Application of UFMylation-Modified YAP as a Target in the Preparation of Drugs for the Treatment of Vascular Aging and / or Reagents for Detection

Abstract

This invention discloses the application of UFMylation-modified YAP as a target in the preparation of therapeutic drugs and / or detection reagents for vascular aging. It is the first to disclose that YAP can undergo UFMylation modification, and that UFM1 and UFMylation-modified YAP increase during vascular endothelial cell senescence. Examples verify that senescent vascular endothelial cells show an increase in UFMylation-modified YAP. Therefore, UFMylation-modified YAP can be used to develop vascular aging detection kits or chips to determine whether the detected blood vessels have undergone aging. This invention provides the molecular mechanism by which UFMylation modification maintains YAP stability and plays an important role in promoting cellular senescence, and identifies previously unrecognized UFMylation-modified YAP as a potential therapeutic target for anti-aging, with broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of UFMylation-modified YAP as a target in the preparation of therapeutic drugs and / or detection reagents for vascular aging. Background Technology

[0002] Cardiovascular disease (CVD) is a leading cause of death worldwide, and vascular dysfunction is a key characteristic of CVD. Blood vessels consist of the intima, media, and adventitia. The intima is primarily composed of a single layer of endothelial cells (ECs) and the basement membrane. Endothelial cells not only act as a barrier between the circulatory system and peripheral tissues but also play crucial physiological roles in vascular homeostasis, maintaining blood flow, regulating vascular tone, pro-inflammatory responses, and angiogenesis. EC dysfunction is a major cause of cardiovascular dysfunction in humans and has been identified as a significant factor in CVD.

[0003] UFMylation is a less studied ubiquitination-like modification used to regulate protein stability and function, which is crucial for maintaining cell development and tissue homeostasis. Key molecules involved in UFMylation modification include ubiquitin folding modifier 1 (UFM1), UFM1-specific cysteine ​​protease 2 (UFSP2), E1 ubiquitin activator 5 (UBA5), E2 UFM1 coupling enzyme 1 (UFC1), and E3 UFM1 ligase 1 (UFL1). As an important mediator of endoplasmic reticulum (ER) stress response, UFMylation plays a protective role in heart failure and inhibits interferon-γ (IFN-γ)-induced macrophage activation, both through the inhibition of ER stress.

[0004] Previous studies have shown that the UFMylation system plays an important role in GPCR recruitment to COPII vesicles, biosynthetic transport, and sorting in the ER via direct interaction between UFMylation and UFBP1. To date, only a few UFMylation substrates have been reported. The substrates of UFMylation and their potential biological relevance, particularly in EC senescence, remain poorly understood. Previous studies have shown that YAP undergoes various post-translational modifications; however, whether it can be modified by UFMylation and the role of this modification in vascular endothelial cell senescence and vascular aging remain unknown. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to address the shortcomings of the prior art by providing the application of UFMylation-modified YAP as a target in the preparation of vascular aging therapeutic drugs and / or detection reagents.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides the application of UFMylation-modified YAP as a target in the preparation of drugs for anti-vascular aging.

[0008] Secondly, this invention provides the application of UFMylation-modified YAP as a target in the preparation of drugs for anti-vascular endothelial cell aging.

[0009] Preferably, the drug exerts its anti-aging effect by reducing UFMylation-modified YAP, the mechanism of which includes inhibiting UFM1 expression or activity, reducing the amount of UFMylation-modified YAP, and thus producing an anti-vascular aging effect.

[0010] Preferably, inhibiting UFMylation-modified YAP improves vascular aging and vascular stiffness.

[0011] Preferably, the method of reducing the amount of UFMylation-modified YAP includes at least one of plasmid transfection, viral infection, gene editing, transcriptional level, translational level, and post-translational modification level.

[0012] Preferably, the method of administration of the drug includes at least one of oral administration, intravenous administration, and intraperitoneal administration.

[0013] Preferably, the drug is any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions, and solutions.

[0014] Thirdly, the present invention provides the application of UFMylation-modified YAP as a target in the preparation of a reagent for detecting vascular aging, characterized in that the reagent is capable of detecting the expression level of UFMylation-modified YAP in tissue cells or body fluids.

[0015] Preferably, the tissue cells include at least one of blood vessels, liver, brain, heart, and kidney. The body fluids include at least one of serum, urine, cerebrospinal fluid, and sweat. The detection reagent is one of a chip and a reagent kit.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] This invention discloses for the first time the correlation between UFMylation-modified YAP and vascular senescence. A positive correlation exists between UFMylation-modified YAP and vascular senescence. Furthermore, it was found that UFMylation modification of YAP promotes its stability, and inhibiting UFM1 can reduce YAP levels, thus combating vascular endothelial cell senescence. UFMylation-modified YAP levels increase during endothelial cell senescence. UFMylation-modified YAP can be used to develop vascular senescence detection kits or chips to determine whether the detected blood vessels are aging. Our findings provide a molecular mechanism by which UFMylation maintains YAP stability and plays a crucial role in promoting cellular senescence, and identify previously unrecognized UFMylation-modified YAP as a potential therapeutic target for anti-aging. Attached Figure Description

[0018] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 UFM1 and YAP are highly expressed in senescent vascular endothelial cells. A shows the immunoblotting results of YAP, UFM1, and senescence-related proteins (Vcam1, p16, p21, p53) in young (PDL10) and senescent (PDL30) human umbilical vein endothelial cells (HUVECs); B shows the immunoblotting results of YAP, UFM1, and senescence-related proteins (Vcam1, p16, p21, p53) in aortic endothelial cells of young and aged mice; C shows the protein quantification analysis results.

[0020] Figure 2 UFMyation-modified YAP was increased in senescent vascular endothelial cells. A shows the results of detecting exogenous YAP UFMyation modification in 293T cells by co-transfection with UFMyation system-related enzymes and YAP protein; B shows the results of detecting endogenous YAP UFMyation modification in aortic endothelial cells isolated from 8-week-old C57BL / 6J mice; C shows the level of UFMyation-modified YAP in senescent endothelial cells analyzed using a neighbor-linking assay with antibodies against YAP and UFM1.

[0021] Figure 3UFMyation modification of YAP can increase its protein stability. In the figures, A represents the reduction of UFMyation system activity in endothelial cells by transfecting HUVECs with siRNA-NC (negative control) or by knocking down the key enzymes UFL1 or UFM1 (key enzymes in UFMyation modification) using siRNA; B represents the results of immunoblotting and quantitative analysis of YAP protein levels; C represents the changes in YAP protein over time in young (PDL10) and aged (PDL30) cells; D represents the quantitative results of C; E represents the changes in YAP protein over time before and after treatment with compound 8.5; and F represents the quantitative results of E.

[0022] Figure 4 Compound 8.5, an inhibitor of UFMyation, improves endothelial cell senescence by reducing the protein level of YAP. In the diagram, A represents the immunoblotting results of YAP, UFM1, and senescence-related proteins (VCAM1, p16, p21, p53); B represents the quantitative analysis results of A; C represents the β-galactosidase staining results for cellular senescence; and D represents the quantitative analysis results of C.

[0023] Figure 5 Compound 8.5, an inhibitor of UFMyation, improved vascular aging in aged mice by reducing YAP protein levels. In the figures, A shows changes in vascular compliance in aged mice after treatment with compound 8.5; B shows changes in pulse wave velocity (PWV) in aged mice after treatment with compound 8.5; C shows Masson and HE staining results of vascular tissue from aged mice; D shows the quantification of collagen deposition in C; and E shows the immunoblotting results of related proteins in aortic endothelial cells of aged mice after treatment with compound 8.5. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0025] 1. YAP was identified as a substrate for UFMylation.

[0026] 1.1 Western blot of proteins ( Figures 1-5 ).

[0027] Tissue or cell lysates were prepared using RIPA buffer containing a mixture of phosphatase inhibitors, PMSF, and complete protease inhibitors. Protein samples were separated by gel electrophoresis using 12% Bis-Tris SDS-Gels. The membrane was then transferred to a nitrocellulose membrane and blocked with 5% skim milk at room temperature for 1 hour. The membrane was then coated with the following primary antibodies: anti-UFM1 (Abcam, Cat#ab109305), anti-YAP (Cell Signaling Technology, Cat#14074), anti-Alpha Tubulin (Proteintech, Cat#66031-1-Ig), anti-p16 INK4A (Cell Signaling Technology, Cat#18769), anti-Flag (Cell Signaling Technology, Cat#14793), anti-p21 (Proteintech, Cat#10355-1-AP), anti-UBA5 (ABclonal, Cat#A15514), anti-VCAM1 (Huabio, Cat#ET1601-18), and anti-UFL1 (Oasis). After incubating overnight at 4°C with Biofarm (Cat#OB-PRB036), anti-p53 (Huabio (Cat#EM1701-91)) and anti-HA (Cell Signaling Technology (Cat#3724), the samples were incubated with horseradish peroxidase (Horseradish Peroxidase, HRP-linked Antibody (Anti-rabbit IgG, HRP-linked Antibody, Cat#7074; Anti-mouse IgG, HRP-linked Antibody, Cat#7076) at room temperature for 1 hour, and then imaging analysis was performed using an ECL luminescence system.

[0028] Depend on Figure 1 It can be seen that the protein levels of UFM1 and YAP are significantly increased in aging vascular endothelium.

[0029] 1.2 Detection of UFMylation Modification in YAP.

[0030] 1.2.1 Detection of UFMylation Modification of Exogenous YAP ( Figure 2 A in the middle.

[0031] HEK293T cells were transiently transfected with UFMylation components (UBA5, UFC1, UFL1, DDRGK1, and UFM1) and Flag-YAP plasmids using Lipofectamine 3000 transfection reagent. Cells were lysed at 100°C using lysis buffer (150 mM Tris-HCl (pH 8), 5% SDS, and 30% glycerol). The lysis buffer was diluted 20-fold with buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 1% NP-40, 2 mM NEM, and protease inhibitor). Immunoprecipitation was then performed using anti-Flag magnetic beads, and UFM1-modified YAP was detected using UFMylation antibody.

[0032] 1.2.2 Detection of UFMylation Modification of Endogenous YAP ( Figure 2 (B in the middle).

[0033] To detect endogenous YAP-UFMylation in mouse aortic endothelial cells, mouse aortas were isolated after euthanasia and left ventricular saline perfusion. Endothelial cells were scraped under a microscope using lysis buffer (150 mM Tris-HCl (pH 8), 5% SDS, and 30% glycerol) and lysed at 100°C. The lysis buffer was diluted 20-fold with buffer A (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 1% NP-40, 2 mM NEM, and protease inhibitor). The supernatant was incubated overnight with anti-YAP antibody or rabbit IgG, followed by incubation with protein A / G magnetic beads at 4°C for 2 hours. After multiple washes with NP-40 buffer, the immunoprecipitate was analyzed by Western blotting with anti-UFM1 antibody.

[0034] 1.2.3 Detection of UFMylation Modification of YAP in Senescent Endothelial Cells using Neighbor-to-Neighbor Connectivity Technique Figure 2 (C in the middle).

[0035] In situ analysis of UFMylation modification of YAP was performed using the Duolink In Situ Red 9 Starter Kit (Cat#DUO92101; Sigma-Aldrich). The experimental procedure was performed according to the manufacturer's instructions. The main steps were as follows: Cells or arterial sections were fixed in 4% paraformaldehyde at room temperature for 15 min, then permeabilized with permeabilization buffer for 30 min; after incubation with primary antibodies (anti-YAP, Huabio, Cat#RT1664; anti-UFM1, Huabio, Cat#HA722571) overnight at 4°C, Duolink PLAProbe (anti-mouse and anti-rabbit probes) was incubated at 37°C for 1 hour. The next step was a ligation step (30 min, 37°C) to allow the antibody-ligated oligonucleotides to form DNA loops, followed by incubation with amplification buffer at 37°C for 100 min. After washing, slides were mounted using Duolink PLA containing DAPI. Imaging and analysis were performed using a Zeiss confocal laser scanning microscope (LSM900).

[0036] Depend on Figure 2 It is known that YAP is modified by UFMyation, and this modification is increased in senescent vascular endothelial cells.

[0037] 2. siRNA transfection ( Figure 3 (AB in the middle).

[0038] RNA transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Cat#13778030). The siRNAs were purchased from Santa Cruz Biotechnology, specifically the following catalog numbers: YAP1 siRNA, Cat#sc-38637; UFM1 siRNA, Cat#sc-76804; UFL1 siRNA, Cat#sc-95134; and Negativecontrol siRNA, Cat#sc-37007. Transfection dosage and timing were performed according to the manufacturer's instructions.

[0039] The results are as follows Figure 3 As shown in AB, reducing the activity of the UFMyation system in cells can significantly reduce the protein level of YAP.

[0040] 2. YAP protein stability experiment ( Figure 3 (CF in the middle).

[0041] Previous studies have shown that UFMyation modification, as a post-translational modification of proteins, can regulate protein stability. Therefore, in this embodiment, cycloheximide (CHX, purchased from Medchemexpress, catalog number: Cat#HY-12320) was used to inhibit protein synthesis in cells and to test protein stability.

[0042] HUVECs were treated with cycloheximide (CHX, 10 μM) for 0, 2, 4, 6, and 8 h to inhibit protein synthesis in the cells, and the level of YAP protein was detected by immunoblotting.

[0043] The results are as follows Figure 3 As shown in CD, the degradation rate of YAP was significantly reduced in senescent endothelial cells (PDL30).

[0044] Furthermore, UFMyation modification was inhibited using the inhibitor compound 8.5, and the results were as follows: Figure 3 As shown in EF, inhibiting UFMyation modification promotes the degradation of YAP.

[0045] 3. H2O2-induced endothelial cell senescence model ( Figure 4 (AB in the middle).

[0046] To clarify the relationship between endothelial cell senescence and UFMyation modification of YAP, this embodiment uses an induced senescence model of endothelial cells induced by H2O2, and verifies the role of the YAP inhibitor verteporfin (purchased from medchemexpress, catalog number: Cat#HY-B0146), an inhibitor of UFMyation modification, in endothelial cell senescence.

[0047] HUVECs were treated with H2O2 (100 μM) for 12 h to induce endothelial cell senescence. Simultaneously, they were treated with either a UFMyation-modified inhibitor compound 8.5 (20 μM) or a YAP inhibitor, verteporfen (0.5 μM). Immunoblot analysis was performed on YAP, UFM1, and senescence-related proteins (Vcam1, p16, p21, p53). Compound 8.5 (UBA5-IN-1) was purchased from Medchemexpress, catalog number: Cat#HY-148266.

[0048] 4. β-galactosidase staining in cell senescence ( Figure 4 (CD in the middle).

[0049] Endothelial cell senescence was induced by treating HUVECs with H2O2 (100 μM) for 12 h, while simultaneously treating them with either a UFMyation-modified inhibitor compound (20 μM) or a YAP inhibitor, verteporfen (0.5 μM). Senescent endothelial cells were detected using a senescence β-galactosidase staining kit (Beyotime Biotechnology, Cat#C0602), following the manufacturer's instructions: cells were fixed in β-galactosidase fixative at room temperature for 15 min, then washed with PBS and incubated overnight at 37°C with staining buffer. Finally, the staining buffer was removed with PBS, and images were captured using a Leica microscope. Image-ProPlus was used to quantify positive cells.

[0050] Depend on Figure 4 It is known that inhibiting UFMyation modification can reduce the protein level of YAP, thereby improving endothelial cell senescence.

[0051] 5. UFMylation-modified inhibitor compound 8.5 can improve vascular aging in aged mice. Figure 5 ).

[0052] To further verify the role of UFMyation modification of YAP in vascular aging, aged mice (C57BL / 6J, 24 months old) were intraperitoneally injected with UFMyation-modified inhibitor compound 8.5 (0.25 mg / kg / d) or PBS. After 3 weeks, vascular aging-related indicators in mice, including vascular compliance, pulse wave velocity (PWV), and vascular collagen deposition, were analyzed to assess vascular aging.

[0053] 5.1 Vascular ultrasound analysis of vascular aging indicators (pulse wave velocity and vascular compliance) Figure 5 (AB in the middle).

[0054] All animal care and experimental procedures were conducted in accordance with international guidelines for laboratory animal care certification. Animal experimental protocols were approved by the Animal Ethics Committee of Hangzhou Normal University. Mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Male C57BL / 6J mice (24 months old) were administered PBS or compound 8.5 (0.25 mg / kg / day) via intraperitoneal injection. Three weeks later, pulse wave velocity (PWV) and vascular strain were assessed using a Vevo3100 imaging system. The specific steps are as follows: 1) After anesthetizing the mice with isoflurane, remove the hair from the neck and abdomen of the mice and fix them on the operating table. Adjust the maintenance dose of isoflurane in the anesthesia device to 1.0%–1.5%; 2) With the ultrasound probe facing the mouse's jaw, display the images of the left carotid artery and aortic arch in B-mode ultrasound and record the blood flow waves at the left common carotid artery, the left carotid bifurcation, and the left common carotid bifurcation of the aorta in PW mode. Then, record the diameter and thickness of the left carotid vessel in M-mode ultrasound; 3) Place the probe on the mouse's abdomen and display the bifurcation of the abdominal aorta and its renal arteries in B-mode ultrasound. Record the blood flow waves at the renal bifurcation of the abdominal aorta in PW mode. Record the aortic diameter and vessel wall thickness in M-mode ultrasound; 4) Calculate ΔT based on the conduction times T1 and T2 measured from the blood flow waves, and measure the distance D from the blood flow wave measurement point. Calculate PWV = D / ΔT. Each of the above data is measured 3–5 times; calculate vascular compliance = (Ds-Dd) / Dd based on the M-mode ultrasound images.

[0055] 5.2 Evaluation of vascular collagen deposition using frozen sections of mouse aorta ( Figure 5 (CD in the middle).

[0056] Mouse aortas were fixed in 4% paraformaldehyde at room temperature for 20 min, then frozen sections were embedded using OCT, and then cut into 5 μm thick sections for Masson staining and HE staining. The modified Masson trichrome staining kit (Cat#G1346) and the hematoxylin and eosin (HE) staining kit (Cat#G1120) purchased from Solarbio were used, and the specific steps were strictly followed according to the instructions.

[0057] The results showed that compound 8.5 significantly improved vascular aging in aged mice, manifested as increased vascular compliance and decreased PWV in the left carotid artery (LCA), aortic arch, and abdominal aorta. Figure 5 (AB in the middle); at the same time, collagen deposition was also significantly reduced ( Figure 5 (CD in the middle). Further, immunoblotting analysis of YAP, UFM1, and aging-related proteins (Vcam1, p16, p21, p53) in mouse aortic endothelial cells revealed that, consistent with the results of cell experiments, inhibition of UFMyation modification of compound 8.5 reduced the levels of UFM1 and YAP in aged mouse endothelial cells, thus improving endothelial cell senescence (CD). Figure 5 (EF in the text).

[0058] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be considered as limitations on the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. For those skilled in the art, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of an agent that inhibits the UFMylation modification of YAP protein in the manufacture of a medicament for the treatment of vascular aging, characterized in that, The agent is compound 8.5 UBA5-IN-1, purchased from medchemexpress, Cat# HY-148266.

Images