A short peptide simulating the c-terminal of rhoe, derivatives and pharmaceutical use thereof in the treatment of ventricular remodeling

Abstract

The application discloses a kind of short peptides simulating RhoE C end, derivative and its pharmaceutical use in treating ventricular remodeling, belong to biological medicine technical field.The short peptide and derivative contain RhoE C end 200-240 linear function domain, can be combined and activated WWP2, remove HGS self-inhibition, start endosome-lysosome degradation pathway.Short peptide sequence is as SEQ ID NO.1, derivative can be coupled with TAT, T7 or RVG membrane penetrating peptide (SEQ ID NO.2-4), and endocytosed to myocardial cell by caveolin high efficiency delivery.The application solves the defects that existing anti-ventricular remodeling drug inhibits protective autophagy flow, and the ability of eliminating toxic protein is limited, can efficiently eliminate damaged mitochondria and misfolded protein, inhibits myocardial hypertrophy, fibrosis, improves cardiac remodeling, provides safe and effective treatment strategy for related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a short peptide and derivative that mimics the C-terminus of RhoE and its pharmaceutical use in the treatment of ventricular remodeling. Background Technology

[0002] Ventricular remodeling is a common pathological basis for the progression of various cardiovascular diseases, such as coronary heart disease, hypertension, and valvular heart disease, to end-stage heart failure, seriously endangering human health. This process involves cardiomyocyte hypertrophy, apoptosis, myocardial fibrosis, and changes in cardiac geometry. Recent studies have shown that defects in cardiomyocyte autophagy play a key role in the occurrence and development of ventricular remodeling. Impaired autophagic flux in cardiomyocytes leads to abnormal accumulation of damaged proteins and organelles in the cytoplasm, inducing cardiomyocyte hypertrophy and thus promoting cardiac remodeling (IntHeartJ.2025;66(4):615-627; MolMedRep.2020;22(2):1342-1350). Therefore, maintaining or restoring the stability of cardiomyocyte autophagic flux and clearing harmful proteins in the cytoplasm are considered potential therapeutic targets for inhibiting myocardial hypertrophy and delaying ventricular remodeling (LifeSci.2021;264:118550).

[0003] Currently, the first-line treatment options for ventricular remodeling in clinical cardiology mainly include angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs). These drugs can effectively inhibit left ventricular dilation and slow down the process of ventricular remodeling by inhibiting the renin-angiotensin-aldosterone system (RAAS). However, these drug treatments for ventricular remodeling have their limitations (AmJPhysiolCellPhysiol.2025Apr1;328(4):C1303-C1317.doi:10.1152 / ajpcell.00753.2024;JAmCollCardiol.2013Jul23;62(4):263-71.doi:10.1016 / j.jacc.2013.02.092;2017Dec23;9(37):24601-24618.doi:10.18632 / oncotarget.23628). On the one hand, cardiac remodeling still progresses in some patients even with standard treatment; on the other hand, recent studies have revealed a potential mechanistic contradiction: the autophagy-lysosomal pathway is an important defense mechanism for cardiomyocytes to maintain homeostasis, while the binding of angiotensin II (AngII), a key effector molecule of the RAAS system, to its type 1 receptor (AT1) is actually one of the important pathways for maintaining the basal autophagic flux of cardiomyocytes.

[0004] Previous research by the applicant has also confirmed that while ACEIs and ARBs alleviate angiotensin II-induced hemodynamic load and myocardial hypertrophy by blocking AT1 receptors, their pharmacological mechanism also blocks AT1 receptor-mediated autophagic flux signaling. This weakens the ability of cardiomyocytes to clear abnormally accumulated proteins in the cytoplasm, thus limiting the anti-ventricular remodeling effect of these drugs (JMolCellCardiol.2018;125:117-128). Traditional RAAS blockers, while improving cardiac load, may interfere with the endogenous "cleaning" mechanism of cardiomyocytes. Novel anti-heart failure drugs, such as angiotensin receptor-neprilysin inhibitors (Entresto), although significantly effective, still involve RAAS inhibition in their core mechanism of action, and similarly cannot avoid the potential negative impact on cardiomyocyte autophagic flux.

[0005] In summary, genetic factors, overweight, and hypertension can all lead to myocardial hypertrophy. Existing drugs such as ACEIs, ARBs, and Entresto all exert their effects by inhibiting angiotensin II type 1 receptors, sharing a common limitation of restricting autophagic influx in cardiomyocytes. This creates a bottleneck in the efficacy of current drug treatments for ventricular remodeling. Therefore, exploring a new approach that directly enhances myocardial autophagic influx and effectively inhibits myocardial hypertrophy without relying on the traditional RAAS blockade pathway, as a supplementary or synergistic treatment to existing traditional anti-cardiac remodeling drugs, has become urgent. Summary of the Invention

[0006] In view of the technical defects of existing anti-ventricular remodeling drugs (ACEI / ARB / Entresto), which can improve hemodynamics, but inhibit the protective autophagic flux of cardiomyocytes by blocking AT1 receptors, resulting in limited ability of drugs to clear intracellular toxic proteins and incomplete anti-remodeling effect, this invention aims to provide a short peptide and derivative that mimics the C-terminus of RhoE and its pharmaceutical use in the treatment of ventricular remodeling.

[0007] To achieve the above objectives, the present invention employs the following technical solution: In a first aspect, the present invention discloses a short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE, wherein the short peptide and its derivatives contain a linear functional domain at position 230-240 of the C-terminus of the RhoE protein, and possess at least one of the following functions: a. By binding to the 2-4 linker region of the E3 ubiquitin ligase WWP2, it can relieve the autoinhibition of WWP2; b. By binding to the UIM domain of the endosome receptor HGS (ESCRT-0), the self-folding inhibition of HGS caused by monoubiquitination in the UIM region is relieved. c. The activated WWP2 polyubiquitinates the VHS domain of the open-state HGS, initiating the endosome-lysosome degradation pathway.

[0008] Preferably, the short peptide and its derivatives can bind to caveolin on the cardiomyocyte membrane and be efficiently delivered to the cardiomyocyte cytoplasm via the caveolin endocytosis pathway, thereby targeting and activating intracellular WWP2.

[0009] Preferably, the amino acid sequence of the short peptide is shown in SEQ ID NO. 1.

[0010] More preferably, the short peptide derivative is obtained by coupling a short peptide with a membrane-penetrating peptide.

[0011] More preferably, the membrane-penetrating peptide is a TAT membrane-penetrating peptide, a T7 permeabilizing peptide, or an RVG peptide.

[0012] As one implementation of the present invention, the amino acid sequence of the short peptide derivative is shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4.

[0013] A second aspect of the present invention discloses a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned short peptide and derivatives of the RhoEC-terminal domain of a small G protein, and a pharmaceutically acceptable carrier, excipient, or diluent, said pharmaceutical composition for the prevention and / or treatment of conditions related to myocardial hypertrophy, myocardial fibrosis, cardiomyocyte apoptosis, or cardiac remodeling.

[0014] A third aspect of the present invention discloses the use of the short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE in the preparation of a medicament for activating the endosome-lysosome degradation pathway and clearing damaged mitochondria or misfolded proteins in cardiomyocytes.

[0015] Furthermore, the drug achieves its function by activating WWP2, relieving HGS autoinhibition, and promoting polyubiquitination of the VHS domain of HGS through the short peptide and its derivatives.

[0016] In a fourth aspect, the invention discloses the use of short peptides and their derivatives that mimic the RhoEC terminal domain of small G proteins in the preparation of medicaments for relieving myocardial hypertrophy, improving cardiac remodeling, or enhancing cardiac function.

[0017] Compared with the prior art, the present invention has the following beneficial effects: This invention provides short peptides and their derivatives that mimic the C-terminal domain of the small G protein RhoE. These peptides are efficiently delivered to the cytoplasm of cardiomyocytes via the caveolin endocytosis pathway, specifically binding to and activating the E3 ubiquitin ligase WWP2, thus relieving its autoinhibitory state. Simultaneously, they bind to the UIM domain of the endosome receptor HGS, relieving the autofolding inhibition of HGS caused by monoubiquitination. The activated WWP2 then performs polyubiquitination modification of the VHS domain of HGS, initiating the endosome-lysosome degradation pathway (small autophagy). This efficiently removes damaged mitochondria and misfolded proteins from the cell, replacing classical autophagy and avoiding the limitations of autophagy-dependent flow. This mechanism does not interfere with the AT1 receptor or the protective autophagy pathway, significantly inhibiting myocardial hypertrophy, myocardial fibrosis, inflammatory infiltration, and cardiomyocyte apoptosis, comprehensively improving cardiac remodeling and enhancing cardiac function. It possesses strong targeting and high delivery efficiency, providing a safer and more effective new therapeutic strategy for myocardial hypertrophy and ventricular remodeling. Attached Figure Description

[0018] Figure 1 The C-terminal (230-240) structure of the small G protein RhoE conforms to typical linear peptide characteristics; Figure 2 The interaction analysis between RhoE and WWP2 was performed, where A is the interaction between Flag-labeled WWP2 and its truncated form and HA-labeled WWP2 (C838A) mutant detected by immunoprecipitation assay; B is the interaction between Flag-labeled WWP2 and its truncated form and 6HIS-labeled RhoE detected by immunoprecipitation assay. Figure 3 The effect of RhoE on the self-inhibitory state of WWP2 is shown in Figure A, where A represents the interaction between exogenous RhoE and WWP2 with enzymatic activity, and B represents the interaction between exogenous RhoE and WWP2 without enzymatic activity (the inactivated form of WWP2). Figure 4 To assess the promoting effect of RhoE on WWP2 self-ubiquitination, AD represents various truncated, deleted, and mutant forms of RhoE transfected with RhoE, and to evaluate the structure and sites of the interaction between RhoE and WWP2. Figure 5 Colocalization analysis of RhoE and WWP2 in cells; Figure 6 The results of co-localization analysis of RhoE-regulated WWP2 and autophagy receptor P62 are shown; where A is the result of immunofluorescence co-localization; B is the quantitative statistics of the relative area of ​​WWP2; and C is the quantitative statistics of co-localization of HGS and WWP2. Figure 7The results show the colocalization analysis of RhoE, WWP2, and P62 forming a ternary complex within cells; where A represents the multichannel immunofluorescence colocalization results; B represents the quantitative analysis of WWP2 and HGS colocalization; and C represents the verification of WWP2 and HGS / RhoE colocalization. Figure 8 RhoE-activated WWP2 promotes autophagic flux in cardiomyocytes and inhibits hypertrophic responses; where A represents the results of immunofluorescence colocalization; and B represents the quantitative statistics of colocalization. Figure 9 Results related to GFP-mRFP-LC3 fluorescently labeled adenovirus transfection of cardiomyocytes; where A represents immunofluorescence colocalization results; and B represents quantitative statistics of colocalization. Figure 10 The results of immunofluorescence colocalization of the short peptide that activates WWP2 and promotes the interaction between the endosomal receptor HGS and WWP2 disclosed in this invention; Figure 11 The results of immunofluorescence colocalization of the short peptides disclosed in this invention promoting the clearance of damaged mitochondria in cardiomyocytes; Figure 12 The results of immunofluorescence colocalization of the short peptides of this invention activating E3 ubiquitin ligase WWP2 and inhibiting cardiomyocyte remodeling. Figure 13 The results of protein electrophoresis of the short peptides of this invention inhibiting cardiomyocyte remodeling are shown. Figure 14 This is the protein electrophoresis result of the present invention, which simulates the short peptide of RhoE to initiate endosome substitution autophagy and inhibit the hypertrophic response of cardiomyocytes; Figure 15 The results show that the colocalization of WWP2 and endosomal receptor HGS in mouse myocardial tissue was promoted by tail vein injection of polylactic acid-glycolic acid copolymer microspheres combined with short peptide P2. Figure 16 This is the result of RhoE regulating the interaction between WWP2 and HGS in cardiomyocytes with defects in autophagy-related genes. Detailed Implementation

[0019] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0020] This study found that the small G protein RhoE can bind to caveolin-3 on the cardiomyocyte membrane, forming vesicles via caveolin endocytosis, thus achieving efficient delivery from the cardiomyocyte membrane to the cytoplasm. Immunofluorescence double labeling and FITC fluorescent labeling experiments confirmed this localization and transport characteristic of RhoE. Further experiments using mass spectrometry, COIP, and ubiquitination confirmed that cytoplasmic RhoE can interact with and activate the E3 ubiquitin ligase WWP2. WWP2 can ubiquitinate substrates such as the endosome receptor HGS, initiating the endosome-lysosome degradation pathway (small autophagy pathway). This pathway, as an important supplement to classical autophagy, can promote the degradation of accumulated proteins in cardiomyocytes and significantly inhibit myocardial hypertrophy.

[0021] In addition, RhoE can also activate WWP2 to ubiquitinate the large autophagy receptor P62 and the small autophagy receptor HGS, further enhancing degradation function and thus inhibiting myocardial remodeling.

[0022] However, directly regulating RhoE expression in cardiomyocytes is difficult in clinical practice, and gene editing technology carries high risks and ethical issues, making its application in clinical practice challenging. Therefore, this invention, through in-depth analysis of the interaction domains and sites between RhoE and WWP2, reveals that, in addition to the N-terminus, the C-terminus of RhoE is also a key structure for its interaction with WWP2, and this structure exhibits typical linear characteristics. Analysis of the structure of the interaction between the C-terminus of the small G protein RhoE and the E3 ubiquitin ligase WWP2 reveals that this structure also possesses typical linear characteristics. Based on this, short peptides, such as... Figure 1 As shown in the figure, the C-terminal (230-240) structure of the small G protein RhoE conforms to the typical characteristics of a linear peptide and is rich in serine (Ser) and threonine (Thr) sites that can interact with the E3 ubiquitin ligase WWP2. This domain can also bind to caveolin-3 on the cardiomyocyte membrane. Based on this, a linear short peptide mimicking the RhoEC terminus was constructed and named P2, Ala-Thr-Asp-Leu-Arg-Lys-Asp-Lys-Ala-Lys-Ser.

[0023] The amino acid sequence of the short peptide (P2) is: ATDLRKDKAKS (SEQ ID NO.1); the C-terminal domain of RhoE can interact with caveolin, therefore, the short peptide can interact with caveolin and be translocated into the cytoplasm of cardiomyocytes via the caveolin endocytosis mechanism.

[0024] To further promote the binding of the short peptide to the cell membrane and enhance its ability to penetrate the cell membrane and be transported from the cell membrane to the cytoplasm, this application designs short peptide derivatives by coupling short peptide P2 with T7 peptide, TAT membrane-penetrating peptide and RVG29 peptide, which have good cell penetration and targeting properties.

[0025] Specifically, the short peptide derivatives include optimized short peptides P2-I (coupled with TAT membrane-penetrating peptide), P2-II (coupled with T7 cell-penetrating peptide), and P2-III (coupled with RVG peptide), whose amino acid sequences are as follows: P2-I: ATDLRKDKAKS-RKKRRQRRR, as shown in SEQ ID NO.2; P2-II: TDLRKDKAKS-AYAAGGR; as shown in SEQ ID NO. 3; P2-III: ATDLRKDKAK-RPGTPCDIFTNSRG, as shown in SEQ ID NO.4.

[0026] The details are shown in Table 1 below: Table 1

[0027] The aforementioned short peptides were synthesized by Shanghai Jier Company using the standard Fmoc solid-phase synthesis method. Preparative reversed-phase high-performance liquid chromatography (RP-HPLC) was used to purify the crude peptides. A C18 column was used with mobile phase A of 0.1% TFA / water and mobile phase B of 0.1% TFA / acetonitrile, using gradient elution at a detection wavelength of 214 nm. The main peak was collected, and after lyophilization, the purity was determined by analytical RP-HPLC. The purity of all short peptides was greater than 95%. The molecular weight was determined using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOFMS), and the results were consistent with the theoretical molecular weight. The purified short peptides were lyophilized into powder and stored at -80°C for later use. Before use, they were dissolved in sterile phosphate-buffered saline (PBS, pH 7.4) to the required concentration.

[0028] This short peptide and its derivatives can mimic RhoE to activate WWP2, promote HGS polyubiquitination and initiate the endosome-lysosome degradation pathway, thereby replacing autophagy to clear accumulated proteins and inhibit myocardial hypertrophy. Therefore, it can overcome the limitations of traditional drugs that cannot clear accumulated proteins due to inhibition of myocardial cell autophagy, and provide a new direction for the treatment of myocardial hypertrophy.

[0029] Example 1: Verification of the interaction between RhoE and WWP2 This embodiment aims to verify, through immunoprecipitation experiments, whether there is a direct interaction between the small G protein RhoE and the E3 ubiquitin ligase WWP2, and to explore whether this interaction depends on the enzyme activity of WWP2.

[0030] (1) Plasmid construction Wild-type WWP2 expression plasmid: The full-length human WWP2 gene (NCBI Reference Sequence: NM_007014.5) was cloned into the pcDNA3.1-Flag eukaryotic expression vector (purchased from Invitrogen, catalog number: V79520) to obtain the Flag-WWP2 (WT) expression plasmid.

[0031] Expression plasmid for the enzyme activity-deficient mutant WWP2: Site-directed mutagenesis was used with the QuikChange site-directed mutagenesis kit (Agilent, catalog number: 200519). The primer sequences were: forward 5'-GCTGCTGTGCACGCCTACAACGTG-3' as shown in SEQ ID NO. 5, and reverse 5'-CACGTTGTAGGCGTGCACAGCAGC-3' as shown in SEQ ID NO. 6. The mutation was verified by sequencing. The cysteine ​​residue at position 838 of the WWP2 catalytic site was mutated to alanine (C838A) to obtain the Flag-WWP2 (C838A) expression plasmid. This mutant loses E3 ubiquitin ligase activity.

[0032] RhoE expression plasmid: The full-length human RhoE gene (NCBI Reference Sequence: NM_005168.5) was cloned into the pcDNA3.1-His eukaryotic expression vector to obtain the His-RhoE expression plasmid. All constructed plasmids were verified to be correct by DNA sequencing.

[0033] (2) Cell culture and transfection Human embryonic kidney HEK293T cells (human embryonic kidney epithelial cell line, purchased from Fanchang Biotechnology Co., Ltd., catalog number: 20240128) were cultured in DMEM high-glucose medium (purchased from Xi'an Kehao Company, catalog number: 20250119) containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin, and incubated at 37°C in a 5% CO2 incubator. 24 hours before transfection, HEK293T cells were seeded into 6-well plates at a density of 3 × 10⁶ cells / well. 5 Cells / pores.

[0034] Transfection groups: The experiment was divided into 4 groups: Control group: Co-transfected with empty vectors pcDNA3.1-Flag and pcDNA3.1-His RhoE+WWP2 (WT) group: co-transfected with His-RhoE and Flag-WWP2 (WT) RhoE+WWP2 (C838A) group: co-transfected with His-RhoE and Flag-WWP2 (C838A) WWP2 (WT) Individual Transfection Group: Individual Transfection Flag-WWP2 (WT) Transfection was performed using the liposome transfection reagent Lipofectamine™ 3000 (Thermo Fisher Scientific, catalog number: L3000001), with a total transfection volume of 2.5 μg of plasmid per well. Fresh complete culture medium was replaced 6 hours after transfection.

[0035] (3) Cell lysis and immunoprecipitation Forty-eight hours after transfection, the culture medium was discarded, and the cells were washed twice with pre-chilled PBS. 200 μL of pre-chilled IP lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, containing protease inhibitors) was added to each well, and the cells were lysed on ice for 30 minutes. The cells were centrifuged at 12,000 rpm for 15 minutes at 4°C, and the supernatant was collected. A small amount was used for BCA protein quantification. 500 μg of total protein was added to 2 μg of anti-Flag antibody or normal mouse IgG (purchased from Yeasen Biotechnology (Shanghai) Co., Ltd., China, catalog number: 36110ES60), and incubated overnight at 4°C. 30 μL of Protein A / G agarose beads were added, and the cells were incubated at 4°C for 4 hours. The agarose beads were washed five times with IP lysis buffer, and 30 μL of 2×SDS loading buffer was added. The cells were boiled for 10 minutes.

[0036] (4) Western blot detection Immunoprecipitates were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. Blocking with 5% skim milk powder for 1 hour, followed by incubation at 4°C overnight with the corresponding primary antibodies (anti-His antibody 1:2000, anti-Flag antibody, purchased from Yeasen Biotechnology, catalog number: 30505ES20, 1:3000). HRP-labeled secondary antibody (purchased from Yeasen Biotechnology, catalog number: 36110ES60, dilution 1:5000) was incubated at room temperature for 1 hour, and detected by ECL chemiluminescence immunoassay.

[0037] Experimental results are as follows Figure 2 As shown, by Figure 2 It is known that the small G protein RhoE can specifically interact with the E3 ubiquitin ligase WWP2, and this interaction is independent of the enzyme activity of WWP2. Both wild-type WWP2 and the enzyme-deficient mutant WWP2 (C838A) can interact with RhoE.

[0038] Example 2: The effect of RhoE on the de-inhibition state of WWP2 This embodiment verifies whether RhoE can relieve the self-inhibition state of WWP2 and promote its own ubiquitination and degradation.

[0039] (1) Plasmids and reagents Expression plasmids: His-RhoE (same as in Example 1), 3FLAG-WWP2 (same as in Example 1), HA-Ubiquitin (ubiquitin expression plasmid, purchased from Addgene, catalog number: #17608). Proteasome inhibitor MG132 (purchased from Merck, catalog number: MG132, M8699).

[0040] Cell processing and grouping: HEK293T cells were divided into two groups: Control group: Transfected with 3FLAG-WWP2+HA-Ubiquitin alone Experimental group: Co-transfected with His-RhoE + 3FLAG-WWP2 + HA-Ubiquitin 24 hours after transfection, MG132 (final concentration 10 μM) was added and the patient was treated for 6 hours to inhibit the degradation of ubiquitinated proteins by the proteasome.

[0041] (2) Immunoprecipitation and detection Cell lysates were collected, and WWP2 protein was enriched with anti-FLAG magnetic beads. The ubiquitination level (using anti-HA antibody, purchased from CST, catalog number: #3724) and total protein level (using anti-WWP2 antibody, purchased from TargetMol, catalog number: TMAY-02489) were detected by Western blotting.

[0042] Experimental results are as follows Figure 3 and 4 As shown, compared with the control group, the total protein level of WWP2 was significantly reduced in the RhoE co-transfected experimental group; compared with the control group, the ubiquitination level of WWP2 was significantly increased in the RhoE co-transfected experimental group. The results indicate that after RhoE binds to WWP2, it relieves the autoinhibitory state of WWP2, activates its E3 ligase activity, leading to WWP2 autoubiquitination and degradation via the proteasome pathway. Figure 4 The results show that the region where RhoE acts on WWP2 is the N-terminus, C-terminus, and CBMs region located in the middle of RhoE.

[0043] Example 3: Colocalization analysis of RhoE and WWP2 in cells This embodiment uses an immunofluorescence co-localization experiment to visually verify the interaction between RhoE and WWP2 at the cellular level.

[0044] Cell lines: H9C2 rat cardiomyocytes (purchased from Sxfcbio Ltd (Xian, China). Contract No.: [Company Name], catalog number: FC-cell-181121A). Antibodies: Mouse anti-RhoE antibody (purchased from CST Biotech, catalog number: #3664), rabbit anti-WWP2 antibody (purchased from CST Biotech, catalog number: #41182). Fluorescent secondary antibodies: Alexa Fluor 488-labeled donkey anti-mouse (green, purchased from Booster Biotech, catalog number: BA1126), Alexa Fluor 555-labeled donkey anti-rabbit (red, purchased from Booster Biotech, catalog number: BA1135), Alexa Fluor 647-labeled donkey anti-goat (purchased from Booster Biotech, catalog number: BA1140).

[0045] H9C2 cells were seeded in laser confocal microscope dishes and cultured for 24 hours. They were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 for 10 minutes, and blocked with 5% BSA for 1 hour. A mixture of mouse anti-RhoE antibody and rabbit anti-WWP2 antibody was added, and the cells were incubated overnight at 4°C. The appropriate fluorescent secondary antibody was added, and the cells were incubated at room temperature in the dark for 1 hour. The nuclei were stained with DAPI for 5 minutes. The cells were observed and photographed using a laser confocal microscope.

[0046] Experimental results are as follows Figure 5 As shown, by Figure 5 As can be seen, immunofluorescence colocalization experiments confirm that RhoE and WWP2 have a specific interaction in cardiomyocytes and can form a complex within the cell.

[0047] Example 4: Regulation of the interaction between RhoE and WWP2 and autophagy receptor P62 This embodiment verifies whether RhoE-activated WWP2 can interact with autophagy receptor P62, and the effect of RhoE expression level on their interaction.

[0048] H9C2 cells were divided into three groups: control group, RhoE overexpression group (HBAD-m-RhoE), and RhoE knockdown group (Ad-shRNA2-RhoE). After cell fixation, permeabilization, and blocking, rabbit anti-WWP2 antibody and mouse anti-P62 antibody were added, and the cells were incubated overnight at 4°C. FITC-labeled goat anti-rabbit IgG (green, labeled WWP2) and CY3-labeled goat anti-mouse IgG (red, labeled P62) were added, and the cells were incubated at room temperature in the dark for 1 hour. Nuclei were stained with DAPI and observed under a laser confocal microscope.

[0049] Experimental results are as follows Figure 6 As shown, by Figure 6It is known that RhoE can promote the interaction between WWP2 and autophagy receptor P62, and this promoting effect depends on the expression level of RhoE.

[0050] Example 5: Formation of a ternary complex of RhoE, WWP2, and P62 This embodiment uses immunofluorescence triple staining to verify whether RhoE, WWP2, and P62 can form a ternary complex within cells.

[0051] H9C2 cells were treated as in Example 4. Mouse anti-RhoE antibody, rabbit anti-WWP2 antibody, and goat anti-P62 antibody were added simultaneously, and the cells were incubated overnight at 4°C. Alexa Fluor 488-labeled donkey anti-goat (green, labeled P62), Alexa Fluor 555-labeled donkey anti-mouse (red, labeled RhoE), and Alexa Fluor 647-labeled donkey anti-rabbit (gray, labeled WWP2) were added, and the cells were incubated at room temperature in the dark for 1 hour. Nuclei were stained with DAPI and observed using a laser confocal microscope.

[0052] Experimental results are as follows Figure 7 As shown, RhoE, WWP2, and P62 can form a ternary complex in cardiomyocytes, suggesting that RhoE may regulate the function of P62 through WWP2.

[0053] Example 6: Effects of RhoE-activated WWP2 on cardiomyocyte autophagy and hypertrophy This embodiment verifies whether RhoE-activated WWP2 can promote autophagic flux in cardiomyocytes and inhibit cardiomyocyte hypertrophy.

[0054] Primary neonatal rat ventricular myocytes (isolated from 1-3 day old SD rats, purchased from the Experimental Animal Center of Xinjiang Medical University). Staining reagent: Acridine orange (AO, for detecting acidic autophagic vesicles, purchased from Sigma, model: 30432-100MG). Adenovirus: GFP-mRFP-LC3 adenovirus (for detecting autophagic flux, purchased from Shanghai Hanheng Company, model: HB-LP2100001, titer: 1×10⁻⁶). 10 PFU / mL).

[0055] Primary neonatal rat ventricular myocytes were divided into three groups: control group (Ad-lacZ), RhoE overexpression group (HBAD-mRibo), and WWP2 knockdown group (Ad-shRNA2-WWP2).

[0056] After 48 hours of treatment, acridine orange (final concentration 1 μg / mL) was added and incubated for 15 minutes. After washing with PBS, the cells were observed under a fluorescence microscope. Acidic autophagosomes showed red fluorescence.

[0057] Cells were infected with GFP-mRFP-LC3 adenovirus (MOI=50) for 24 hours, then treated according to their groups and cultured for another 24 hours before being observed under a laser confocal microscope. GFP-positive and mRFP-positive spots (yellow) represent autophagosomes, while GFP-negative and mRFP-positive spots (red) represent autolysosomes.

[0058] Experimental results are as follows Figure 8 and Figure 9 As shown, RhoE-activated WWP2 can promote autophagic flux in cardiomyocytes, and this promoting effect depends on the expression of WWP2.

[0059] Example 7: Activating WWP2 and promoting the interaction between endosomal receptor HGS and WWP2 This embodiment verifies whether a short peptide simulating the C-terminal domain of RhoE can activate the E3 ubiquitin ligase WWP2.

[0060] The preliminary experiments in this application simulated the characteristics of RhoE's N-terminus, C-terminus, and Core GTP-binding region CBMs motif to construct short peptides, which were used in the grouped short peptide experiments of this embodiment and the following embodiments.

[0061] Among them, Peptide1 is abbreviated as P1, which means that short peptide 1 is at the N-terminus of RhoE. The sequence of P1 is: MKERRASQKLSSKSIMDPNQ. Peptide2, abbreviated as P2, is short peptide 2. It is located at the C-terminus of WEI RhoE. The sequence of P2 is: SQRATKRISHMPSRPELSAVATDLRKDKAKS (where 230-240 is the most critical region). Peptide3, or P3 for short, is a short peptide with questionable efficacy. It is constructed by mimicking the CBM motif of the RhoE Core GTP-binding region. The sequence of P3 is: ENYVPTVFENYT.

[0062] Primary cardiomyocytes in the logarithmic growth phase were harvested at a concentration of 2 × 10⁻⁶. 5Seeds were placed at a density of cells / well in confocal microplates and in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin-antibiotic mixture. The culture was incubated at 37°C in a 5% CO2 incubator until confluence reached 70%–80%. Experimental groups included: Null group: cultured in normal medium without additional treatment; HG+PA group: H / P+Peptide1 (P1); H / P+Peptide2 (P2); H / P+Peptide3. After each treatment, the culture medium was discarded, and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed three times with PBS. Cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min, and washed three times with PBS. Cells were blocked with 5% BSA at room temperature for 1 h. A primary antibody mixture was added: rabbit anti-HGS antibody (1:200, FITC-labeled) and mouse anti-WWP2 antibody (1:200, CY3-labeled), and incubated overnight at 4°C. The primary antibody was discarded, and the cells were washed three times with PBS. The corresponding fluorescent secondary antibody (1:500) was added, and the cells were incubated at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI at room temperature for 5 min, and the slides were mounted with anti-fluorescence quenching mounting solution.

[0063] Images were acquired using a laser confocal scanning microscope, with the following channels set: DAPI (blue, cell nucleus), FITC (green, HGS), and CY3 (red, WWP2).

[0064] See results Figure 10 The results showed that compared with the Null group, the colocalization ratio of HGS and WWP2 in the HG+PA group was significantly increased. Compared with the HG+PA group, after intervention with short peptide 1 and short peptide 3, the colocalization signal of green fluorescence (HGS) and red fluorescence (WWP2) was further significantly enhanced, while the colocalization enhancement effect after intervention with short peptide 2 was weaker. This suggests that short peptide 1 and short peptide 3 can lead to a significant increase in the colocalization of green and red fluorescent molecules, which can effectively activate WWP2 and promote its interaction with the endosome receptor HGS.

[0065] Example 8: Promoting the clearance of damaged mitochondria in cardiomyocytes This embodiment is to verify that the short peptide of the present invention can promote the clearance of damaged mitochondria in cardiomyocytes and inhibit the hypertrophic response of cardiomyocytes.

[0066] Primary cardiomyocytes in logarithmic growth phase were seeded in confocal dishes and cultured routinely until confluence reached 70%–80%. Pretreatment: Add 10 g of [a specific chemical compound] to a final concentration of [a specific chemical compound]. -6 Treatment with mol / L FCCP (mitochondrial uncoupling agent) for 2 h induced mitochondrial damage in cardiomyocytes; Group intervention: Null group: FCCP pretreatment followed by replacement with normal culture medium, continued culturing for 4h, 20h, and 36h; Peptide1 (P1) group: continued culturing for 4h, 20h, and 36h; Peptide2 (P2) group. Mitochondrial fluorescent labeling and imaging: After the intervention of each group, HBmTur-Mito mitochondrial fluorescent probe (final concentration 100 nmol / L) was added and incubated at 37℃ in the dark for 30min; after washing 3 times with PBS, fixation was performed with 4% paraformaldehyde for 15min, cell nuclei were counterstained with DAPI for 5min, and slides were mounted with anti-fluorescence quenching mounting solution; images were acquired using a laser confocal microscope (40× objective lens), with damaged mitochondria labeled with red fluorescent markers and cell nuclei labeled with blue fluorescent markers, scale bar at 25μm.

[0067] The results are as follows Figure 11 As shown, mitochondrial clearance dynamics: Compared with the Null group, the Peptide1 and Peptide2 groups showed a significant reduction in red mitochondrial fluorescence signals after 20h and 36h of intervention, with almost complete clearance at 36h, suggesting that short peptides can efficiently promote the degradation and clearance of damaged mitochondria; the Null group still retained a large amount of mitochondrial fluorescence signals at 36h, indicating slow clearance of damaged mitochondria. Cell morphology changes: The Null group showed significant cell hypertrophy (enlarged cell body and irregular nuclear morphology) over time, while the Peptide1 and Peptide2 groups maintained good cell morphology, and the hypertrophic response was significantly inhibited, further verifying that short peptides can inhibit cardiomyocyte hypertrophy. This indicates that the present invention can effectively promote the clearance of damaged mitochondria in cardiomyocytes and inhibit the hypertrophic response of cardiomyocytes.

[0068] Example 9: Activating WWP2 and significantly inhibiting cardiomyocyte remodeling response This embodiment is to verify that the short peptide of the present invention can activate WWP2 and significantly inhibit the remodeling response of cardiomyocytes.

[0069] Primary cardiomyocytes in logarithmic growth phase were seeded in confocal dishes and cultured to 70%–80% confluence. Group interventions were performed: HG+PA group; H / P+Peptide1 (P1) group; and H / P+Peptide2 (P2) group. Immunofluorescence staining was performed. After each group treatment, the culture medium was discarded, and the cells were washed three times with PBS. Cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed three times with PBS. Cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 min, and washed three times with PBS. Cells were blocked with 5% BSA at room temperature for 1 h. Primary antibody (mouse anti-α-SMA antibody, 1:200, CY3 labeled) was added, and the cells were incubated overnight at 4°C. The primary antibody was discarded, and the cells were washed three times with PBS. The corresponding fluorescent secondary antibody (1:500) was added, and the cells were incubated at room temperature in the dark for 1 h. Cell nuclei were counterstained with DAPI at room temperature for 5 min, and the slides were mounted with anti-fluorescence quenching mounting medium.

[0070] Confocal microscopy observation and result analysis: Images were acquired using a laser confocal scanning microscope (40× objective lens). Channels were set as follows: DAPI (blue, cell nucleus) and CY3 (red, α-SMA), with a scale bar of 25 μm. Ten fields of view were randomly selected from each group. ImageJ software was used to quantitatively analyze the fluorescence intensity of α-SMA and the proportion of stress fibers formed. The experiment was independently repeated three times.

[0071] See results Figure 12 The results showed that, compared with the HG+PA group, intervention with short peptide 1 and short peptide 2 significantly reduced the formation of stress fibers of α-SMA in cardiomyocytes, significantly reduced fluorescence intensity, and significantly inhibited cytoskeleton remodeling, suggesting that short peptides can effectively inhibit cardiomyocyte remodeling response by activating WWP2.

[0072] Western Blot experiment: Equal amounts of protein (30 μg / well) were subjected to SDS-PAGE electrophoresis (8% separating gel), with a constant voltage of 80V stacking gel and 120V separating gel; the protein was transferred to a PVDF membrane using a semi-dry transfer method, and blocked with 5% skim milk at room temperature for 1 h; primary antibodies were added: rabbit anti-α-SMA (1:1000), rabbit anti-HSP70 (1:5000, internal control 1), and rabbit anti-Tubulin (1:5000, internal control 2), and incubated overnight at 4℃; after washing the membrane, HRP-labeled secondary antibody (1:5000) was added, and incubated at room temperature for 1 h; development was performed using ECL chemiluminescence method, and the gray values ​​of the bands were quantified using ImageJ software. Using HSP70 / Tubulin as an internal control, the relative expression level of α-SMA was calculated: compared with the Null group, the expression of α-SMA in the HG+PA group was significantly increased, suggesting that metabolic stress induces cardiomyocyte remodeling; compared with the HG+PA group, the expression of α-SMA decreased in a dose-dependent manner after intervention with Peptide1 (P1), Peptide2 (P2), and Peptide3 (P3), which was consistent with the immunofluorescence results, further confirming that short peptides can inhibit cardiomyocyte remodeling response by activating WWP2.

[0073] See results Figure 13 The results showed that compared with the Null group, the expression of α-SMA in the HG+PA group was significantly increased, suggesting that metabolic stress induces cardiomyocyte remodeling. Compared with the HG+PA group, the expression of α-SMA decreased in a dose-dependent manner after intervention with Peptide1, Peptide2 and Peptide3, which was consistent with the immunofluorescence results, further confirming that short peptides can inhibit cardiomyocyte remodeling response by activating WWP2.

[0074] Example 10: Western Blot Validation of Initiating Endosome Alternative Autophagy and Inhibiting Cardiomyocyte Hypertrophy This embodiment aims to verify that a short peptide mimicking RhoE can initiate the endosome-lysosome pathway to replace autophagy and inhibit the hypertrophic response of cardiomyocytes.

[0075] Primary cardiomyocytes in logarithmic growth phase were seeded in 6-well plates and cultured to 70%–80% confluence. Group interventions were performed: Null group: cultured in normal medium; PE group: treated with phenylephrine (PE) at a final concentration of 10 μmol / L; PE + Peptide2 group (P2); PE + Peptide2 (P2) + CH group: PE + short peptide 2 intervention was followed by the addition of an autophagy inhibitor CH (such as chloroquine) to verify that the short peptide effect was independent of the classical autophagy pathway. Protein sample preparation and Western blotting: After each group intervention, cells were washed three times with pre-chilled PBS, lysed on ice for 30 min with RIPA lysis buffer containing protease inhibitors, centrifuged at 12000 rpm for 15 min at 4°C, and the supernatant was collected for protein concentration determination using the BCA method. Equal amounts of protein (30 μg / well) were subjected to SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h. Primary antibodies were added: rabbit anti-ANF (1:1000, a marker of myocardial hypertrophy) and rabbit anti-HSP90 (1:5000, an internal control). The membranes were incubated overnight at 4°C. After washing the membranes, HRP-labeled secondary antibody (1:5000) was added and the membranes were incubated at room temperature for 1 h. The membranes were then developed using ECL and the grayscale values ​​were quantified using ImageJ.

[0076] For the results analysis, please refer to [link / details]. Figure 14 The relative expression level of ANF was calculated using HSP90 as an internal reference: compared with the Null group, the ANF expression in the PE group was significantly increased, indicating that PE successfully induced cardiomyocyte hypertrophy; compared with the PE group, the ANF expression in the PE+Peptide2 group was significantly decreased, indicating that short peptide 2 can effectively inhibit cardiomyocyte hypertrophy; after adding the autophagy inhibitor CH, the inhibitory effect of short peptide 2 on ANF was still significant, indicating that the anti-hypertrophy effect of short peptide does not depend on the classical autophagy pathway, but is achieved by initiating the endosome-lysosome pathway instead of autophagy.

[0077] Example 11: In vivo verification of the effects of RhoE-WWP2-HGS and short peptide intervention. This embodiment aims to verify the regulatory effect of RhoE on WWP2-HGS in vivo and clarify the impact of CH intervention on this axis and cardiac remodeling, providing in vivo experimental evidence for short peptide-targeted WWP2-HGS treatment of myocardial hypertrophy.

[0078] Animal Model and Grouping: A mouse model of cardiomyocyte-specific overexpression of RhoE (H11-RhoE:αMHC-Cre) was constructed and randomly divided into two groups: Model group: H11-RhoE:αMHC-Cre mice, treated with an equal amount of solvent as a control; Intervention group: H11-RhoE:αMHC-Cre+CH mice, treated with an autophagy inhibitor CH (such as chloroquine) for 4 weeks.

[0079] Immunofluorescence staining and imaging: After the intervention, mice were sacrificed, heart tissue was isolated, fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned; dewaxed to water, antigen retrieval was performed with 0.01M citrate buffer, and blocked with 5% BSA for 1 h; primary antibodies were added: rabbit anti-WWP2 (1:200, CY3 labeled, red) and rabbit anti-HGS (1:200, FITC labeled, green), and incubated overnight at 4℃; after washing the membrane, the corresponding fluorescent secondary antibody (1:500) was added, and incubated at room temperature in the dark for 1 h; cell nuclei were counterstained with DAPI for 5 min, and the slides were mounted with anti-fluorescence quenching mounting solution; large field of view (20×) and high magnification (600×) images of the heart were acquired using a laser confocal microscope to observe the localization and co-localization of WWP2 and HGS.

[0080] See results Figure 15 As shown in the figures, WWP2 in the model group exhibited a dense punctate / filamentous distribution within cardiomyocytes, with strong and widespread signal intensity. In the intervention group (+CH), WWP2 signal intensity was significantly reduced, and the distribution was more diffuse, suggesting that CH can downregulate the expression and accumulation of WWP2 in myocardial tissue. In the model group, HGS showed diffuse cytoplasmic aggregation with punctate clusters, highly overlapping with WWP2. In the intervention group, HGS signal intensity shifted to a strong fibrous / myofibrillary distribution, with reduced punctate clusters, suggesting that CH remodeled the subcellular localization of HGS. In the model group, the yellow colocalization signal of WWP2 and HGS was abundant and dense, indicating a strong interaction between the two in myocardial tissue. In the intervention group, the yellow colocalization signal was significantly reduced, and the red and green signals were separated, indicating that CH can disrupt the RhoE-mediated WWP2-HGS interaction.

[0081] Based on the above research content, combined with Figure 16 The illustrated mechanism diagram presents the core action chain of this invention: In cardiomyocytes with defects in autophagy-related genes, a short peptide mimicking the RhoEC terminus enters the cytoplasm via caveolin endocytosis, binds to and activates the E3 ubiquitin ligase WWP2, and relieves its autoinhibition; the activated WWP2 further regulates the endosome receptor HGS, and through polyubiquitination modification of the VHS domain of HGS, initiates the endosome-lysosome degradation pathway, replacing classical autophagy to clear damaged mitochondria and misfolded proteins, ultimately reducing myocardial hypertrophy, fibrosis, inflammatory infiltration and apoptosis, improving cardiac remodeling and enhancing cardiac function.

[0082] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE, characterized in that, The short peptide and its derivatives contain a linear functional domain at position 230-240 of the C-terminus of the RhoE protein, and possess at least one of the following functions: a. By binding to the 2-4 linker region of the E3 ubiquitin ligase WWP2, it can relieve the autoinhibition of WWP2; b. By binding to the UIM domain of the endosome receptor HGS (ESCRT-0), the self-folding inhibition of HGS caused by monoubiquitination in the UIM region is relieved. c. The activated WWP2 polyubiquitinates the VHS domain of the open-state HGS, initiating the endosome-lysosome degradation pathway.

2. The short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE according to claim 1, characterized in that, The short peptides and their derivatives can bind to caveolin on the cardiomyocyte membrane and be efficiently delivered to the cardiomyocyte cytoplasm via the caveolin endocytosis pathway, thereby targeting and activating intracellular WWP2.

3. The short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE according to claim 1, characterized in that, The amino acid sequence of the short peptide is shown in SEQ ID NO.

1.

4. The short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE according to claim 3, characterized in that, The short peptide derivative is obtained by coupling a short peptide with a membrane-penetrating peptide.

5. The short peptide and its derivatives that mimic the RhoEC terminal domain of a small G protein according to claim 4, characterized in that, The membrane-penetrating peptide is TAT ​​membrane-penetrating peptide, T7 permeabilizing peptide, or RVG peptide.

6. The short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE according to claim 5, characterized in that, The amino acid sequence of the short peptide derivative is shown in SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO.

4.

7. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of a short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE as described in any one of claims 1-6, and a pharmaceutically acceptable carrier, excipient, or diluent, for the prevention and / or treatment of conditions related to myocardial hypertrophy, myocardial fibrosis, cardiomyocyte apoptosis, or cardiac remodeling.

8. The use of the short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE as described in any one of claims 1-6 in the preparation of a medicament for activating the endosome-lysosome degradation pathway and clearing damaged mitochondria or misfolded proteins in cardiomyocytes.

9. The application according to claim 8, characterized in that, The drug achieves its function by activating WWP2, relieving HGS autoinhibition, and promoting the polyubiquitination of the VHS domain of HGS through the short peptide and its derivatives.

10. The use of the short peptide and its derivatives that mimic the C-terminal domain of the small G protein RhoE as described in any one of claims 1-6 in the preparation of a medicament for relieving myocardial hypertrophy, improving cardiac remodeling, or enhancing cardiac function.

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