi activates at least one signaling pathway of the TBK1-FBXO3-TMEM192-TAX1BP1 signaling cascade.

[0043] In the present invention, the test substance determined to be a lysophagy activator is one that activates TBK1, phosphorylates FBXO3, ubiquitines TMEM192, or activates TAX1BP1.

[0044] In the present invention, the term "test substance" refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. The sample includes, but is not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0045] In addition, the present invention provides a pharmaceutical composition for treating or preventing lysosomal dysregulation by containing a lysophagy activator. Here, the lysophagy activator may be a substance screened through the method according to the present invention.

[0046] In the present invention, lysosomal dysregulation may be selected from the group consisting of lysosomal storage disorders, neurodegenerative diseases, diabetes, or cancer.

[0047] In the present invention, lysosomal storage disorders include, but are not limited to, Tay-Sachs disease, Fabry disease, Pombe disease, mucopolysaccharidosis, Niemann-Pick disease, Gaucher disease, Hunter syndrome, alpha-mannosidosis, aspartylglucosamineuria, cholesterol ester accumulation disease, chronic hexosaminidase A deficiency, cystinosis, Danone disease, Farber disease, fucoside accumulation, galactosialidosis, or Baton disease including late infant Baton disease and adolescent Baton disease.

[0048] In the present invention, neurodegenerative diseases include, but are not limited to, neurogenic ceroid liporucinosis, Alzheimer's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and Parkinson's disease, as well as diseases such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), cortical-basal degeneration (CBD), or dementia (DLB) with Lewy bodies.

[0049] In the present invention, diabetes includes, but is not limited to, type 1 diabetes or type 2 diabetes, and is particularly type 2 diabetes.

[0050] In the present invention, cancer may be a solid tumor or a blood cancer, and may be a primary cancer or a metastatic cancer. The cancer includes, but is not limited to, cervical cancer, breast cancer, squamous cell carcinoma, colorectal cancer, gastric cancer, liver cancer, skin cancer, prostate cancer, mammary gland cancer, nasopharyngeal adenocarcinoma, lung cancer, or brain tumor.

[0051] In the present invention, the term "active ingredient" refers to a component that exhibits the desired activity alone or can exhibit activity together with a carrier that is inactive itself.

[0052] In one embodiment of the present invention, the composition of the present invention may be a pharmaceutical composition for treating or preventing lysosomal dysregulation containing a lysophagi activator.

[0053] According to one embodiment of the present invention, the lysophagi activator is characterized by being included in an amount of 1 to 80 weight% based on the total weight of the composition. At this time, if the content is less than 1 weight%, the therapeutic and preventive effects of lysosome dysregulation, which are the intended effects of the present invention, cannot be obtained, and if it exceeds 80 weight%, the effect may be inefficient as it is not proportional to the increase in content, and there is a problem that the stability of the formulation is not ensured.

[0054] The composition of the present invention may further include a suitable carrier, excipient, or diluent commonly used in addition to the active ingredient.

[0055] The pharmaceutical composition according to the present invention may include a pharmaceutically acceptable carrier in addition to a lysophagy activator. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is one that is commonly used in formulations and includes, but is not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0056] The pharmaceutical composition of the present invention may additionally include, in addition to the above components, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0057] In this specification, the term "prevention" refers to any act of suppressing or delaying the progression of lysosomal dysregulation by administering a composition of the present invention.

[0058] In this specification, the term “treatment” means inhibition of the development of lysosomal dysregulation; alleviation of lysosomal dysregulation; and elimination of lysosomal dysregulation.

[0059] The pharmaceutical composition of the present invention can be administered orally or parenterally.

[0060] Suitable dosages of the pharmaceutical composition of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, patient's age, body weight, gender, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. The dosage of the pharmaceutical composition of the present invention is generally within the range of 0.001 to 1000 mg / kg, preferably 0.05 to 200 mg / kg, and more preferably 0.1 to 100 mg / kg for adults. In addition, in the case of a topical preparation, it is preferable to apply an amount of 1.0 to 3.0 ml once to five times a day for at least one month for adults. However, the above dosages do not limit the scope of the present invention.

[0061] The pharmaceutical composition of the present invention may be prepared in a unit dose form or contained in a multi-dose container by formulation using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily carried out by a person skilled in the art. The formulation may be in the form of a solution, suspension, syrup, or emulsion in an oil or aqueous medium, or may be in the form of an excipient, powder, powder, granule, tablet, or capsule, and may additionally include a dispersant or a stabilizer.

[0062] The composition of the present invention may be a functional food composition for improving lysosomal dysregulation.

[0063] The term "functional food" as used in the present invention refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc., using raw materials or ingredients that have functional properties useful to the human body. Here, "functionality" means obtaining useful effects for health purposes, such as regulating nutrients or physiological actions regarding the structure and function of the human body. The health functional food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and ingredients commonly added in the industry. Furthermore, unlike general pharmaceuticals, it has the advantage of being made from food ingredients, thus avoiding side effects that may occur with long-term use of pharmaceuticals, and can be highly portable.

[0064] The food composition according to the present invention may additionally include, in addition to a lysophagy activator as an active ingredient, ingredients that are typically added during food manufacturing, such as proteins, carbohydrates, fats, nutrients, seasonings, and flavorings.

[0065] Examples of the above carbohydrates are monosaccharides, e.g., glucose, fructose, etc.; disaccharides, e.g., maltose, sucrose, oligosaccharides, etc.; and polysaccharides, e.g., dextrin, cyclodextrin, etc., which are conventional sugars and sugar alcohols such as xylitol, sorbitol, erythritol, etc. As flavoring agents, natural flavoring agents [taumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

[0066] For example, when the food composition of the present invention is prepared as a drink, in addition to the lysophagy activator, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, Eucommia ulmoides extract, jujube extract, licorice extract, etc. may be additionally included.

[0067] Another aspect of the present invention provides a method for preventing or treating lysosomal dysregulation, comprising the step of administering a composition of the present invention to an individual.

[0068] In the present invention, the term "individual" refers to any animal other than humans that has developed or may develop a lysosomal dysregulation disorder, and the individual can be efficiently treated by administering the composition of the present invention to an individual suspected of having a lysosomal dysregulation disorder.

[0069] In the present invention, the term "administration" means introducing the composition of the present invention to an individual suspected of having a lysosomal dysregulation by any appropriate method. The route of administration may be various oral or parenteral routes as long as it can reach the target tissue, as described above.

[0070] The present invention will be explained in detail below through examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[0071]

[0072] Examples

[0073] Example 1. Materials and Method

[0074] 1. Reagents

[0075] Bafilomycin A1 and L-leucil-L-leucine methyl ester (LLOMe) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). BC-1215, GSK8612, and MRT67307 were purchased from MedChemExpress (Monmouth Junction, New Jersey, USA). Short interfering RNAs (siRNAs) specifically targeting human ATG5 (5'-FRONT-FRONT-3'); FBXO3 (#1, 5'-FBXO3-3'), (#2, 5'-FBXO3-3'); TMEM192 (#1, 5'-CCAGUAUCACCCACAGCAAA-3'; SEQ No. 1), (#2, 5'-CUAUUUCAAGCCUAGAAGA-3'; SEQ No. 2); SQSTM1 (5'-GCAUUGAAGUUGAUAUCGAUUU-3'; SEQ No. 3); TAX1BP1(#1, 5'-CAAAGAAAUUGCUGACAAA-3'; Sequence No. 4), (#2, 5'-CAAAGAAAUUGCUGACAAA-3'; Sequence No. 5), (#3, 5'-AAGAAGAACTGTTAAAGTTAA-3'; Sequence No. 6); TBK1(#1, 5'-CCAUGUGGGAGUUUAUACA-3'; Sequence No. 7), (#2, 5'-GCAGUUGUUUCUCUGUAU-3'; Sequence No. 8); FBXO2(#1, 5'-GATGAGAGCGTCAAGAAGT-3'; Sequence No. 9), (#2, 5'-CAGTTCTACTTCCTGAGCA-3'; Sequence No. 10); CUL4A(#1, 5'-GCACAGAUCUUCCGUUUA-3'; SEQ No. 11), (#2, 5'-GAAGAUUAACACGUGCUGG-3'; SEQ No. 12); UBE2QL1(5'-AAGCUGAAGCUACCUUU-3'; SEQ No. 13); and negative scrambled siRNA(5'-CCUACGCCACCAAUUUCGU-3'; SEQ No. 14) were synthesized at Genolution (Seoul, South Korea).

[0076] 2. Plasmid

[0077] Dr. Peter K. Kim (University of Toronto, Canada) provided pLAMP1-GFP and mCherry-NBR1. pEGFP-SQSTM1 was provided by Dr. Li Yu (Tsinghua University, China). pEGFP-VAMP3, pmCherry-Gal3, pRK5-HA-Ubiquitin-K6, and pOPTN-EGFP were purchased from Addgene (42310, 85662, 22900, and 27052; deposited by Dr. Thierry Galli, Dr. Hemmo Myer, Dr. Sandra Weller, and Beatrice Yue, respectively). TBK1-FLAG and TAX1BP1-FLAG were obtained from GenScript. HA-FBXO3 wild type (WT), ΔF-box, ΔSUKH, and ΔApaG were provided by Dr. Zhi-Xiong Jim Xiao (Sichuan University, China). pTMEM192-FLAG and pEGFP-TMEM192 were kindly provided by Dr. Fu-Ming Tsai (Taipei Tzu Chi Hospital, Taiwan). mCherry-SQSTM1 was provided by Dr. Masaaki Komatus (Juntendo University, Japan).

[0078] For mCherry-SEpHluorin-Galectin 3, Galectin 3 was subcloned into mCherry-SEpHluorin(Addgene, 32001). TBK1 and FBXO3 were subcloned into the pEGFP-C1 vector. TMEM192K201,211,237,246,254R and FBXO3 V221I Site-directed mutations were performed using the Muta-Directed Mutagenesis Kit (Intron Biotechnology) to induce mutations. The specificity of the mutations was confirmed through direct sequencing. The TMEM192 mutant was subcloned into the PCR3.1-Flag vector, and the FBXO3 mutant was subcloned into the pEGFP-C1 vector.

[0079] 3. Cell Culture and Establishment of Stable Cell Line

[0080] HepG2 cells were purchased from the American Type Culture Collection. To generate stable cell lines, HepG2 cells were transfected with GFP-Gal3 or CH-Gal3 using Lipofectamine 2000 (Invitrogen, Carlsbad, California, USA) according to the manufacturer's protocol. Stable transfected cells were selected using 1 mg / ml of G418 for 10 days (Invitrogen). After inoculating individual cells, stable clones were selected using a fluorescence microscope (Olympus, Tokyo, Japan). All cells were cultured at 37°C in a 5% CO2 incubator and maintained in Dulbecco's modified Eagle's medium (DMEM; WELGENE, Gyeongsan, South Korea) supplemented with 10% FBS (WELGENE) and 1% penicillin-streptomycin (WELGENE).

[0081] 4. Generation of FBXO3 knockout cell lines using CRISPR / Cas9

[0082] FBXO3 was knocked out in HepG2 cell lines produced at UBIGENE (Guangzhou, China). Exon 1 of the FBXO3 gene was specifically edited using CRISPR / Cas9 technology. To target this region, human FBXO3 oligonucleotides (gRNA1R: 5'-AGTCCAAAAAGGATAAGATGAGG-3' (SEQ No. 15); gRNA2R: 5'-TAGGGTCAGCGGCGCCGTCTCGG-3' (SEQ No. 16)) were inserted, annealed, and ligated into the YKO-RP003 vector (UBIGENE). Then, the YKO-RP003-hFBXO3[gRNA] plasmid containing the target sgRNA sequence was transfected into HepG2 cells using the Neon™ Transfection System (Thermo Fisher Scientific). Transfected cells were screened using puromycin for 2 to 3 days. After screening antibiotics, cells were inoculated individually. A single FBXO3 KO clone was selected after 2 to 4 weeks and verified by Sanger sequencing.

[0083] 5. Cell-based ubiquitination compound library screening

[0084] HepG2 / GFP-Gal3 cells were seeded into 96-well plates for cell-based ubiquitination compound library screening. After 24 hours, approximately 5 to 100 μM of the TargetMol ubiquitination compound library (TargetMol, Wellesley Hills, MA, USA), including 78 ubiquitination-associated small molecules targeting proteasomes, E1 / E2 / E3 enzymes, DUB, and p97, was treated in each well with LLOMe. GFP-Gal3 spots in the cells were monitored using a fluorescence microscope (Olympus). The experiment was repeated three times.

[0085] 6. Western Blotting

[0086] For immunoblotting, cells were lysed using cell lysis buffer, and 2x Laemmli sample buffer (Bio-Rad, Hercules, CA, USA) was added to the lysate. Total protein was quantified using Bradford solution (Bio-Rad) according to the manufacturer's instructions. Samples were separated by SDS-PAGE and transferred to PVDF membranes (Bio-Rad). After blocking with 4% skim milk in TBST (25 mM Tris-base, 140 mM NaCl, 0.05% Tween® 20 [Sigma-Aldrich]) (MBcell, Seoul, Korea), the membranes were irradiated with the following primary antibody: anti-LC3 purchased from NOVUS Biologicals (Centennial, Colorado, USA); Anti-FLAG, anti-p-TBK1 (Ser172), anti-TBK1, and anti-SQSTM1 antibodies purchased from Cell Signaling Technology (Danvers, Massachusetts, USA); anti-GFP, anti-FBXO3, and anti-HA antibodies purchased from Santa Cruz Biotechnology (Dallas, Texas, USA); anti-TMEM192 and anti-TAX1BP1 antibodies purchased from Proteintech (Rosemont, Illinois, USA); and anti-α-ACTIN (ACTA1) and anti-FLAG antibodies purchased from Sigma-Aldrich. The membranes were incubated with HRP-conjugated secondary antibodies (Cell Signaling Technology). Signals were detected using Luminograph I (ATTO, Tokyo, Japan).

[0087] 7. Immunoprecipitation

[0088] For the immunoprecipitation assay, cells were homogenized in RIPA buffer containing a protease inhibitor (GenDEPOT) (50 mM Tris-HCl, pH 7.5, 150 mM sodium chloride, 0.5% sodium deoxycholate, 1% Triton X-100, 0.1% SDS, and 2 mM EDTA; Intron Biotechnology, Seoul, South Korea) at 4°C for 1 hour. For the exogenous IP assay, the supernatant was immunoprecipitated with the following antibodies: anti-GFP-agarose and anti-HA-agarose antibodies purchased from Santa Cruz Biotechnology; and anti-FLAG-agarose antibody purchased from Abcam. For the endogenous IP assay, the supernatant was immunoprecipitated with the following antibodies: anti-FBXO3 antibody purchased from Santa Cruz Biotechnology; The anti-TMEM192 antibody obtained from Proteintech was incubated overnight at 4°C with a Protein G plus / Protein A agarose suspension (Santa Cruz Biotechnology). After incubation, the samples were washed twice with RIPA buffer at 4°C and 2x Laemmli sample buffer (Bio-Rad) was added. All samples were analyzed by Western blot as described in the Western blotting section above.

[0089] 8. Phos-tag SDS-PAGE

[0090] Phos-tag TM In the case of SDS-PAGE, Mn 2+ Phos-tag containing TM Acrylamide (Wako, Osaka, Japan) was used. Before polymerization, Phos-tag acrylamide (35 μM) and MnCl2 (7 μM) were added to the separation gel. Electrophoresis was performed under constant current conditions (25 mA per gel). After electrophoresis, the gels were gently washed four times for 20 minutes each with a transfer buffer containing 10 mM EDTA. After washing, the gels were immersed in a transfer buffer without EDTA for 20 minutes and then transferred to a PVDF membrane (Bio-Rad).

[0091] 9. Fluorescence Microscope

[0092] Cells were treated with various chemicals or siRNA and fixed with 4% paraformaldehyde for 10 minutes. Then, samples were observed using a fluorescence microscope (Olympus). The number of Gal3 spots was analyzed using Image J software (NIH, Bethesda, MD, USA). Data analysis was performed using GraphPad Prism 10 (GraphPad Software, San Diego, California, USA).

[0093] 10. Confocal Microscope

[0094] HepG2 cells were cultured in cover glasses and co-transfected with the plasmid pEGFP-TMEM192 and adapters mCherry-SQSTM1, TAX1BP1-FLAG, mCherry-NBR1, and pOPTN-EGFP. After 24 hours, the cells were treated with LLOMe for an additional 2 hours. Next, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 minutes. For permeability, the cells were incubated with 0.1% Triton X-100 for 2 minutes and blocked with 2% BSA in PBS at RT for 1 hour. After blocking, the cells were incubated overnight with the anti-FLAG antibody (Sigma-Aldrich). Then, the cells were washed with PBS and incubated overnight with the Alexa Fluor 555 Goat anti-mouse IgG antibody (Thermo Fisher, Waltham, MA, USA). Fluorescence images of TMEM192 co-localized with TAX1BP1 were obtained using a confocal laser scanning microscope (Carl Zeiss, LSM 800).

[0095] 11. Statistical Analysis

[0096] Data were obtained from at least three independent experiments and expressed as mean ± SEM. All statistical analyses were performed using GraphPad Prism 10 software. Results were statistically evaluated using the t-test. Differences of p < 0.05 were considered significant.

[0097] Example 2. FBXO3 inhibition impairs Gal3 spot clearance and lysophagy flux.

[0098] The inventors developed a cell-based image screening system using Enhanced Green Fluorescent Protein (EGFP)-Gal3 in HepG2 cells (HepG2 / GFP-Gal3 cells) and identified a novel lysophage mechanism by performing screening using a library of ubiquitinated compounds in combination with the lysosomal agent LLOMe.

[0099] Specifically, to quantify cells containing lysophages, HepG2 / CH-Gal3 cells were co-treated with LLOMe and Baf or BC-1215 for 2 hours. Then, the LLOMe was washed with fresh medium and incubated with Baf or BC-1215 for 24 hours. Fluorescence images were obtained using a fluorescence microscope (Olympus). mCherry and SEpHluorin double-positive Gal3 spots (mCherry + / SEpHluorin + ) and mCherry positive / SEpHluorin negative Gal3 spots (mCherry + / SEpHluorin - The number of ) was calculated using Image J (NIH). The obtained data was analyzed using GraphPad Prism 10.

[0100] Through screening, BC-1215 was identified as the most potent new lysophage modulator (Fig. 1).

[0101] To verify these results, HepG2 / GFP-Gal3 cells were co-treated with LLOMe and bafilomycin A1 (Baf), which blocks autophagosome-lysosome fusion. As shown in Figure 2a, BC-1215 significantly disrupted the clearance of damaged lysosomes in LLOMe-treated cells, which was similar to that of Baf treatment.

[0102] However, BC-1215 treatment alone at various concentrations did not affect GFP-Gal3 spot formation (Fig. 2a; Fig. 3a, b). This indicates that BC-1215 specifically targets lysophages in LLOMe-treated cells.

[0103] BC-1215 is a highly selective inhibitor of FBXO3 that binds to the ApaG domain of FBXO3 and interferes with its interaction with substrates. 33,34 To further investigate the role of FBXO3 in regulating GFP-Gal3 spot clearance, the inventors transfected HepG2 / GFP-Gal3 cells with scrambled siRNA or specific siRNA targeting FBXO3, and then treated them with LLOMe. Depletion of FBXO3 resulted in a significant reduction in GFP-Gal3 spot clearance, which is due to FBXO2, CUL4A, and UBE2QL1 26,27,35 Or it was similar to other well-known lysophagy regulators, including autophagy-associated 5 (ATG5), which is an autophagy regulator (Fig. 2b, c; Fig. 4).

[0104] Since SEpHluorin, one of the EGFP variants, is more sensitive to pH than EGFP, a novel lysophage flux monitoring system was established using mCherry-SEpHluorin-Galectin 3 (CH-Gal3) in HepG2 cells (HepG2 / CH-Gal3 cells). Consistently, mCherry+ / SEpHluorin-Gal3 spots decreased, whereas mCherry+ / SEpHluorin+-Gal3 spots increased in cells treated with Baf or BC-1215 (Fig. 2d). Furthermore, LC3-II protein levels were higher in cells treated with LLOMe and Baf or BC-1215 than in cells treated with each compound alone (Fig. 2e).

[0105] Taken together, these results show that inhibition of FBXO3 interferes with lysosome clearance and lysophage flux in cells treated with LLOMe.

[0106] Example 3. FBXO3 mediates the ubiquitination of the lysosomal membrane protein TMEM192 in response to lysosomal injury.

[0107] To further explain the involvement of FBXO3 in lysophage, potential lysosomal membrane protein targets for ubiquitination in LLOMe-treated cells were identified through immunoprecipitation analysis.

[0108] We found that FBXO3 did not interact directly with LAMP1 but showed binding affinity for VAMP3 (Fig. 5a, b). Despite the enhanced interaction between FBXO3 and VAMP3, VAMP3 ubiquitination was only slightly affected in LLOMe-treated cells (Fig. 54c). This suggests that FBXO3 does not primarily mediate lysophagy through LAMP1 or VAMP3.

[0109] Next, the inventors shifted their focus to TMEM192, a potential target of FBXO3. As shown in Figures 5 and 6, the interaction between FBXO3 and TMEM192 was further upregulated by LLOMe treatment (Figs. 5d, e; Figs. 6a, b). Notably, the ubiquitination of TMEM192 was significantly enhanced in LLOMe-treated cells compared to untreated control cells (Fig. 5f). This revealed that TMEM192 is a firm ubiquitinated target of FBXO3 in LLOMe-treated HepG2 cells.

[0110] In addition, deletion of the SUKH or ApaG domain of FBXO3 resulted in a slight decrease in TMEM192 binding, while deletion of the F-box region resulted in a subtle increase in the interaction between mutant FBXO3 and TMEM192 (Fig. 6c, d).

[0111] TMEM192 contains several putative ubiquitinated lysine residues, including K201, K211, K237, K246, and K254. 25 To determine whether lysine residues of TMEM192 were ubiquitinated in response to LLOMe, TMEM192 mutants that inhibited ubiquitination were generated by substituting all lysine candidates with arginine. A TMEM192 mutant with a single lysine residue maintained ubiquitination in LLOMe-treated cells, whereas a mutant with substitutions at all five lysine sites showed reduced TMEM192 ubiquitination (data not shown, Fig. 6e).

[0112] Importantly, both chemical and genetic inhibition of FBXO3 effectively inhibited TMEM192 ubiquitination in LLOMe-treated cells (Fig. 6f, g). Furthermore, depletion of TMEM192 significantly disrupted GFP-Gal3 spot clearance induced by LLOMe treatment (Fig. 7).

[0113] In summary, the above experimental results suggest that FBXO3 regulates the ubiquitination of TMEM192 in response to lysosome damage.

[0114] Example 4. SQSTM1 and TAX1BP1 interact with TMEM192 as autophagy adapters in response to lysosome damage.

[0115] Next, we investigated the autophagic adapters involved in recognizing ubiquitinated TMEM192 in response to lysosome damage. Co-localization assays between TMEM192 and the adapter proteins NBR1 or OPTN showed no substantial changes upon LLOMe treatment (Fig. 8a, b).

[0116] The interaction between NBR1 and TMEM192 was slightly reduced by LLOMe treatment, and TMEM192 did not show significant binding to OPTN (Fig. 8c, d).

[0117] These results suggest that NBR1 and OPTN may not be the major autophagy adapters in TMEM192-mediated lysophagy. The inventors further investigated other potential adapters and found that both SQSTM1 and TAX1BP1 showed significantly increased co-localization with TMEM192 in LLOMe-treated cells (Figs. 9a to c).

[0118] Furthermore, the interaction between SQSTM1 or TAX1BP1 and TMEM192 was enhanced by LLOMe treatment (Figs. 9d, e). This implies that they are involved as autophagy adapters in TMEM192-mediated lysophagi. Knockdown of SQSTM1 and TAX1BP1 reduced LLOMe-induced GFP-Gal3 spot clearance (Figs. 9f to h).

[0119] Therefore, SQSTM1 or TAX1BP1 acts as an autophagy adapter that recognizes ubiquitinated TMEM192 in response to lysosome damage.

[0120] Example 5. TBK1 activation of LLOMe mediates FBXO3 phosphorylation

[0121] To explore the upstream regulatory mechanisms regulating FBXO3, we focused on TBK1 in LLOMe-mediated lysophagi using the TBK1 inhibitors MRT67307 and GSK8612.

[0122] Treatment with the inhibitor reduced the clearance of GFP-Gal3 spots induced by LLOMe (Fig. 10a). Furthermore, TBK1 phosphorylation was significantly increased by LLOMe treatment, but disappeared when treated with a TBK1 inhibitor (Fig. 10b). Notably, when TBK1 was depleted, the clearance of GFP-Gal3 spots also decreased (Fig. 11). Taken together, these results indicate that TBK1 activation is essential for efficient lysophagi.

[0123] Next, we investigated the possibility that TBK1 mediates FBXO3 activity as an upstream regulator. Co-immunoprecipitation studies using GFP-TBK1 and HA-FBXO3 demonstrated an interaction between TBK1 and FBXO3, which was further enhanced upon lysosome damage (Fig. 10c).

[0124] Phos-tag to investigate FBXO3 phosphorylation TM SDS-PAGE analysis was performed. Notably, FBXO3 was phosphorylated upon LLOMe treatment and decreased upon co-treatment with a TBK1 inhibitor (Fig. 10d). These observations indicate that TBK1 regulates FBXO3 phosphorylation in response to lysosomal damage.

[0125] Example 6. Effect of the TBK1-FBXO3-TMEM192-TAX1BP1 axis on LLOMe-induced lysophagy

[0126] We investigated the importance of the TBK1-FBXO3-TMEM192-TAX1BP1 axis in lysophages because TBK1 played a crucial role in mediating the phosphorylation of FBXO3 in response to lysosomal damage (Fig. 10), and FBXO3 promoted interaction with TMEM192 and subsequent ubiquitination, followed by the recruitment of the autophagy adapter TAX1BP1 (Figs. 6 and 9). Notably, the interaction between FBXO3 and TMEM192, which was increased by LLOMe treatment, was reduced by FBXO3 or TBK1 inhibitors (Fig. 12). Furthermore, the increased ubiquitination of TMEM192 induced by LLOMe was reduced upon treatment with TBK1 inhibitors (Fig. 13a). Moreover, the recruitment of TAX1BP1 to TMEM192 was reduced by FBXO3 and TBK1 inhibitors (Fig. 13b).

[0127] In addition, we investigated whether this axis affects the recruitment of the autophagy machinery. After LLOMe treatment, LC3 co-localized with TMEM192, but chemical inhibition by FBXO3 and TBK1 reduced the recruitment of LC3 to TMEM192 (Fig. 13c).

[0128] Taken together, these results strongly suggest the importance of the TBK1-FBXO3-TMEM192-TAX1BP1 axis in lysophagy regulation.

[0129] Example 7. Association of FBXO3 and LLOMe-induced lysophages in FBXO3 knockout HepG2 cells

[0130] The inventors generated FBXO3 knockout (KO) cell lines in HepG2 cells using CRISPR / Cas9 technology (Fig. 14a). The inventors applied LLOMe treatment to FBXO3 wild-type (WT) and KO HepG2 cells transiently expressing GFP-Gal3.

[0131] According to the results of the inventors' study, the clearance of GFP-Gal3 spots induced by LLOMe was reduced in FBXO3 KO cells compared to WT cells (Fig. 14b). In addition, the LLOMe-induced increase in TMEM192 induced by LLOME was significantly lower in FBXO3 KO cells (Fig. 14c), and as a result, the recruitment of TAX1BP1 to TMEM192 in FBXO3 KO cells was reduced compared to WT cells (Fig. 14d). These results indicate that FBXO3 KO interferes with lysophage by inhibiting the ubiquitination of TMEM192 and the recruitment of TAX1BP1.

[0132] In addition, by analyzing the human SNP database, we confirmed the naturally occurring polymorphism of FBXO3 in Val221Ile (V221I), which exhibits a loss-of-function mutation. 33,36 To evaluate the relevance of this FBXO3 mutation in lysophages, we reintroduced WT or FBXO3 V221I into FBXO3 KO cells and found that the delayed clearance of Gal3 spots observed in KO cells was rescued by FBXO3 WT reconstitution. However, when FBXO3 V221I was reintroduced, the clearance was still reduced (Fig. 14e).

[0133] In addition, TMEM192 ubiquitination was restored in cells reconstituted with FBXO3 WT, but remained damaged in cells reconstituted with FBXO3 V221I (Fig. 14f). These results further support the important role of FBXO3 in the degradation of damaged lysosomes overall.

[0134] Discussion

[0135] Maintaining the integrity of lysosomes is essential for cellular homeostasis and physiological functions. This integrity is strictly controlled by biosynthesis, repair, and degradation. When lysosomes are damaged, the TFEB and ESCRT-III complex is activated to replace the damaged lysosome and repair the lysosomal membrane. In cases of severe damage, ruptured lysosomes are removed via lysophages. However, the precise regulatory mechanisms controlling lysophages are not yet fully understood. The damage sensor kinase-E3 ligase-membrane protein ubiquitination-adapter protein recruitment scenario is well-documented in mitophagy. In this process, PINK1 kinase recognizes mitochondrial damage and recruits the E3 ligase parkin, leading to the polyubiquitination of some mitochondrial membrane proteins, including VDAC1, and the recruitment of autophagy adapter proteins such as SQSTM1. However, a cascading scenario similar to PINK1-PRKN-VDAC1-SQSTM1 has not yet been characterized in lysophages. In the study according to the present invention, screening of ubiquitination-related compounds was performed to discover a novel regulatory mechanism of lysophagy, the TBK1-FBXO3-TMEM192-TAX1BP1 axis scenario (Fig. 15). This newly identified lysophagy pathway is similar to mitophagy. The results of the study highlight the central role of the new axis in the degradation of ruptured lysosomes.

[0136] TMEM192 has been widely used to purify lysosomes due to its superior localization to lysosomes compared to other proteins. 28,37 It is worth noting that previous studies have shown TMEM192 to be a potential ubiquitinated membrane-penetrating protein for lysosomal damage involving specific lysine residues. 25 The protein structural topology showed that both the N-terminal and C-terminal tails of TMEM192 are located on the cytoplasmic side of the membrane. 38Five related lysine residues (201, 211, 237, 246, and 254) are located at the C-terminus of TMEM192. To identify the important polyubiquitin lysine residues involved in lysophage, the inventors performed point mutations to replace each lysine with arginine to prevent ubiquitination. However, individual substitution mutants of TMEM192 (lysine-arginine) were found to still be ubiquitinated (data not shown), suggesting that multiple lysine residues may be involved as ubiquitin targets. According to this concept, ubiquitination of TMEM192 was completely stopped, particularly when all lysine residues were mutated simultaneously (see Fig. 6e). From these results, it becomes clear that ubiquitination of TMEM192, which is part of the lysophage process, depends on multiple lysine residues.

[0137] Recently, cell membrane rupture has been identified as a stress factor that induces the release of cytotoxic contents, such as acidic lysosomal lumen compartments, and cell death. 12 For this reason, the ability to recognize membrane damage is important for cellular homeostasis. It has been proven that TBK1 plays an essential role as a membrane damage sensor kinase. 39 During an adenovirus infection, the adenovirus enters the cell via endocytosis, destroys the endosome membrane, and reaches the cytoplasm. 40 Then, TBK1 is activated via phosphorylation at Ser172 and accumulates at the infiltration site of the endosome, activating selective autophagy (i.e., xenophage) of invading pathogens. 39 Gal8 primarily recognizes pathogen-induced endosome ruptures and is important for the local activation of TBK1. 41 TBK1 has also been reported to be highly associated with lysophages. TBK1, activated in response to lysosome damage, accumulates in ruptured lysosomes, and inhibiting TBK1 reduces lysophage flux. 28To date, research on TBK1 function has focused on regulating adapter proteins during autophagy. However, recent reports have demonstrated that TBK1 acts as an upstream sensor kinase for membrane damage. In this study, the inventors also identified TBK1 as a lysosomal damage sensor kinase. TBK1 acts as an upstream sensor kinase following lysosomal damage and recruits FBXO3. TBK1 regulates FBXO3 activity through phosphorylation and FBXO3-mediated lysophagy flux. Nevertheless, further analysis is required to identify TBK1-dependent phosphorylation residues in FBXO3. In conclusion, these findings demonstrate the essential roles of TBK1 and FBXO3 in lysophagi.

[0138] F-box proteins generally play an important role in recognizing specific substrates modified by phosphorylation. 42,43 Only a few, including FBXO27, FBXO2, and FBXO6, have been proposed as lysophagy modifiers. 25,35 These F-box proteins are characterized by the ability to specifically bind to glycoproteins modified with gomannose N-glycans and can recognize damaged lysosomes. Nevertheless, considering that more than 70 F-box proteins are activated in response to various stimuli, other F-box proteins could also serve as potential lysophage regulators. Among them, it has been strongly suggested that FBXO3 acts as an important lysophage regulator. Analysis of the human SNP database indicates that FBXO3 possesses a non-synonymous C / T polymorphism, a naturally occurring mutation in Val221Ile (V221I). The loss-of-function FBXO3 V221I variant significantly reduces its ability to polyubiquitide substrates. 33 Loss-of-function mutations also reduce lysophage flux and ubiquitination of lysosomal membrane proteins compared to WT upon lysosome rupture, suggesting that FBXO3 is an important regulator in TMEM192-mediated lysophages (Fig. 14).

[0139] In conclusion, the findings of the present invention provide valuable insights into molecular pathways that protect lysosome integrity and cell health, and provide potential targets for therapeutic interventions for diseases associated with lysophagi and lysosomal dysfunction.

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[0184]

[0185] The present invention can easily determine the efficacy and utility of substances having lysophage-regulating activity based on the newly identified TBK1-FBXO3-TMEM192-TAX1BP1 axis, a lysophage regulatory pathway. The present invention can effectively screen substances with excellent effects for the prevention, alleviation, improvement, or treatment of lysosomal dysregulation. By providing an in-depth understanding of lysosomal dysregulation, the present invention can present new approaches to therapies, including innovative therapeutic agents.

Claims

1. As a method for screening lysophage activators, 1) A step of preparing a HepG2 cell line expressing the TBK1, FBXO3, TMEM192, TAX1BP1 genes and Enhanced Green Fluorescent Protein (EGFP); 2) A step of inducing lysophage activation by treating the prepared HepG2 cell line with a lysosomotropic agent without or with the test substance; 3) a step of capturing a fluorescence image of the cells and measuring the number of spots exhibiting the green fluorescence of EGFP; and 4) A step of determining a test substance that reduces the number of spots exhibiting green fluorescence, compared to the case where a lysosomal agonist was administered without the test substance, as a lysophage modulator that activates lysophages. A method including 2. In Paragraph 1, The above lysosomal homing agent is L-leucyl-L-leucine methyl ester (LLOMe), method.

3. In Paragraph 1, A method characterized in that, in step 2) above, lysophage activation is induced through the TBK1-FBXO3-TMEM192-TAX1BP1 signaling cascade consisting of i) activation of TBK1, ii) phosphorylation of FBXO3, iii) ubiquitination of TMEM192, and iv) activation of TAX1BP1.

4. In Paragraph 3, A method further comprising the step of determining whether a test substance determined to be a lysophagy activator that activates the lysophagy activates at least one signaling pathway of the TBK1-FBXO3-TMEM192-TAX1BP1 signaling cascade.

5. In Paragraph 3, A method characterized in that the test substance determined to be a lysophage activator that activates the above lysophage activates TBK1, phosphorylates FBXO3, ubiquitines TMEM192, or activates TAX1BP1.

6. In Paragraph 1, A method characterized in that, in step 3) above, the fluorescence image of the cell is obtained using a confocal microscope.

7. In Paragraph 1, A screening method in which the expression levels of the above TBK1, FBXO3, TMEM192, and TAX1BP1 are measured by one or more methods selected from the group consisting of reverse transcription-polymerase chain reaction (RTPCR), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunohistochemistry, microarray, western blotting, and flow cytometry (FACS).

8. A pharmaceutical composition for treating or preventing lysosomal dysregulation containing a lysophagy activator.

9. In Paragraph 8, A pharmaceutical composition characterized in that the above lysophagi activator activates TBK1, phosphorylates FBXO3, ubiquitines TMEM192, or activates TAX1BP1.

10. In Paragraph 8, A pharmaceutical composition in which the above-mentioned lysosomal dysregulation is selected from the group consisting of lysosomal storage disorders, neurodegenerative diseases, diabetes, and cancer.

11. A functional food composition for improving or preventing lysosomal dysregulation containing a lysophagy activator.

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