Hsc70 protein k128r mutant and application thereof in regulating cma activity

Abstract

The application discloses an HSC70 protein K128R mutant and application thereof in regulating CMA activity. The application discloses application of ubiquitination modification of an HSC70 protein at a K128 site in regulating chaperone-mediated autophagy. It is found for the first time that the HSC70 protein is subjected to K11-connected polyubiquitination under metabolic stress, and the modification site is a lysine at the 128th position (K128). The modification is mediated by a ROS-ATM-Atg7-TRIM21 signal axis, and is a necessary condition for the combination of the HSC70 and a lysosome membrane receptor LAMP2A to be enhanced and CMA to be activated. By constructing a K128R mutant, it is proved that the modification loss can block the response of CMA to metabolic stress. The application provides a new diagnostic marker and a drug target for CMA-related diseases (such as tumors and neurodegenerative diseases), and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to an HSC70 protein K128R mutant and its application in regulating CMA activity. Background Technology

[0002] Chaperone-mediated autophagy (CMA) is a process of selectively degrading intracellular proteins, crucial for maintaining protein homeostasis and adapting to metabolic stress. Unlike the non-selective degradation of macroautophagy, CMA specifically degrades cytoplasmic soluble proteins containing KFERQ-like motifs. This selective recognition process relies on heat shock cognate protein 70 (HSC70)—HSC70 specifically recognizes the KFERQ-like motif in substrate proteins and delivers the substrate to the receptor protein LAMP2A (Lysosome-associated membrane protein type 2A) on the lysosomal membrane, forming a translocation complex that ultimately allows the substrate to enter the lysosomal lumen for degradation. CMA is upregulated under stress conditions such as oxidative stress, hypoxia, starvation, and exposure to toxic compounds. Cells activate CMA to degrade unwanted proteins to provide amino acids while simultaneously clearing damaged proteins to maintain cellular homeostasis.

[0003] As a core protein in the CMA pathway, HSC70's expression level, localization, and interactions with other proteins directly determine CMA activity and substrate selection specificity. Studies have shown that HSC70 not only participates in CMA but also in microautophagy and chaperone-assisted selective autophagy (CASA), playing multiple roles in maintaining protein homeostasis. Therefore, elucidating the regulatory mechanisms of HSC70 is crucial for understanding the activation process of CMA.

[0004] Post-translational modifications of proteins are crucial for regulating protein function. Ubiquitination, a classic post-translational modification, has garnered increasing attention in recent years for its non-degradation functions (such as altering protein activity, protein-protein interactions, and subcellular localization) in addition to its classic proteasomal degradation function. In CMA regulation, ubiquitination is primarily believed to occur at the substrate protein level—post-translational modifications of the substrate (including phosphorylation, acetylation, ubiquitination, palmitoylation, and SUMOylation) can affect its binding affinity to HSC70, thereby regulating the efficiency of substrate entry into the CMA pathway. Furthermore, studies have shown that when soluble substrates cannot be correctly folded by the HSC70 / CHIP system, they can be ubiquitinated and degraded via the proteasome.

[0005] However, there are currently no reports on whether HSC70 itself undergoes ubiquitination modification at specific sites or with specific linkage types, and whether such modification participates in regulating CMA activity. Existing techniques only focus on the function of HSC70 as a molecular chaperone in recognizing substrates, and the impact of substrate modification on its binding to HSC70. However, whether post-translational modifications of HSC70 itself, particularly ubiquitination, respond to cellular stress signals (such as metabolic stress) and thus regulate its binding to LAMP2A, thereby affecting the degree of CMA activation, remains a crucial scientific question. Elucidating the fine-tuned regulatory mechanisms of HSC70 ubiquitination modification will provide new molecular targets for targeting CMA in the treatment of related diseases (such as tumors and neurodegenerative diseases). Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a K128R mutant of the HSC70 protein and its application in regulating CMA activity. The disclosure of this invention reveals the specific ubiquitination modification of HSC70 under metabolic stress, clarifies its modification sites and types, and demonstrates the key regulatory role of this modification in HSC70 function and CMA activity.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solutions.

[0008] This invention discloses the application of a regulator of ubiquitination level at the K128 site of HSC70 protein in the preparation of a reagent for regulating the activity of cell CMA in vitro.

[0009] Furthermore, the regulator modulates the K11-linked polyubiquitination level of the lysine residue at position 128 of the HSC70 protein.

[0010] Furthermore, the regulator modulates the binding of HSC70 to LAMP2A protein, thereby upregulating or downregulating cellular CMA activity.

[0011] Furthermore, the product is a medicine for the prevention and / or treatment of diseases related to abnormal autophagy activity mediated by molecular chaperones.

[0012] Preferably, the diseases associated with abnormal autophagy activity mediated by the molecular chaperone include at least one of tumors, neurodegenerative diseases, metabolic diseases, and aging-related diseases.

[0013] The present invention also discloses an HSC70 protein mutant, characterized in that the mutant is an HSC70 K128R mutant obtained by mutating the lysine residue at position 128 of the wild-type HSC70 protein to arginine; the mutant cannot undergo ubiquitination modification at the K128 site and can inhibit chaperone-mediated autophagy activity.

[0014] This invention also discloses the application of a substance that regulates the ubiquitination level at the K128 site of the HSC70 protein in the preparation of drugs for treating CMA-related diseases.

[0015] Furthermore, the substance is a small molecule compound, peptide, or nucleic acid that targets TRIM21, Atg7, or ATM.

[0016] This invention also discloses a method for screening candidate substances that regulate CMA activity, characterized by comprising the following steps: (1) Contact the candidate substance with cells expressing HSC70; (2) Detect the ubiquitination level at the HSC70 K128 site; (3) If the ubiquitination level at this site changes, the candidate substance is a potential substance that regulates CMA activity.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows.

[0018] This invention is the first to clearly demonstrate that the lysine residue at position 128 (K128) of the HSC70 protein can undergo K11-linked polyubiquitination modification. It confirms that this modification is a non-degradable modification that does not affect the stability of the HSC70 protein, but only specifically regulates its CMA-related functions. This fills the research gap on the regulation of CMA activity by post-translational modifications of HSC70 and provides a new perspective for elucidating the multidimensional functional regulation of HSC70.

[0019] This invention is the first to demonstrate that ubiquitination at the HSC70 K128 site is the core molecular switch for cells to sense metabolic stress and dynamically regulate CMA activity. It clarifies the complete upstream ROS-ATM-Atg7-TRIM21 regulatory signal axis and elucidates the whole-chain regulatory mechanism from metabolic stress signal reception to CMA activation, greatly deepening the understanding of CMA activity regulation in this field.

[0020] The ubiquitination target at the HSC70 K128 site provided by this invention can achieve specific regulation of CMA activity, which is completely different from the intervention method of directly targeting the full-length HSC70 protein. It will not interfere with the basic molecular chaperone function and other physiological functions of HSC70, fundamentally solving the technical defects of strong off-target effects and large side effects of existing technologies, and providing a safe and effective new target for precise regulation of CMA.

[0021] This invention confirms that the ubiquitination level at the HSC70 K128 site is directly positively correlated with intracellular CMA activity, and can serve as a novel diagnostic biomarker for diseases related to abnormal CMA activity, for early screening, subtyping diagnosis, and efficacy evaluation. At the same time, by targeting and regulating the ubiquitination level at this site, CMA activity can be precisely upregulated or downregulated, providing a new direction for drug development for the treatment of various major diseases such as tumors, neurodegenerative diseases, metabolic diseases, and aging-related diseases, and has extremely high clinical translational potential. Attached Figure Description

[0022] Figure 1 The HSC70 protein exhibits K11-linked polyubiquitination at the K128 site. Specifically, AC represents the interaction between TRIM21 and HSC70 intracellularly; D indicates that TRIM21 is a ubiquitin ligase that promotes increased HSC70 ubiquitination levels in vitro; E shows that overexpression of TRIM21 increases intracellular HSC70 K11-linked polyubiquitination levels; F indicates that knockdown of TRIM21 decreases intracellular HSC70 K11-linked polyubiquitination levels; G indicates that K11-linked polyubiquitination of HSC70 occurs at the K128 site; H shows that the K128 site of HSC70 is highly conserved across different species; and I indicates that TRIM21 promotes K11-linked polyubiquitination of the HSC70 protein at the K128 site.

[0023] Figure 2 The ROS-ATM-Atg7-TRIM21 axis regulates ubiquitination at the HSC70 K128 site under metabolic stress. In this study, A represents Atg7 as a ubiquitin-activating enzyme that promotes increased HSC70 ubiquitination levels in vitro; B represents Atg7 overexpression as increasing intracellular HSC70 K11-linked polyubiquitination levels; C represents Atg7 knockdown as decreasing intracellular HSC70 K11-linked polyubiquitination levels; D represents Atg7 promoting K11-linked polyubiquitination of HSC70 protein at the K128 site; E represents starvation as promoting K11-linked polyubiquitination of HSC70; F represents starvation as promoting K11-linked polyubiquitination of HSC70 protein at the K128 site; G represents the ROS inhibitor NAC as significantly blocking starvation-induced HSC70 ubiquitination; H represents the inhibition of starvation-induced HSC70 protein ubiquitination after ATM knockdown; I represents the inhibition of starvation-induced HSC70 protein ubiquitination after Atg7 knockdown; and J represents the inhibition of starvation-induced HSC70 protein ubiquitination after TRIM21 knockdown.

[0024] Figure 3Ubiquitination at the HSC70 K128 site promotes CMA activity. Specifically, A indicates that starvation enhances LAMP2A binding to HSC70 but not to the HSC70 K128R mutant protein; B indicates that starvation-induced binding of LAMP2A to HSC70 protein is inhibited after TRIM21 knockdown; and C indicates an increase in the number of KFERQ-eRFP spots (representing increased CMA activity) in wild-type HSC70 cells under starvation conditions, while this phenomenon is inhibited in HSC70 K128R mutant cells. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the above-described subject matter of the present invention to the following embodiments; all technologies implemented based on the content of the present invention fall within the scope of the present invention.

[0026] Unless otherwise specified, all reagents and materials used in this invention are commercially available.

[0027] I. Materials and Methods.

[0028] 1. Cell culture.

[0029] HEK293 and HCT116 cells were purchased from the Shanghai Cell Bank, Chinese Academy of Sciences. HEK293 cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum, and HCT116 cells were cultured in McCoy's 5A medium containing 10% fetal bovine serum. All cells were cultured in a cell incubator at 37°C with 5% CO2.

[0030] 2. Plasmid construction, transfection, and lentiviral infection.

[0031] The Myc-Atg7 and HA-TRIM21 plasmids are from our previous research. The Flag-LAMP2A, Myc-LAMP2A, and Myc-HSC70 plasmids were provided by Wuhan Miaoling Biotechnology Co., Ltd. The HA-Ub(K11), Flag-HSC70 wild-type, and K128R mutant plasmids were provided by Shanghai Jikai Gene Chemical Co., Ltd. The HSC70 K128R plasmid was generated by site-directed mutagenesis using the wild-type HSC70 plasmid as a template and the site-directed mutagenesis primers (forward: 5'-GACAAAGATGCGCGAAATTGCAGAAGC-3', reverse: 5'-AGAACCATAGAAGACACC-3') according to the MutanBEST Kit (TaKaRa, R401) instructions. The TRIM21-specific siRNA was provided by Guangzhou Ruibo Biotechnology Co., Ltd. (sequence 5'-GAUGGUGUCUGCUAUUGUATT-3'), and the Atg7-specific siRNA was provided by Shanghai Sangon Biotech Co., Ltd. (sequence 5'-GGUCAAAGGACGAAGAUAAdTdT-3'). Transfection with these siRNAs was performed using JetPrime transfection reagent and the corresponding Jet buffer according to the manufacturer's instructions. Plasmid transfection was performed using Biobest or Lipo3000 transfection reagents according to the manufacturer's instructions. Cells infected with lentivirus HBLV-KFERQ-eRFP-N1 (Hanheng Biotechnology, HH20231121DY-LP01) were screened for stable overexpression using puromycin (1ug / mL) to identify cell lines.

[0032] 3. Western blot analysis (WB) and co-immunoprecipitation (Co-IP).

[0033] Cells were collected and lysed using lysis buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate), with protease inhibitors added to the lysis buffer. After lysis at 4°C for 30 min, the mixture was centrifuged at 13500 rpm for 20 min at 4°C, and the supernatant was collected. Subsequently, protein quantification was performed using the Coomassie Brilliant Blue G-250 method or the BCA method, and protein samples were prepared based on the quantification results.

[0034] For immunoprecipitation, cell lysis and input sample preparation were performed in the same manner as in Western blot experiments. Primary antibody was then added to the protein lysis buffer and incubated at 4°C for 1 h. A / G magnetic beads (Santa Cruz, sc-2003) were then added and incubated overnight at 4°C. The next day, the magnetic beads were washed three times with PBS, then boiled in 2×Loading solution for 10 min, and the supernatant was collected. Subsequently, based on the molecular weight of the target protein, protein samples were subjected to polyacrylamide gel electrophoresis on 8% and 10% SDS-PAGE gels and transferred to PVDF membranes (Millipore, IPVH00010). After blocking with 5% bovine serum albumin (BSA) for 1 h at room temperature, the samples were washed three times with TBST and then incubated overnight at 4°C with primary antibody. Afterward, the samples were washed three times with TBST and then incubated with HRP-conjugated secondary antibody at room temperature for 1 h. Finally, after three washes, the bands were detected using an enhanced chemiluminescence detection kit (ThermoFisherScientific, 32106) and visualized using a DNR Western blot detection system.

[0035] 4. In vitro ubiquitination assay.

[0036] (1) Plasmid transfection and protein purification: Myc-HSC70, Myc-Atg7 and HA-TRIM21 plasmids were transfected into HEK293 cells and cultured for 48 h before cell lysis. The corresponding tag fusion proteins were purified by immunoprecipitation using agarose beads conjugated with anti-Myc, anti-Flag or anti-HA antibodies, respectively. (2) Construction of ubiquitination reaction system: The purified protein was added to the ubiquitination reaction system according to the experimental design. The system contained Mg-ATP, ubiquitin, ubiquitin activator (E1), ubiquitin conjugate (E2), ubiquitin ligase (E3) and reaction buffer. All components were obtained from Abcam E3 Ligase Auto-Ubiquitylation Assay kit (catalog number ab139469). (3) Reaction and detection: The reaction system was incubated at 37℃ for 1 h, and then an appropriate amount of protein loading buffer was added. After incubation at 100℃ for 10 min, Western blot was performed. Changes in HSC70 ubiquitination levels were detected using anti-ubiquitin antibodies to assess the ubiquitination activities of TRIM21 and Atg7.

[0037] 5. Immunofluorescence (IF).

[0038] (1) Cell spreading: Place a cell spreader of appropriate diameter into a 12-well plate in advance, and seed an appropriate number of cells according to the experimental design. Wait for the cells to adhere. (2) Cell fixation: Remove the cells, discard the culture medium, and wash the cells three times with room temperature PBS. Then, add 4% paraformaldehyde to fix the cells at room temperature for 20 min. (3) Permeabilization: After washing three times with PBS, add 0.25% Triton X-100 to treat the cells for 15 min to achieve cell membrane permeabilization. (4) Blocking: After washing three times with PBS, block with 5% BSA blocking solution for 1 h. (5) Primary antibody incubation: After blocking, incubate overnight at 4℃ with anti-TRIM21 antibody and anti-HSC70 antibody. (6) Secondary antibody incubation and nuclear staining: After washing three times with PBS, incubate the sample for 1 h with fluorescently labeled secondary antibody of the corresponding species (1:400 dilution) at room temperature in the dark. After washing three times with PBS again, add DAPI staining solution to stain the nucleus for 5 min. (7) Mounting and observation: Add mounting medium to the marked slide, then take out the slide and invert it onto the slide. The entire process should be done in the dark until the slide is completely dry. Then observe it using a laser confocal microscope.

[0039] 6. CMA activity detection experiment.

[0040] To investigate the effect of HSC70 K128 ubiquitination on chaperone-mediated autophagy (CMA) activity under metabolic stress, HEK293 cells stably expressing the KFERQ-eRFP reporter gene (infected with HBLV-KFERQ-eRFP-N1 lentivirus) were first constructed. HSC70 wild-type (WT) and HSC70 K128R mutant plasmids were then overexpressed. Cells were seeded in 12-well plates and divided into a control group (normal culture) and a metabolic stress group (starvation treatment). After treatment, immunofluorescence analysis was performed using confocal microscopy, and the number of KFERQ-eRFP spots was counted to reflect the CMA activity level.

[0041] II. Experimental Results.

[0042] Result 1: TRIM21 promotes polyubiquitination of HSC70 at the K11 linker at K128 (corresponding to...) Figure 1 ).

[0043] To investigate whether TRIM21 acts as a ubiquitin ligase (E3) for HSC70, we first examined the binding between HSC70 and TRIM21. HEK293 cells were harvested and lysed, and proteins were enriched using IgG antibody, HSC70 antibody, and A / G beads. Western blot analysis was then performed to detect the binding between TRIM21 and HSC70. Figure 1A). HEK293 cells were collected and lysed. Proteins were enriched using IgG antibody, TRIM21 antibody, and A / G beads. Western blot was used to detect the binding of HSC70 and TRIM21. Figure 1 B). The co-localization of HSC70 and TRIM21 cells was detected using immunofluorescence assays. HEK293 and HCT116 cells were seeded in 12-well plates with pre-placed slides. After cell adhesion, immunofluorescence experiments were performed using confocal microscopy. The scale bar was 10 μm. Figure 1 C). The results all indicate that HSC70 and TRIM21 interact.

[0044] To further clarify the ubiquitination effect of TRIM21 on HSC70, we transfected HEK293 cells with Myc-HSC70 and HA-TRIM21 plasmids, respectively, and lysed the cells after 48 h of culture. The corresponding tag fusion proteins were purified by immunoprecipitation using agarose beads conjugated with anti-Myc or anti-HA antibodies, respectively. A ubiquitination reaction system was constructed and incubated at 37°C for 1 h. Then, an appropriate amount of protein loading buffer was added, and the mixture was incubated at 100°C for 10 min before Western blot analysis. Figure 1 D). Experimental results show that TRIM21 is a ubiquitin ligase of HSC70.

[0045] To further determine the effect of TRIM21 overexpression on HSC70 ubiquitination, HA-Ub(K11) plasmid, Flag-HSC70 plasmid, and HA-TRIM21 plasmid were transfected. The medium was changed 4-6 h after transfection, and cells were harvested and lysed after 48 h. Proteins were enriched using Flag antibody and A / G beads, and Western blot was used to detect changes in HSC70 ubiquitination levels. The results showed that TRIM21 overexpression can promote K11-type ubiquitination in HSC70 cells. Figure 1 E).

[0046] To further determine the effect of TRIM21 knockdown on HSC70 ubiquitination, we silenced TRIM21 using small interfering RNA. After 24 h, we transfected cells with HA-Ub(K11) plasmid and Flag-HSC70 plasmid. Cells were harvested and lysed after 24 h, and immunoprecipitation experiments were performed. Proteins were enriched using Flag antibody and A / G beads. Western blot analysis was used to detect changes in HSC70 ubiquitination levels. The results showed that TRIM21 knockdown reduced K11-type ubiquitination in HSC70. Figure 1 F).

[0047] To further clarify the specific ubiquitination site of HSC70, we overexpressed Flag-HSC70 wild-type plasmid, Flag-HSC70 K25R plasmid, Flag-HSC70 K128R plasmid, Flag-HSC70 K188R plasmid, and Flag-HSC70 K361R plasmid in HEK293 cells, respectively. After 48 h, cells were collected, and proteins were enriched using Flag antibody and A / G beads. Western blot was then used to detect the ubiquitination level of HSC70. The results showed that when the K128 site of HSC70 was mutated to arginine (K128R), its ubiquitination level was significantly reduced. Figure 1 G). Meanwhile, the K128 site of HSC70 is highly conserved across different species ( Figure 1 H).

[0048] To further clarify the regulation of TRIM21 on ubiquitination at the K128 site of HSC70 protein, we transfected HEK293 cells with HA-Ub(K11) plasmid, HA-TRIM21 plasmid, and Flag-HSC70 wild-type plasmid / Flag-HSC70 K128R plasmid. Medium was changed 4-6 h after transfection, and cells were harvested and lysed after 48 h. Proteins were enriched using Flag antibody and A / G beads, and Western blot was used to detect the ubiquitination of HSC70 and its mutant HSC70 K128R protein. The results showed that overexpression of TRIM21 promoted enhanced polyubiquitination at the K11 linker of HSC70. This enhanced ubiquitination effect disappeared when the K128 site of HSC70 was mutated to arginine (K128R). Figure 1 I).

[0049] All of the above results indicate that TRIM21, as an E3 ubiquitin ligase, specifically binds to HSC70 and catalyzes K11-linked polyubiquitination at its K128 site.

[0050] Result 2: Under metabolic stress, the ROS-ATM-Atg7-TRIM21 axis regulates ubiquitination at the HSC70 K128 site (corresponding to...). Figure 2 ).

[0051] Next, we look for upstream signals of TRIM21 ubiquitination HSC70. Figure 2 The results of in vitro ubiquitination experiments in A suggest that Atg7 may act as the ubiquitin activator (E1) of HSC70 to promote the ubiquitination of HSC70. Figure 2 Overexpression of Atg7 in B increases the level of K11-type ubiquitination in HSC70; Figure 2 Knockdown of Atg7 in C reduces the level of K11-type ubiquitination in HSC70; Figure 2In D, the promoting effect of Atg7 on HSC70 ubiquitination disappeared after mutation at the HSC70 K128 site. These results indicate that Atg7, acting as E1, promotes K11-linked polyubiquitination at the HSC70 K128 site.

[0052] We starve the cells to simulate metabolic stress. Figure 2 The results showed that starvation treatment time-dependently upregulated the K11-type ubiquitination level of HSC70; Figure 2 The results showed that in cells expressing the HSC70-K128R mutant, the starvation-induced enhancement of HSC70 ubiquitination was essentially eliminated, confirming that the K128 site is a necessary site for this modification to occur. Figure 2 The results showed that pretreatment with the ROS scavenger NAC significantly blocked starvation-induced HSC70 ubiquitination, indicating that ROS is a key upstream molecule in this signaling pathway. Figure 2 The results showed that in ATM-knockdown cells, the starvation-induced HSC70 ubiquitination effect disappeared, further confirming that ATM is an essential element in this pathway. Figure 2 The results showed that in Atg7 knockdown cells, starvation-induced HSC70 ubiquitination levels were significantly reduced; similarly, Figure 2 In J, starvation-induced HSC70 ubiquitination was also inhibited in TRIM21 knockdown cells.

[0053] The above results indicate that under metabolic stress, intracellular ROS levels increase, which in turn activates ATM-Atg7-TRIM21 sequentially, ultimately mediating ubiquitination at the HSC70 K128 site, forming a complete “ROS-ATM-Atg7-TRIM21-HSC70 K128” signaling axis.

[0054] Result 3: Ubiquitination at HSC70 K128 site promotes CMA activity (corresponding to...) Figure 3 ).

[0055] To investigate whether HSC70 K128 ubiquitination affects chaperone-mediated autophagy (CMA) under metabolic stress, we transfected HEK293 cells with Myc-LAMP2A plasmid, Flag-HSC70 wild-type plasmid / Flag-HSC70K128R plasmid. The medium was changed 4-6 h after transfection, followed by 8 h of starvation. The proteins were enriched using Flag antibody and A / G beads. Western blot was used to detect the binding of HSC70 and its mutant HSC70-K128R to LAMP2A. The results showed that under metabolic stress, LAMP2A binding to HSC70 was enhanced, while binding to the HSC70 K128R mutant protein was not enhanced. Figure 3 A).

[0056] To further clarify the effect of TRIM21 on chaperone-mediated autophagy (CMA) under metabolic stress, we silenced TRIM21 using small interfering RNA. After 24 h, we transfected the Flag-LAMP2A and Myc-HSC70 plasmids, subjected the cells to 8 h of starvation, and performed immunoprecipitation experiments. We enriched the protein using Flag antibody and A / G beads, and Western blot was used to detect changes in the binding level of HSC70 to LAMP2A. The results showed that knocking down TRIM21 significantly inhibited the enhanced binding of LAMP2A to HSC70 protein induced by metabolic stress. Figure 3 B).

[0057] To investigate the effect of HSC70 K128 site ubiquitination on chaperone-mediated autophagy (CMA) activity under metabolic stress, HEK293 cells were infected with a virus carrying the reporter gene KFERQ-eRFP and seeded in 12-well plates. Immunofluorescence analysis was performed using confocal microscopy, with the number of KFERQ-eRFP spots representing CMA activity. The results showed that in cells expressing wild-type HSC70 (HSC70-WT), starvation treatment significantly increased the number of KFERQ-eRFP spots, indicating CMA activation. However, in cells expressing the mutant HSC70-K128R, starvation treatment did not significantly increase the number of KFERQ-eRFP spots, essentially blocking CMA activation. Figure 3 C).

[0058] These results indicate that ubiquitination at the HSC70 K128 site is a key molecular switch for activating CMA under metabolic stress. Ubiquitination at this site promotes the binding of HSC70 to LAMP2A, thereby driving CMA activation; blocking ubiquitination at this site (K128R mutation) or blocking the upstream regulatory enzyme (TRIM21 silencing) will prevent CMA from responding to metabolic stress.

[0059] In summary, this invention, for the first time, fully elucidates a complete signaling pathway from metabolic stress (ROS production) to CMA functional activation, with the core being K11-linked polyubiquitination at the HSC70 K128 site. This modification, mediated by the ROS-ATM-Atg7-TRIM21 signaling axis, is a necessary condition for enhanced HSC70-LAMP2A binding and CMA activation under metabolic stress. This discovery not only elucidates a novel mechanism for CMA activity regulation but also provides the HSC70 K128 site and its upstream regulatory enzymes (TRIM21, Atg7, ATM) as novel drug targets and diagnostic biomarkers for intervention in CMA-related diseases.

[0060] The above description is merely a preferred embodiment of the present invention and is not intended to limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of regulators of ubiquitination levels at the K128 site of HSC70 protein in the preparation of reagents for regulating cellular CMA activity in vitro.

2. The application according to claim 1, characterized in that, The regulator modulates the level of K11-linked polyubiquitination at the 128th lysine residue of the HSC70 protein.

3. The application according to claim 1, characterized in that, The regulator modulates the binding of HSC70 to LAMP2A protein, thereby upregulating or downregulating cellular CMA activity.

4. The application according to claim 1, characterized in that, The reagent is a tool for screening candidate drugs for the prevention and / or treatment of diseases related to abnormal autophagy activity mediated by molecular chaperones.

5. The application according to claim 4, characterized in that, The diseases associated with abnormal autophagy activity mediated by the molecular chaperone include at least one of tumors, neurodegenerative diseases, metabolic diseases, and age-related diseases.

6. An HSC70 protein mutant, characterized in that, The mutant is the HSC70 K128R mutant obtained by mutating the lysine residue at position 128 of the wild-type HSC70 protein to arginine; the mutant cannot undergo ubiquitination modification at the K128 site and can inhibit chaperone-mediated autophagy activity.

7. The application of a substance that regulates the ubiquitination level at the K128 site of the HSC70 protein in the preparation of drugs for treating CMA-related diseases.

8. The application according to claim 7, characterized in that, The substance is a small molecule compound, peptide, or nucleic acid that targets TRIM21, Atg7, or ATM.

9. A method for screening candidate substances that regulate CMA activity, characterized in that, Includes the following steps: (1) Contact the candidate substance with cells expressing HSC70; (2) Detect the ubiquitination level at the HSC70 K128 site; (3) If the ubiquitination level at this site changes, the candidate substance is a potential substance that regulates CMA activity.

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