Application of UFMylation modified YAP as target in preparation of vascular aging treatment medicine and / or detection reagent

Abstract

The invention discloses application of UFMylation modified YAP as a target in preparation of a vascular aging treatment medicine and / or a detection reagent, discloses that the YAP can be subjected to UFMylation modification, and the UFM1 and the UFMylation modified YAP are increased when vascular endothelial cells are aged for the first time, and verifies that the aged vascular endothelial cells and the UFMylation modified YAP are increased in the embodiment, so that the application of the UFMylation modified YAP as the target in the preparation of the vascular aging treatment medicine and / or the detection reagent can be realized, and the application of the UFMylation modified YAP as the target in the preparation of the vascular aging treatment medicine and / or the detection reagent in the preparation of the vascular aging treatment medicine and / or the detection reagent in the preparation of the vascular aging treatment medicine and / or the detection reagent. The UFMylation modified YAP can be used for developing a blood vessel aging detection kit or chip so as to judge whether the detected blood vessel is aged or not. The invention provides a molecular mechanism which is used for maintaining the stability of the YAP through UFMylation modification and plays an important role in promoting cell senescence, and determines that the UFMylation modified YAP which is not recognized previously is a potential treatment target for resisting senescence, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and specifically to the use of UFMylation-modified YAP as a target in the preparation of vascular aging therapeutic drugs and / or detection reagents. Background Art

[0002] Cardiovascular disease (CVD) is the leading cause of death worldwide, of which vascular dysfunction is a key feature of CVD. Blood vessels are composed of the intima, media, and adventitia. The intima is mainly composed of a single layer of endothelial cells (EC) and a basement membrane. Endothelial cells not only act as a barrier between the circulation and peripheral tissues, but also play important physiological roles in vascular homeostasis, maintaining blood flow, regulating vascular tone, proinflammatory responses, and neovascularization. EC dysfunction is the main cause of cardiovascular dysfunction in humans and has been identified as an important factor in CVD.

[0003] UFMylation modification is a less studied ubiquitination-like modification used to regulate protein stability and function, which is essential for maintaining cell development and tissue homeostasis. The core molecules involved in UFMylation modification include ubiquitin fold modifier 1 (UFM1), UFM1-specific cysteine ​​protease (UFSP2), E1 ubiquitin activating enzyme 5 (UBA5), E2 UFM1 conjugating enzyme 1 (UFC1) and E3 UFM1 ligase 1 (UFL1). As an important mediator of endoplasmic reticulum (ER) stress response, UFMylation's protective effect in heart failure and inhibition of interferon-γ (IFN-γ)-induced macrophage activation are mediated by inhibiting ER stress.

[0004] Previous studies have shown that the UFMylation system has important functions in GPCR recruitment to COPII vesicles, biosynthetic trafficking, and sorting in the ER through direct interaction between UFMylation and UFBP1. So far, only a few UFMylation substrates have been reported. The substrates of UFMylation and their potential biological relevance, especially in ECs aging, remain poorly understood. Previous studies have shown that YAP has multiple protein post-translational modifications, but whether it can be modified by UFMylation and the role of this modification in vascular endothelial cell aging and vascular aging remain unknown. Summary of the invention

[0005] In view of this, the purpose of the present invention is to address the deficiencies in the prior art and provide an 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 purpose, the technical solution adopted by the present invention is as follows:

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

[0008] In a second aspect, the present invention provides the use of UFMylation-modified YAP as a target in the preparation of drugs for resisting vascular endothelial cell aging.

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

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

[0011] Preferably, it is characterized in that the method of reducing the amount of UFMylation-modified YAP includes at least one of plasmid transfection, viral infection, gene editing, transcription level, translation level, and post-translational modification level.

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

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

[0014] In a third aspect, the present invention provides an application of UFMylation-modified YAP as a target in the preparation of a reagent for detecting vascular aging, characterized in that the reagent can detect the expression level of UFMylation-modified YAP in tissue cells or in body fluids.

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

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

[0017] The present invention discloses for the first time that UFMylation-modified YAP is correlated with vascular aging, that UFMylation-modified YAP is positively correlated with vascular aging, and that UFMylation modification of YAP promotes its stability, and that inhibition of UFM1 can reduce the level of YAP to resist endothelial cell aging. UFMylation-modified YAP increases during endothelial cell aging, and UFMylation-modified YAP can be used to develop vascular aging detection kits or chips to determine whether the detected blood vessels are aging. Research has found that our discovery provides a molecular mechanism by which UFMylation maintains YAP stability and plays an important role in promoting cell aging, and identifies previously unrecognized UFMylation-modified YAP as a potential therapeutic target for anti-aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 UFM1 and YAP are highly expressed in aged vascular endothelial cells. A is the immunoblotting result of YAP, UFM1 and aging-related proteins (Vcam1, p16, p21, p53) in young (PDL10) and aged (PDL30) human umbilical vein endothelial cells (HUVECs); B is the immunoblotting result of YAP, UFM1 and aging-related proteins (Vcam1, p16, p21, p53) in young and old mouse aortic endothelial cells; C is the result of protein quantitative analysis.

[0020] Figure 2 The increase of UFMyation-modified YAP in aged vascular endothelial cells. A is the result of detecting UFMyation modification of exogenous YAP by co-transfecting UFMyation system-related enzymes and YAP protein in 293T cells; B is the result of detecting UFMyation modification of endogenous YAP by isolating aortic endothelial cells from 8-week-old C57BL / 6J mice; C is the level of UFMyation-modified YAP in aged endothelial cells analyzed by proximity ligation experiment using antibodies to YAP and UFM1.

[0021] Figure 3UFMyation modification of YAP can increase its protein stability. Among them, A is the result of HUVECs transfected with siRNA-NC (negative control), or using siRNA to knock down the key enzyme UFL1 or UFM1 (key enzyme modified by UFMyation) to reduce the activity of the UFMyation system in endothelial cells; B is the result of immunoblotting and quantitative analysis of YAP protein levels; C is the result of the change of YAP protein over time in young (PDL10) and aged (PDL30) cells; D is the quantitative result of C; E is the result of the change of YAP protein over time before and after treatment with compound 8.5; F is the quantitative result of E.

[0022] Figure 4 The UFMyation-modified inhibitor compound 8.5 improves endothelial cell senescence by reducing the protein level of YAP. A is the immunoblotting result of YAP, UFM1 and senescence-related proteins (VCAM1, p16, p21, p53), B is the quantitative analysis result of A, C is the β-galactosidase staining result of cell senescence, and D is the quantitative analysis result of C.

[0023] Figure 5 Compound 8.5, an inhibitor modified by UFMyation, improves vascular aging in aged mice by reducing the protein level of YAP. A is the result of the change in vascular compliance of aged mice after treatment with compound 8.5, B is the result of the change in pulse wave velocity (PWV) of aged mice after treatment with compound 8.5, C is the result of Masson and HE staining of vascular tissues of aged mice, D is the quantification result of collagen deposition in C, and E is the result of immunoblotting of related proteins in aortic endothelial cells of aged mice after treatment with compound 8.5. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

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

[0026] 1.1 Western Blot Figure 1-Figure 5 ).

[0027] Tissue or cell lysates were prepared with RIPA buffer containing phosphatase inhibitors, PMSF and complete protease inhibitor cocktail. 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 h. The membrane was then incubated 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), anti-UFL1 (Oasis Biotechnology, Cat#ET1601-17). Biofarm, Cat#OB-PRB036), anti-p53 (Huabio, Cat#EM1701-91) and anti-HA (Cell Signaling Technology, Cat#3724) were incubated at 4°C overnight, and then incubated with horseradish peroxidase (Horseradish Peroxidase, HRP-secondary 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 imaged and analyzed using the ECL luminescence system.

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

[0029] 1.2 Detection of UFMylation modification of YAP.

[0030] 1.2.1 Detection of UFMylation modification of exogenous YAP ( Figure 2 A).

[0031] HEK293T cells were transiently transfected with plasmids of UFMylation components (UBA5, UFC1, UFL1, DDRGK1, and UFM1) and Flag-YAP using Lipofectamine 3000 transfection reagent, and the cells were lysed at 100 degrees using lysis buffer (150mMTris-HCl (pH8), 5% SDS, and 30% glycerol), and the lysate was diluted 20 times with buffer A (50mM Tris-HCl pH 8.0, 150mMNaCl, 10mM imidazole, 1% NP-40, 2mM NEM, and protease inhibitors). Anti-Flag magnetic beads were then used for immunoprecipitation, and UFMylation-modified YAP was detected using UFM1 antibody.

[0032] 1.2.2 Detection of UFMylation modification of endogenous YAP ( Figure 2 B).

[0033] To detect endogenous YAP-UFMylation in mouse aortic endothelial cells, mouse aortas were isolated after euthanasia and left ventricular saline perfusion, and mouse aortic endothelial cells were scraped under a microscope using lysis buffer (150mM Tris-HCl (pH8), 5% SDS, and 30% glycerol) and lysed at 100 degrees. The lysate was diluted 20 times with buffer A (50mM Tris-HCl pH 8.0, 150mM NaCl, 10mM imidazole, 1% NP-40, 2mM NEM and protease inhibitors). The supernatant was incubated with anti-YAP antibody or rabbit IgG overnight and then incubated with proteinA / G magnetic beads at 4°C for 2 hours. After multiple washes with NP-40 buffer, the immunoprecipitates were analyzed by Western blotting with anti-UFM1 antibody.

[0034] 1.2.3 Proximity ligation technique to detect UFMylation modification of YAP in senescent endothelial cells ( Figure 2 C).

[0035] The UFMylation modification of YAP was analyzed in situ using Duolink In Situ Red 9 Starter Kit (Cat#DUO92101; Sigma-Aldrich). The experimental operation was performed according to the instructions. The main steps were: cells or arterial sections were fixed with 4% paraformaldehyde at room temperature for 15 minutes, and the membrane was permeabilized with permeabilization solution for 30 minutes; after incubation with primary antibodies (anti-YAP, Huabio, Cat#RT1664; anti-UFM1, Huabio, Cat#HA722571) at 4°C overnight, DuolinkPLAProbe (anti-mouse and anti-rabbit probes) was incubated at 37°C for 1 hour. The next connection step (30 minutes, 37°C) allowed the antibody-linked oligonucleotides to form DNA circles, and then incubated with amplification buffer at 37°C for 100 minutes. After washing, DuolinkPLA containing DAPI was used to cover the slides. Imaging and analysis were performed using a Zeiss confocal laser scanning microscope (LSM900).

[0036] Depend on Figure 2 It can be seen that YAP has UFMyation modification, and this modification is increased in aged vascular endothelial cells.

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

[0038] RNA transfection was performed using Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Cat#13778030), and siRNA was purchased from Santa Cruz Biotechnology, with the following specific catalog numbers: YAP1 siRNA, Cat#sc-38637; UFM1 siRNA, Cat#sc-76804; UFL1 siRNA, Cat#sc-95134; Negativecontrol siRNA, Cat#sc-37007. The transfection dose and time were as recommended in the 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 ).

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

[0042] Cycloheximide (CHX, 10uM) was used to treat HUVECs for 0, 2, 4, 6, and 8 hours to inhibit protein synthesis in cells, and the level of YAP protein was detected by immunoblotting.

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

[0044] Further, the UFMyation modification inhibitor compound 8.5 was used to inhibit the UFMyation modification, and the results were as follows Figure 3 As shown in EF in Figure 3, inhibition of UFMyation modification promotes the degradation of YAP.

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

[0046] In order to clarify the relationship between endothelial cell senescence and UFMyation modification of YAP, this example uses H2O2 to induce an induced senescence model of endothelial cells, and verteporfin (purchased from medchemexpress, Cat#HY-B0146), an inhibitor of YAP, and the role of UFMyation-modified inhibitor compound 8.5 in endothelial cell senescence.

[0047] HUVECs were treated with H2O2 (100uM) for 12h to induce endothelial cell senescence, and the UFMyation-modified inhibitor compound 8.5 (20uM) or YAP inhibitor verteporfin (0.5uM) were treated at the same time, and YAP, UFM1 and senescence-related proteins (Vcam1, p16, p21, p53) were analyzed by immunoblotting. Compound 8.5 (UBA5-IN-1) was purchased from medchemexpress, Cat#HY-148266.

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

[0049] HUVECs were treated with H2O2 (100uM) for 12h to induce endothelial cell senescence, and UFMyation-modified inhibitor compound 8.5 (20uM) or YAP inhibitor verteporfin (0.5uM) were given at the same time. Senescent endothelial cells were detected using a cell senescence β-galactosidase staining kit (Beyotime Biotechnology, Cat#C0602) according to the instructions: cells were fixed in β-galactosidase fixative at room temperature for 15 minutes, then washed with PBS and incubated with staining buffer at 37°C overnight. 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 can be seen that inhibiting UFMyation modification can reduce the protein level of YAP and thus improve endothelial cell senescence.

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

[0052] In order to further verify the role of UFMyation modification of YAP in vascular aging, in this example, old 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, the vascular aging-related indicators of the mice including vascular compliance, pulse wave velocity (PWV) and vascular collagen deposition were analyzed to evaluate vascular aging.

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

[0054] All animal care and experimental procedures were performed in accordance with the guidelines of the International Laboratory Animal Care Accreditation. The animal experimental protocol was approved by the Animal Ethics Committee of Hangzhou Normal University. Mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Male C57BL / 6J mice (24 months) were intraperitoneally injected with PBS or compound 8.5 (0.25 mg / kg / d). Three weeks later, the Vevo3100 imaging system was used to evaluate pulse wave velocity (PWV) and vascular strain. The specific steps are as follows: 1) After the mouse is anesthetized with isoflurane, the hair on the neck and abdomen of the mouse is removed and the mouse is fixed on the operating table, and the maintenance amount of isoflurane in the anesthesia device is adjusted to 1.0% to 1.5%; 2) The ultrasound probe is directed toward the mouse's lower jaw, and the left carotid artery and aortic arch images are displayed in B-mode ultrasound mode, and the blood flow waves of the left common carotid, the left carotid bifurcation, and the bifurcation of the left common carotid aorta are recorded in PW mode, and then the diameter and thickness of the left carotid blood vessels are recorded in M-mode ultrasound mode; 3) The probe is placed on the mouse's abdomen, and the bifurcation of the abdominal aorta and its renal arteries is displayed in B-mode ultrasound mode, and the blood flow waves at the renal bifurcation of the abdominal aorta are recorded in PW mode. The aortic diameter and vascular wall thickness are recorded in M-mode ultrasound mode; 4) △T is calculated based on the conduction time T1 and T2 measured by the blood flow wave, and the distance D of the blood flow wave measurement point is measured, and PWV=D / △T is calculated. Each of the above data is measured 3-5 times; vascular compliance=(Ds-Dd) / Dd is calculated based on the M-mode ultrasound image.

[0055] 5.2 Assessment of vascular collagen deposition in mouse aorta cryosections Figure 5 in the CD).

[0056] Mouse aorta was fixed with 4% paraformaldehyde at room temperature for 20 min, embedded in OCT, and then cut into 5 μm thick slices for Masson staining and HE staining, using the modified Masson trichrome staining kit (Cat#G1346) and hematoxylin and eosin (HE) staining kit (Cat#G1120) purchased from Solebow, respectively. The specific steps were carried out strictly according to the instructions.

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

[0058] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, 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 regarded as the protection scope of the present invention.

Claims

1. Application of UFMylation-modified YAP as a target in the preparation of anti-vascular aging drugs.

2. Application of UFMylation-modified YAP as a target in the preparation of anti-aging drugs for vascular endothelial cells.

3. The use according to claim 1 or 2, characterized in that: The drug reduces the amount of UFMylation-modified YAP and lowers the protein level of YAP, thereby producing an anti-vascular aging effect.

4. The use according to claim 3, characterized in that: The method of reducing the amount of UFMylation-modified YAP includes at least one of plasmid transfection, viral infection, gene editing, transcription level, translation level, and post-translational modification level.

5. The use according to claim 1 or 2, characterized in that: The administration method of the drug includes at least one of oral administration, intravenous injection and intraperitoneal injection.

6. The use according to claim 1 or 2, characterized in that: The drug is in any pharmaceutically acceptable dosage form, including at least one of tablets, capsules, injections, granules, suspensions and solutions.

7. Use of UFMylation-modified YAP as a target in the preparation of a reagent for detecting vascular aging, characterized in that: The reagent is used to detect the expression amount of UFMylation-modified YAP in tissue cells or body fluids.

8. Use of UFMylation-modified YAP as a target in the preparation of a reagent for detecting vascular endothelial cell senescence, characterized in that: The reagent is used to detect the expression amount of UFMylation-modified YAP in tissue cells or body fluids.

9. The use according to claim 7 or 8, characterized in that: The tissue cells include at least one of blood vessels, liver, brain, heart, and kidney.

10. The use according to claim 7 or 8, characterized in that: The body fluid includes at least one of serum, urine, cerebrospinal fluid, and sweat.

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