Deacetylated Septin4 protein and its pharmaceutical use

Abstract

The present invention provides a deacetylated modified Septin4 protein and its pharmaceutical use. The present invention provides a deacetylated modified Septin4 protein or an active fragment thereof. Compared with the wild-type Septin4 protein, the deacetylated modified Septin4 protein or its active fragment comprises an amino acid sequence as shown in SEQ ID NO:1, wherein the lysine is deacetylated. The present invention also provides the use of the deacetylated modified Septin4 protein or its active fragment or the pharmaceutical composition in the preparation of a drug for preventing or treating hypertensive renal injury. The applicant has combined the post-translational modification of acetylated proteins with hypertensive renal injury for the first time, providing new ideas and research directions for designing hypertensive kidney treatment plans and targeted drugs.

Description

Technical Field

[0001] The present application relates to the field of medicines for preventing or treating hypertensive renal injury. Specifically, the present application relates to a deacetylated modified Septin 4 protein or an active fragment thereof, and the present application also relates to a pharmaceutical composition comprising the deacetylated modified Septin 4 protein or an active fragment thereof, and the use of the deacetylated modified Septin 4 protein or an active fragment thereof for preventing or treating hypertensive renal injury. Background Art

[0002] Hypertension is one of the most common cardiovascular diseases and is a major public health problem worldwide. Structural and functional changes in arteries occur during aging, which may be caused by hypertension and lead to cardiovascular events and end-stage renal disease. Hypertension is a major risk factor for a rapid decline in glomerular filtration rate (GFR) and the development of chronic kidney disease (CKD) in patients with kidney disease. Untreated hypertension can damage the kidneys by causing glomerulosclerosis and arteriolar sclerosis.

[0003] Currently, the main drugs for hypertensive nephropathy include RAAS inhibitors, angiotensin-converting enzyme (ACE) inhibitors, and angiotensin II receptor blockers (ARBs), which mainly affect the control of blood pressure (BP). However, strict BP control does not delay the onset of end-stage renal disease (ESRD) and significant deterioration of renal function. Therefore, it is necessary to further study the molecular mechanism of hypertensive nephropathy in order to develop new targeted drugs and clinical treatment methods.

[0004] Septin4 belongs to the Septins GTP-binding protein family and is associated with cell division, apoptosis, vesicle transport and other cellular processes. Septin4_vi2, as a pro-apoptotic protein, participates in various apoptotic processes. Fas, etoposide, staurosporine and arabinoside have the ability to induce apoptosis by binding to Septin4 / XIAP (X-linked inhibitor of apoptosis protein). These stimuli can increase the expression level of Septin4, thereby promoting cell apoptosis. Septin4 can participate in various diseases by inducing cell apoptosis, such as regulating the survival of stem cells that are crucial for intestinal homeostasis and regeneration. Therefore, Septin4 is currently considered to be an important marker protein for organ damage.

[0005] However, it is still unclear whether Septin 4 plays a role in hypertensive renal injury. There is no research on the correlation between Septin 4 and hypertensive renal injury in the prior art. Summary of the invention

[0006] The technical solution of this application is proposed based on the following research:

[0007] The applicant discovered that the new substrate of SIRT2 is the apoptosis-related factor Septin4. The applicant first confirmed that the respective acetyltransferase / deacetylase activities of CREB binding protein (CBP) / SIRT2 regulate the acetylation of Septin4-Lys174. Deacetylation of Septin4 K174 can rescue renal podocyte damage in Septin4 knockdown cells. These findings indicate that the novel SIRT2-regulated deacetylation pathway mediates the function of Septin4 in the occurrence and development of hypertensive renal damage. In addition, the applicant also found that deacetylation of Septin4 K174 by SIRT2 plays an important role in hypertensive renal damage. The findings of the present application provide a new research direction for the treatment and prevention of hypertensive nephropathy.

[0008] Therefore, the purpose of this application is achieved through the following technical solutions:

[0009] The first aspect of the present invention provides a deacetylated modified Septin 4 protein or an active fragment thereof. Compared with the wild-type Septin 4 protein, the deacetylated modified Septin 4 protein or an active fragment thereof comprises an amino acid sequence as shown in SEQ ID NO: 1, wherein the lysine is deacetylated.

[0010] SEQ ID NO:1

[0011] MDRSLGWQGNSVPEDRTEAGIKRFLEDTTDDGELSKFVKDFSGNASCHP PEAKTWASRPQVPEPRPQAPDLYDDDLEFRPPSRPQSSDNQQYFCAPAPLSPSARPRSPWGKLDPYDSSEDDKEYVGFATLPNQVHRKSVKKGFDFTLMVAGESGL GKSTLVNSLFLTDLYRDRKLLGAEERIMQTVEITKHAVDIEEKGVRLRLTIVDT PGFGDAVNNTECWKPVAEYIDQQFEQYFRDESGLNRKNIQDNRVHCCLYFISPFGHGLRPLDVEFMKALHQRVNIVPILAKADTLTPPEVDHKKRKIREEIEHFGIK IYQFPDCDSDEDEDFKLQDQALKESIPFAVIGSNTVVEARGRRVRGRLYPWGIVEVENPGHCDFVKLRTMLVRTHMQDLKDVTRETHYENYRAQCIQSMTRLVVK ERNRNKLTRESGTDFPIPAVPPGTDPETEKLIREKDEELRRMQEMLHKIQKQM KENY

[0012] According to the deacetylated Septin4 protein or its active fragment described in the present invention, the lysine deacetylation is achieved by deacetylation modification using lysine deacetylase (KDACs).

[0013] The second aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a deacetylated Septin4 protein or an active fragment thereof.

[0014] According to the pharmaceutical composition of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable diluent, excipient and / or carrier.

[0015] The third aspect of the present invention provides a preparation, wherein the preparation deacetylates the Septin 4 protein, and the deacetylated Septin 4 protein or its active fragment comprises the amino acid sequence shown in SEQ ID NO: 1, wherein the lysine is deacetylated.

[0016] The preparation according to the present invention, wherein the preparation comprises lysine deacetylase.

[0017] The fourth aspect of the present invention provides the use of the deacetylated Septin4 protein or its active fragment in the preparation of a drug for preventing or treating hypertensive renal injury.

[0018] According to the use of the present invention, the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.

[0019] The fifth aspect of the present invention provides use of the pharmaceutical composition or preparation in the preparation of a medicament for preventing or treating hypertensive renal injury.

[0020] According to the use of the present invention, the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.

[0021] Compared with the prior art, the present application has the following beneficial effects: the applicant combines the post-translational modification of acetylated proteins with hypertensive kidney damage for the first time, providing new ideas and research directions for the design of hypertensive kidney treatment plans and targeted drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The following describes the implementation scheme of the present application in detail with reference to the accompanying drawings, wherein:

[0023] Figure 1 SIRT2 is shown to be involved in angiotensin II (AngII)-induced renal podocyte injury;

[0024] Among them, (A) Clusters of Sirtuin protein expression profiles in the hearts of mice infused with saline or Ang II on day 14. (B, D) The expression level of SIRT2 in the renal podocytes of mice was measured after 48 days of stimulation with different AngII concentrations. (C, E) Quantitative data are mean ± SD, *P < 0.05, **P < 0.01; *** P < 0.001. (F) Identification of interacting proteins of SIRT2 by mass spectrometry. (G) Cell lysates were immunoprecipitated with anti-SIRT2 antibodies and then immunoblotted with Septin4 antibodies. (H) Transfection of Septin4 plasmids carrying the Flag was performed, and the total lysate was immunoprecipitated (IP) with anti-Flag antibodies and detected with SIRT2 antibodies. (I) PLA traversal was performed to determine the interaction between SIRT2 and Septin4. The arrow indicates the presence of interaction.

[0025] Figure 2 Shown is SIRT2 binding to the GTPase domain of Septin4, which is targeted by SIRT2-dependent deacetylation of lysine 174

[0026] Among them, (A) full-length Flag-tagged-Septin4 or four truncated Flag-Septin4 plasmids were transfected. Total lysates were subjected to IP with anti-Flag antibody and western blotted with SIRT2 antibody. (B) Septin4 contains three functional domains, including N-terminus, C-terminus and GTPase domain. (C) Cells treated with TSA (0.5μM, 16h) and NAM (5mM, 4h) were immunoprecipitated with acetylation antibody and detected with Septin4 antibody. (D) Endogenous Septin4 interacts with endogenous CBP. E Flag-tagged CBP, P300, p300 / CBP-associated factor (PCAF) or Myc-tagged GCN5 were overexpressed, and Septin4 acetylation was immunoprecipitated with anti-acetylated lysine antibody (Ac-K) and detected with Septin4 antibody. (F) SIRT2WT (wild type) or H187YQ167A (Mut, mutant) tagged with Flag was overexpressed. Septin4 acetylation was immunoprecipitated with an anti-acetylated lysine antibody (Ac-K) and detected with a Septin4 antibody. (G) Normal control and shSIRT2 cells treated with or without AGK2 (20 μM, 24 hours) were immunoprecipitated with an acetylated antibody and detected with a Septin4 antibody. (H) CBP labeled with a tag was co-transfected with Septin4 WT or K174R (Mut) labeled with a tag. Acetylation of Septin4 was detected by IP using an anti-acetylated lysine antibody (Ac-K). (I) Myc-tagged SIRT2 was co-transfected with Flag-tagged Septin4 WT or K174R (Mut). Acetylation of Septin4 was detected using an anti-acetylated lysine antibody (Ac-K).

[0027] Figure 3 SIRT2 was shown to alleviate AngII-induced renal podocyte injury by deacetylating Septin4.

[0028] Among them, (A) Myc-tagged SIRT2 plasmid was transfected with or without AngII. The total lysate was IPed with anti-Myc antibody and detected with Septin4 antibody. (B) Myc-tagged SIRT2 plasmid was transfected into renal podocyte-shSIRT2 cells with or without AngII. The total lysate was IPed with acetylated antibody and detected with Septin4 antibody. (C) The SIRT2 plasmid labeled with the tag was transfected into renal podocyte-shSIRT2 cells. The cells were treated with or without 10-5mol / LAngII for 48 hours. Cleaved-PARP1 was evaluated by westernbolt. (D) Quantitative data mean ± SD, *P<0.05, **P<0.01. (E) The viability of renal podocytes was measured by CCK8 assay. Data are expressed as mean ± SD, *P<0.05, **P<0.01. (G) Renal podocytes were stained with anti-phalloidin-FITC antibody. The cell nuclei were stained with DAPI. Scale bar, 50 μm. (F) Data are expressed as mean ± SD, **P < 0.01; ***P < 0.001.

[0029] Figure 4 Septin4, shown to be involved in AngII-induced renal podocyte injury, is dependent on Septin4-K174, which is regulated by SIRT2.

[0030] (A) Septin4 WT or K174R plasmids with a Flag tag were transfected into renal podocytes-shSeptin4 cells. Renal podocytes were treated with NaCl or 10-5mol / LAngII for 48 hours. Cleaved-PARP1 and Caspase3 were evaluated by western bolt. (B) Quantitative data, mean ± SD, ***P < 0.001. (C) Renal podocytes were stained with anti-phalloidin-FITC antibody. Cell nuclei were stained with DAPI. Scale bar, 50 μm. (E) Data represent mean ± SD, *P < 0.05. (D) The viability of renal podocytes was measured by CCK8 assay. Data are represented as mean ± SD, *P < 0.05, **P < 0.01.

[0031] Figure 5 It was shown that SIRT2 knockdown mice showed high acetylation level of Septin4 and significantly aggravated AngII-induced hypertensive renal injury.

[0032] (A) Total protein was obtained from kidney tissues of SIRT2-WT and SIRT2- / - mice 14 days after infusion of AngII (1.5 mg / kg / min). (A, D) Western blotting was performed to evaluate the expression levels of SIRT2, Cleaved-PARP1 and Cleaved-Caspase3. (EF) Quantification of Western blot data is shown as mean ± SD (***###P < 0.001, each group of mice, n = 7). (B) Total lysates of kidney tissue were IPed with Septin4 antibody and Western blotted with SIRT2 antibody. (C) Total lysates of kidney tissue were IPed with acetylation antibody and Western blotted with Septin4 antibody. (G) HE staining was performed to evaluate the degree of glomerular edema. Arrow, tubular edema. Scale bar, 20 μm. (I) Data are expressed as mean ± SD, (***P < 0.001, each group of mice, n = 7). (H) AZAN staining was performed to evaluate the content of extracellular matrix secretion in the glomerulus. Arrow, extracellular matrix of the glomerulus (blue). Scale bar, 20 μm. (J) Data are expressed as mean ± SD, (***###P<0.001, each group of mice, n=7). (K) PAS staining was performed to evaluate glomerulosclerosis, arrow, segmental sclerosis of the glomerulus. Scale bar, 20 μm. (M) Data are expressed as mean ± SD, (***###P<0.001, each group of mice, n=7). (L) Mass staining was performed to evaluate the degree of glomerular fibrosis. Arrow, glomerular fibrosis. Scale bar, 20 μm. (N) Data are expressed as mean ± SD, (***###P<0.001, each group of mice, n=7).

[0033] Figure 6 It is shown that SIRT2 transgenic (super) mice exhibited low acetylation levels of Septin4 and significantly alleviated AngII-induced hypertensive renal injury.

[0034] (A, C) Total protein was obtained from kidney tissues of WT and SIRT2 transgenic mice 14 days after AngII (1.5 mg / kg / min) infusion. Western-blot was performed to evaluate the expression levels of Flag-tagged SIRT2, Cleaved-PARP1 and Cleaved-Caspase3. (D) Quantification of Western blot data is shown as mean ± SD (***###P < 0.001, mice in each group, n = 7). (B) Total lysates of kidney tissue were IPed with acetylated antibodies and western blotted with Septin4 antibodies. (E) HE staining was performed to assess the degree of glomerular edema. Arrows, tubular edema. Scale bar, 20 μm. (G) Data are expressed as mean ± SD, (***P < 0.001, mice in each group, n = 7). (F) AZAN staining was performed to assess the content of extracellular matrix secretion in glomeruli. Arrows, extracellular matrix of glomeruli (blue). Scale bar, 20 μm. (H) Data are expressed as mean ± SD, (***###P<0.001, each group of mice, n=7). (I) PAS staining was performed to evaluate glomerular sclerosis. Arrows, glomerular segmental sclerosis (lavender). Scale bar, 20 μm. (K) Data are expressed as mean ± SD, (***###P<0.001, each group of mice, n=7). (J) Mass staining was performed to evaluate the degree of glomerular fibrosis. Arrows, glomerular fibrosis (blue). Scale bar, 20 μm. (L) Data are expressed as mean ± SD, (***###P<0.001, each group of mice, n=7). DETAILED DESCRIPTION

[0035] The present application is further described below in conjunction with the accompanying drawings and examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0036] Flag-P300, Flag-CBP, and Myc-GCN5 plasmids were obtained from Fudan University, Shanghai;

[0037] Flag-PCAF plasmid was obtained from Shenzhen University;

[0038] SIRT2 wild-type and SIRT2 knockout (SIRT2- / -) mice with exon 5–8 deletion were obtained from Shanghai Bio-Model Biological Science and Technology Development Corporation;

[0039] SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice were purchased from Shanghai Biological Model Biological Science and Technology Development Corporation;

[0040] Example 1 Septin4-K174R reduces AngII-induced vascular endothelial cell injury, apoptosis and ROS accumulation.

[0041] 1. Materials and Methods

[0042] 1.1 SIRT2 knockout and transgenic mice

[0043] SIRT2 wild-type and SIRT2 knockout (SIRT2- / -) mice with exon 5-8 deletion were obtained from Professor Chuxia Deng (University of Macau). SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice were established by Shanghai Bio-Model Biological Science and Technology Development Corporation.

[0044] All animals were housed under pathogen-free conditions. All experiments were performed using 8-10 week old male mice. In the NaCl and AngII infusion model (A9525, sigma, USA), SIRT2 wild-type and SIRT2 knockout (SIRT2- / -) mice (each group, N=7), as well as SIRT2 wild-type and SIRT2 transgenic (super) mice (each group, N=7) were implanted with osmotic mini pumps according to the manufacturer's instructions (AlZET osmotic pumps, DURECT Corporation, Cupertino, CA). An incision was made in the middle of the shoulder and an osmotic mini pump was implanted. NaCl or AngII (1.5 mg / kg / day) was injected into mice via a mini pump for 14 days (Alzet, 2002 model). Blood pressure was measured daily by the tail cuff method. SIRT2 knockout and SIRT2 transgenic efficiency were measured by Western blotting at the study endpoint.

[0045] All animal treatments were in compliance with the animal welfare regulations of China Medical University. The animal study protocol was approved by the Animal Discipline Committee of China Medical University.

[0046] 1.2 Immunohistochemistry (IHC) analysis

[0047] Mouse kidney tissues were immersed in 4% (V / V) paraformaldehyde for 4 h and then transferred to 70% (V / V) ethanol. The individual tissues were placed in a processing box, dehydrated through a continuous alcohol gradient, and then embedded in paraffin blocks. Kidney tissue sections with a thickness of 5 μm were cut, dewaxed with xylene, and rehydrated by immersion in decreasing concentrations of ethanol and then washed in PBS. The sections were then stained with hematoxylin and eosin (HE), Azan Trichrome kit (AZT-K-250, American Biognost, USA), PAS (G1285, Solarbio, China) or Massion's Trichrome staining kit (G1340, Solarbio, China) according to the operating manual. After staining, the sections were dehydrated in increasing concentrations of ethanol and xylene.

[0048] 1.3 Cell culture

[0049] Human podocytes were purchased from Bena Culture Collection (Beijing, China) and cultured in serum-free McCoy's 5A medium (modified) with L-glutamine (Biological Industries). HEK293T cells were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and cultured in high-glucose Dulbecco's modified Eagle's medium (Israel Bioindustry, 01-052-1). The cells were incubated with 10% fetal bovine serum (FBS) (CLARK, Australia), penicillin (100 U) and streptomycin (100 μg / ml) in 5% CO. 2 The cells were cultured at 37°C in a humidified atmosphere.

[0050] 1.4 Antibodies and Reagents

[0051] The antibodies used in this application include polyclonal rabbit anti-SIRT2 (S8447, Sigma), polyclonal goat anti-Septin4 (ab166788, abcam), monoclonal mouse anti-Flag (GNI4110-FG, GNI, Japan), monoclonal mouse anti-Myc (immunoprecipitation: 2276S, Cell Signaling; immunoblotting: GNI4110-MC, GNI, Japan), monoclonal mouse anti-GAPDH (10494-1-AP, Proteintech), polyclonal rabbit anti-CBP (7389S, Cell Signaling), anti-acetylated-lysine (9441S, Cell Signaling), polyclonal rabbit anti-cleaved PARP (5625S, Cell Signaling), polyclonal rabbit anti-cleaved Caspase3 (19677-1-AP, Proteintech).

[0052] AngII (A9525) was purchased from Sigma, AGK2 (B7323) was purchased from Apexbio, nicotinamide (NAM, S1899) and trichostatin A (TSA, S1045) were purchased from Selleck. PI (propidium iodide, ST511) was from Beyotime. Cell counting kit 8 (CCK8, B34304, Bimake, USA) was from Selleck. Phalloidin-FITC (AAT-23102) was from Bioquest.

[0053] 1.5 Plasmid construction and transfection

[0054] Human full-length Septin4 (Gene ID: 5414) and Septin4 carrying K174R mutation (GeneChem, China) were cloned into 3Flag GV141 vector, and four truncated Septin4 plasmids were constructed, which contained different domains: Septin4 N-terminal domain with flag tag; Septin4 C-terminal domain with flag tag; Septin4 C-terminal and catalytic GTPase domain with flag tag. Full-length human SIRT2 (Gene ID: 22933) was cloned into pCMV-Myc-N (TAKARA, Japan) and pcDNA3.1-flag / HA. SIRT2 H187Y, Q167A mutant plasmids were generated using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, CA, USA). Flag-P300, Flag-CBP and Myc-GCN5 were obtained from Qunying Lei (Shanghai Medical University, China). Flag-PCAF was obtained from Weiguo Zhu (Shenzhen University, Shenzhen, China). Plasmid transfection was performed using Lipofectamine 3000 (Invitrogen, California, USA) according to the manufacturer's instructions. Cells were harvested 36-48 hours after transfection.

[0055] 1.6 Plasmid construction, antibodies and reagents

[0056] SIRT2 and Septin4 shRNA lentiviruses were purchased from GeneChem. Construction of stable gene knockdown cell lines was performed. In brief, lentiviruses were collected from HEK293T cells according to the manufacturer's instructions. Lentivirus particles were mixed with 5×PEG-itTMSolution (System Biosciences, USA). Freshly plated cells in 6-well culture plates were infected with lentiviruses. Stable cell lines were selected with puromycin (10 μg / ml) for 7 days. Finally, the infection efficiency of target cells was confirmed by Western blotting.

[0057] SEQ ID NO:2 shSirt2 target sequence 22296: TGCTCATCAACAAGGAGAA

[0058] SEQ ID NO:3 shSirt2 target sequence 22297: TAAGCTGGATGAAAAAAGAGAA

[0059] SEQ ID NO:4 shSirt2 target sequence 22298: CAACCATCTGTCACTACTT

[0060] SEQ ID NO:5 shSeptin4 target sequence 72648: ccTAAAGGAAAGCATCCCATT

[0061] SEQ ID NO:6 shSeptin4 target sequence 72649: ccTAAAGGAAAGCATCCCATT

[0062] SEQ ID NO:7 shSeptin4 target sequence 72650: ccTAAAGGAAAGCATCCCATT

[0063] 1.7 Immunoprecipitation and immunoblotting

[0064] For acetylated immunoprecipitation, cells were washed three times with phosphate-buffered saline (PBS) and lysed with marker lysis buffer (137 mM NaCl, 10 mM NaF, 50 mM Tris-HCl (pH 7.6), 1 mM EDTA, 0.1 mM Na 3 VO 4 , 10% glycerol, 1% Nonidet P-40 (NP-40) and 1mM PMSF (protease inhibitor)). In addition, 5μM TSA and 20mM NAM were added to the cell lysis buffer. The cell lysate was incubated with anti-FlagAffinity Gel (B23102, biotool, USA) at 4°C for 12 hours, or with appropriate antibodies at 4°C for 3 hours, and then incubated with ProteinA / G immunoprecipitation magnetic beads (B23202, biotool) at 4°C for 12 hours. The protein-antibody complex was then washed 3 times with cold label lysis buffer at 4°C and eluted with SDS loading.

[0065] 1.8 Acetylation assay

[0066] Cells were treated with TSA (5 μM, 16 h) and NAM (5 mM, 4 h) and then harvested and lysed for immunoprecipitation and western blot analysis. In addition, to further investigate SIRT2-induced Septin4 deacetylation, cells were incubated with SIRT2-specific inhibitor AGK2 (10 μM) for 24 h.

[0067] 1.9 Cell proliferation analysis

[0068] The cells were plated at 3 × 10 3The cells were seeded in triplicate in a 96-well plate at a density of 10 cells / well. Basic McCoy's 5A medium (90 μl) and CCK8 staining solution (10 μl) were added to the cells for 2 hours at 37° C. The absorbance at 450 nm was measured using an absorbance reader (TECAN, Switzerland).

[0069] 1.10 FITC-phalloidin assay

[0070] The cells were transiently transfected with plasmids for 24 h. The next day, cells were plated at 3 × 10 4 The density of cells / well was seeded into 24-well plates. After 24 hours, the cells were induced by AngII at an appropriate concentration for 48 hours. The medium was then removed and the cells were washed twice with PBS preheated at 37°C according to the instructions of Bioquest and measured using phalloidin-FITC (AAT-23102). Cells were imaged using a fluorescence microscope (Olympus).

[0071] 1.11 PLA analysis

[0072] according to PLA analysis was performed using the Insitu–Fluorescence Manual (DUO9210-1-1KT, Sigma-Aldrich). Cells on slides were fixed with 4% PFA for 15 min. Subsequently, slides were permeabilized with Triton X-100 for 15 min. Blocking solution was added to each slide, and the slides were incubated in a preheated humidity chamber at 37°C for 30 min. Slides with diluted primary antibodies were incubated overnight at 4°C. Primary antibody solution was aspirated from the slides and washed in 1x WashBufferA. PLA probe solution was added and incubated in a preheated humidity chamber at 37°C for 1 h. PLA probe solution was removed from the slides and washed with 1x Wash Buffer A. Ligation solution with ligase was added and incubated in a preheated humidity chamber at 37°C for 30 min. Ligation solution was aspirated from the slides and washed in 1x Wash Buffer A. Amplification-polymerase solution was added to each sample and incubated at 37°C for 100 minutes in a preheated humidity chamber. Finally, the amplification-polymerase solution was knocked out from the slide and washed in 1x wash buffer B, followed by washing in 0.01x wash buffer B. Duolink Insitu Mounting Medium with DAPI was mounted on the slide with a cover glass. Cells were imaged using a fluorescence microscope (Olympus).

[0073] 1.12 Statistical analysis

[0074] Data are expressed as mean ± standard deviation (SD). Homogeneity of variance was assessed by F test (group pairs). Shapiro-Wilk test was performed to assess normality of data. Differences between groups were assessed using two-tailed Student's t test for continuous variables (expressed as mean ± SD). One-way ANOVA, two-way ANOVA and indicative nonparametric tests were performed to compare differences between multiple groups. P values ​​were adjusted for multiple comparisons if applicable. All statistical analyses were performed using SPSS version 22.0 software (SPSS Inc, Chicago, IL, USA), and P < 0.05 was considered statistically significant.

[0075] 2. Results and Analysis

[0076] 2.1 SIRT2 participates in AngII-induced renal podocytes by interacting with damage-associated protein-Septin4 damage.

[0077] To determine the expression profile of Sirtuins subunits in damaged kidneys, the applicants attenuated hypertensive renal injury in wild-type (WT) mice by Ang II infusion and verified the Sirtuins subunit expression levels obtained by iTRAQ / TMT / label free analysis. The expression levels of Sirtuins subunit proteins were further quantified by LC-PRMMS analysis performed at Shanghai Applied Protein Technology Co., Ltd. The applicants found that among the seven Sirtuins subunit proteins, SIRT2 was the most upregulated in injured kidneys 14 days after Ang II infusion ( Figure 1 A). The results suggest that SIRT2 plays an important role in hypertensive renal injury.

[0078] To further confirm the role of SIRT2 in hypertensive renal injury, different concentrations of AngII were used to induce renal podocyte injury in human podocytes in vitro ( Figure 1 B). The applicant found that the expression of SIRT2 gradually increased in this concentration gradient ( Figure 1 B, D). Consistent with previous results, SIRT2 was also highly expressed in Ang II-induced mice ( Figure 1 C, E).

[0079] To further explore the mechanism of SIRT2 in hypertensive renal injury, the applicant used mass spectrometry ( Figure 1F) Potential protein-interacting molecules were identified. In addition to the known target proteins that are dependent on SIRT2 for deacetylation, the applicant also focused on the Septin4 protein associated with apoptosis. First, the applicant studied the protein interaction between endogenous SIRT2 and Septin4 by co-immunoprecipitation (Co-IP) (Figure 1G). In addition, the SIRT2-Septin4 interaction was also confirmed by exogenous overexpression of Flag-tagged Septin4 ( Figure 1 Next, Flag-tagged-Septin4 was transfected into podocytes, and the interaction between Flag-tagged-Septin4 and SIRT2 was confirmed by PLA analysis ( Figure 1 I).

[0080] Therefore, the above results confirm that Septin4 is a new interacting protein of SIRT2, and SIRT2 may be involved in AngII-induced renal podocyte injury by interacting with Septin4.

[0081] 2.2 SIRT2 binds to the GTPase domain of Septin4, which in turn binds to the GTPase domain of Septin4 Lysine 174 is a target for SIRT2-dependent deacetylation.

[0082] According to the UniProt database, Septin4 contains three functional domains, including the N-terminus, the C-terminus, and the GTPase domain ( Figure 2 B). Using endogenous SIRT2 and full-length Flag-tagged-Septin4 or various truncated Flag-Septin4 plasmids, the applicants demonstrated that SIRT2 binds to the GTPase domain of Septin4 ( Figure 2 A). These data indicate that SIRT2 interacts with the GTPase domain of Septin4, and AngII enhances the binding. Next, the applicant verified whether SIRT2 could regulate the acetylation activity of Septin4. The acetylation level of Septin4 increased after treatment with trichostatin A (TSA) and nicotinamide (NAM), two commonly used deacetylase inhibitors that inhibit histone deacetylases HDAC I and III and deacetylases of the Sirtuins family (Figure 2C). Next, to identify the acetyltransferase of Septin4, four acetyltransferases were transfected separately, including p300 (E1A binding protein, 300kDa), CBP, PCAF (p300 / CBP-associated factor) or GCN5 (KAT2A). The applicant found that overexpression of CBP, but not overexpression of other acetyltransferases, significantly enhanced the acetylation level of Septin4 ( Figure 2E). In addition, endogenous CBP bound to Septin4 (Figure 2D). Therefore, CBP was shown to be an acetyltransferase for Septin4. Next, to confirm that SIRT2 can deacetyl Septin4, the applicant constructed a stable SIRT2 knockdown cell line using three shRNA fragments. The applicant found that the 22297 fragment produced the best knockdown efficiency (not shown), so the applicant used normal control and shSIRT2 cells with or without 20μmol / LAGK2 (a commonly used SIRT2-specific inhibitor). Consistent with previous results, the acetylation level of Septin4 was higher in shSIRT2 cells or cells treated with AGK2 compared with normal control cells ( Figure 2 G). Next, overexpression of wild-type (WT) SIRT2 reduced endogenous Septin4 acetylation, whereas transfection of a catalytically inactive mutant of SIRT2 (H187YQ167A) had no effect ( Figure 2 F). To investigate the specific site on Septin4 that is deacetylated by SIRT2, the applicant then used site-directed mutagenesis to mutate the lysine (K) 174 acetylation site to arginine (R, non-acetylatable mutant). Wild-type (WT)-Septin4 or K174R mutant plasmids were transfected together with Flag control or Flag-CBP plasmids. Arginine substitution of K174 resulted in the disappearance of Septin4 acetylation with or without CBP, while CBP increased the deacetylation level of WT-Septin4 (Figure 2H). Similarly, arginine substitution of K174 resulted in the disappearance of Septin4 acetylation with or without SIRT2 overexpression compared to WT-Septin4 ( Figure 2 I). These findings suggest that CBP is an acetyltransferase for Septin4 K174, a target for SIRT2-dependent deacetylation.

[0083] 2.3 SIRT2 alleviates AngII-induced renal podocyte injury by deacetylating Septin4.

[0084] In order to fully understand the role of SIRT2-Septin4 signal transduction in hypertensive renal injury, the applicant confirmed that AngII caused an increase in the binding between SIRT2 and Septin4 in renal podocytes ( Figure 3 A). In addition, AngII induced a downregulation of the acetylation level of Septin4, which was increased in shSIRT2 renal podocytes, but after re-expression of SIRT2 in shSIRT2 renal podocytes, the acetylation level of Septin4 was restored ( Figure 3 B) Subsequently, the applicant used normal control and shSIRT2 renal podocytes, with or without 10 -5ShSIRT2 cells showed an increase in cleaved-PARP1 levels in response to renal podocyte injury, whereas transient re-expression of WT-SIRT2 in SIRT2-deficient renal podocytes rescued this injury ( Figure 3 CD). Consistent with these findings, cytoskeletal disassembly was more extensive in shSIRT2 renal podocytes, whereas transient re-expression of WT-SIRT2 in SIRT2-depleted renal podocytes rescued this disassembly ( Figure 3 F, G). Similar results were obtained using CCK8 analysis ( Figure 3 E) In conclusion, SIRT2 can alleviate AngII-induced renal podocyte injury, and Septin4 may be involved in the response.

[0085] 2.4 Septin4 involved in AngII-induced renal podocyte injury depends on Septin4-K174 regulated by SIRT2.

[0086] The applicant's findings indicate that SIRT2 regulates Septin4 through deacetylation of K174. However, the role of deacetylation of Septin4 by SIRT2 in hypertensive renal injury remains unclear. Therefore, the applicant constructed stable Septin4 knockdown (shSeptin4) renal podocytes using three shRNA fragments and confirmed that the knockdown efficiency of the 72650 fragment was the highest; therefore, subsequent experiments used a stable knockdown cell line. In addition, the applicant detected the effect of 10 - 5 In the knockdown renal podocytes of 1 mol / L AngII, the expression of Cleaved-PARP1 and Cleaved-Caspase3 in shSeptin4, transient re-expression of WT-Septin4 and K174R-Septin4 (in the form of simulated SIRT2 deacetylated Septin4) induced renal podocyte injury. As shown in Figure 4A-B, the levels of Cleaved-PARP1 and Cleaved-Caspase3 after WT-Septin4 re-expression were higher than those of shSeptin4, while there was no significant difference between the transient re-expression of K174R in Septin4-deficient renal podocytes and shSeptin4 renal podocytes. Consistent with previous results, the cytoskeletal collapse after WT-Septin4 re-expression in shSeptin4 renal podocytes was greater than that in shSeptin4 renal podocytes, while there was no difference between the transient re-expression of K174R-Septin4 in shSeptin4 cells and shSeptin4 renal podocytes ( Figure 4 C, E). Similar results were obtained using CCK8 analysis ( Figure 4D) In ​​summary, SIRT2 alleviates AngII-induced renal podocyte injury by deacetylation of Lys174 Septin4.

[0087] 2.5 SIRT2 knockout mice showed high acetylation levels of Septin4 and significantly aggravated AngII-induced Hypertensive renal damage.

[0088] Hypertension can lead to progressive kidney damage; in the early stages, renal volume and tubular epithelial cell swelling and glomerular mesangial matrix deposition increase. In order to investigate the role of SIRT2 in hypertensive renal injury. AngII was infused for 2 weeks using an osmotic minipump to establish a hypertensive renal injury model in SIRT2-WT and SIRT2- / -C57BL / 6 mice in vivo. The applicant found that after AngII-induced hypertensive injury, the expression of SIRT2 in SIRT2-WT kidney tissue increased significantly ( Figure 5 A, E), while SIRT2- / - mice do not express SIRT2.

[0089] In addition, the interaction between SIRT2 and Septin4 was detected by co-immunoprecipitation in hypertensive renal injury mice ( Figure 5 B) and the acetylation level of Septin4 ( Figure 5 C) As shown in the results, consistent with the results in podocytes, AngII induced an increase in their interaction, while the acetylation level of Septin4 was increased in SIRT2 knockout mice.

[0090] The applicant then evaluated whether hypertensive renal injury was accompanied by changes in the expression of injury-related proteins. Compared with the SIRT2-WT group, the levels of Cleaved-PARP1 and Cleaved-Caspase3 in the SIRT2- / - group were significantly increased ( Figure 5 D, F). Therefore, SIRT2 gene knockout mice have aggravated cell apoptosis in hypertensive renal injury. Therefore, the applicant believes that SIRT2 may be related to hypertensive renal injury in vivo. Next, the applicant evaluated the role of SIRT2 in tubular epithelial cell edema and excessive glomerular mesangial matrix by H&E staining and AZAN trichrome staining in the early stage of hypertensive injury. As expected, H&E and Azan trichrome staining showed that compared with SIRT2-WT mice, SIRT2 knockdown after AngII induction significantly aggravated the degree of tubular edema and increased the area of ​​glomerular mesangial matrix ( Figure 5 GH). Subsequently, glomerular sclerosis and renal fibrosis may occur in the late stage of renal injury. PAS and Masson staining were performed to evaluate the degree of glomerular sclerosis and renal fibrosis in SIRT2-WT and SIRT2- / - mice. Figure 5As shown in KL, after hypertensive renal injury, the segmental sclerosis and fibrosis areas in SIRT2- / - mice were larger than those in SIRT2-WT mice (P<0.001) ( Figure 5 MN). These results indicate that SIRT2 knockdown significantly aggravates glomerular sclerosis and fibrosis in advanced hypertensive renal injury.

[0091] In conclusion, SIRT2 knockdown exacerbated AngII-induced hypertensive renal injury by deacetylating Septin4.

[0092] 2.6 SIRT2 transgenic (super) mice show low acetylation levels of Septin4 and can significantly alleviate AngII Kidney damage caused by hypertension.

[0093] To further explore the role of SIRT2 in hypertensive renal injury, SIRT2 transgenic mice were used to verify the above experiments. Figure 6 As shown in A, SIRT2 transgenic (super) mice were successfully constructed. The acetylation level of Septin4 was detected in hypertensive renal injury mice by co-immunoprecipitation method ( Figure 6 B). The acetylation level of Septin4 was reduced in SIRT2 transgenic (super) mice. In addition, the amount of cleaved-PARP1 and cleaved-Caspase3 was significantly reduced in SIRT2 transgenic (super) group compared with the WT group ( Figure 6 CD). Therefore, SIRT2 transgenic (super) mice showed attenuated apoptosis in hypertensive renal injury. Subsequently, H&E and Azan trichrome staining showed that transfection of SIRT2 (super) significantly alleviated the degree of AngII-induced renal tubular edema and increased the area of ​​glomerular mesangial matrix compared with WT mice ( Figure 6 EH). After hypertensive renal injury, the segmental sclerosis and fibrosis areas in SIRT2 transgenic (super) mice were smaller than those in wild-type mice (P<0.001) ( Figure 6 IL). Therefore, SIRT2 transgenic (super) alleviated AngII-induced hypertensive renal injury. This further proves that Septin4-dependent deacetylation regulation of SIRT2 can alleviate hypertensive renal injury.

[0094] discuss

[0095] Discussion and Conclusion

[0096] The applicant's findings indicate that deacetylation of Septin4-K174 can rescue renal podocyte damage in Septin4 knockout renal podocytes. In addition, SIRT2 knockout mice showed high acetylated levels of Septin4 and significantly aggravated hypertensive renal injury caused by AngII. However, SIRT2 transgenic (super) mice had low levels of Septin4 acetylation and had the opposite effect in hypertensive renal injury caused by AngII. These observations reveal a novel SIRT2-regulated deacetylation pathway that mediates the role of Septin4 in hypertensive renal injury. In addition, deacetylation of Septin4 at K174 provides a theoretical basis for designing treatment regimens and targeted drugs.

[0097] SIRT2 is an NAD+-dependent class III histone deacetylase that plays an important role in endothelial cells and heart-related diseases. Specific inhibitors of SIRT2, AGK2, reduce H 2 O 2 induced endothelial cell toxicity. In addition, activated SIRT2 signaling alleviates DOX-induced cardiotoxicity. SIRT2-deficient mice experience spontaneous heart failure and exhibit cardiac hypertrophy, remodeling, fibrosis, and dysfunction with age. SIRT2 activation protects the heart from aging-related and isoproterenol-induced pathological cardiac hypertrophy by inhibiting NFAT transcription factors. However, there is no evidence that SIRT2 plays a role in hypertensive renal injury.

[0098] Using iTRAQ / TMT / label free analysis and LC-PRMMS analysis, the applicant discovered that SIRT2 is involved in hypertensive renal injury for the first time. Here, the applicant reports that in the early stages of hypertensive renal injury, SIRT2 knockout mice exhibited significantly aggravated tubular edema, accompanied by excessive secretion of glomerular extracellular matrix. However, SIRT2 transgenic (super) mice can alleviate hypertensive renal injury. In addition, glomerular sclerosis and renal fibrosis were significantly aggravated in the late stage. These results confirm that SIRT2 plays a protective role in hypertensive renal injury. The upregulation of SIRT2 plays an important role in adipocytes and HUVEC cells under oxidant stimulation. Similarly, in the applicant's study, SIRT2 has a role in the renal podocyte injury model. Re-expression of SIRT2 rescued cytoskeletal disintegration in SIRT2 knockdown cells. In addition, SIRT2 regulates many common substrates that depend on NAD+ deacetylase activity, including FoxO1, FoxO3 and NF-κB. SIRT2 promotes the activity of AMPK by deacetylating the upstream kinase LKB132 of AMPK, thereby protecting the heart from AngII-induced hypertrophic stimulation. The applicant discovered a new apoptosis-related protein downstream of SIRT2 Septin4. In addition, AngII significantly increased the expression of Septin4 after the loss of SIRT2. These results indicate that Septin4 may be involved in the hypertensive renal injury response.

[0099] Septin4 is currently considered an important marker protein for organ damage. ARTs (Septin4 isform2) can participate in various diseases by inducing apoptosis, for example by regulating stem cell survival in the ISC niche. In addition, Septin4 plays a vital role in apoptosis and can alleviate liver fibrosis by promoting apoptosis. However, the role of Septin4 and signal transduction SIRT2-Septin4 in hypertensive nephropathy remains unknown. The applicant confirmed that the respective acetyltransferase / deacetylase activities of CBP / SIRT2 regulate the acetylation of Septin4-Lys174. In addition, the applicant found that deacetylation of Septin4 K174 can rescue renal podocyte damage in Septin4 knockdown cells. .

[0100] In summary, the applicant has identified for the first time an acetylation-dependent regulatory mechanism that controls Septin4 function in hypertension. Septin4 deacetylation can prevent hypertensive nephropathy. The applicant's findings suggest that Septin4 may be crucial in SIRT2-mediated hypertension-related diseases, providing a potential mechanism for SIRT2 to act as a protective factor in hypertensive nephropathy. These observations further suggest the potential utility of targeting Septin4 K174 deacetylation to treat hypertensive nephropathy.

[0101] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solution of the present application rather than to limit it. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present application. Sequence Listing <110> Sun Yingxian Zhang Ying Zhang Naijin <120> Deacetylated modified Septin4 protein and its pharmaceutical use <141> 2022-03-18 <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 478 <212> PRT <213> Artificial Sequence <400> 1 Met Asp Arg Ser Leu Gly Trp Gln Gly Asn Ser Val Pro Glu Asp Arg 1 5 10 15 Thr Glu Ala Gly Ile Lys Arg Phe Leu Glu Asp Thr Thr Asp Asp Gly 20 25 30 Glu Leu Ser Lys Phe Val Lys Asp Phe Ser Gly Asn Ala Ser Cys His 35 40 45 Pro Pro Glu Ala Lys Thr Trp Ala Ser Arg Pro Gln Val Pro Glu Pro 50 55 60 Arg Pro Gln Ala Pro Asp Leu Tyr Asp Asp Asp Leu Glu Phe Arg Pro 65 70 75 80 Pro Ser Arg Pro Gln Ser Ser Asp Asn Gln Gln Tyr Phe Cys Ala Pro 85 90 95 Ala Pro Leu Ser Pro Ser Ala Arg Pro Arg Ser Pro Trp Gly Lys Leu 100 105 110 Asp Pro Tyr Asp Ser Ser Glu Asp Asp Lys Glu Tyr Val Gly Phe Ala 115 120 125 Thr Leu Pro Asn Gln Val His Arg Lys Ser Val Lys Lys Gly Phe Asp 130 135 140 Phe Thr Leu Met Val Ala Gly Glu Ser Gly Leu Gly Lys Ser Thr Leu 145 150 155 160 Val Asn Ser Leu Phe Leu Thr Asp Leu Tyr Arg Asp Arg Lys Leu Leu 165 170 175 Gly Ala Glu Glu Arg Ile Met Gln Thr Val Glu Ile Thr Lys His Ala 180 185 190 Val Asp Ile Glu Glu Lys Gly Val Arg Leu Arg Leu Thr Ile Val Asp 195 200 205 Thr Pro Gly Phe Gly Asp Ala Val Asn Asn Thr Glu Cys Trp Lys Pro 210 215 220 Val Ala Glu Tyr Ile Asp Gln Gln Phe Glu Gln Tyr Phe Arg Asp Glu 225 230 235 240 Ser Gly Leu Asn Arg Lys Asn Ile Gln Asp Asn Arg Val His Cys Cys 245 250 255 Leu Tyr Phe Ile Ser Pro Phe Gly His Gly Leu Arg Pro Leu Asp Val 260 265 270 Glu Phe Met Lys Ala Leu His Gln Arg Val Asn Ile Val Pro Ile Leu 275 280 285 Ala Lys Ala Asp Thr Leu Thr Pro Pro Glu Val Asp His Lys Lys Arg 290 295 300 Lys Ile Arg Glu Glu Ile Glu His Phe Gly Ile Lys Ile Tyr Gln Phe 305 310 315 320 Pro Asp Cys Asp Ser Asp Glu Asp Glu Asp Phe Lys Leu Gln Asp Gln 325 330 335 Ala Leu Lys Glu Ser Ile Pro Phe Ala Val Ile Gly Ser Asn Thr Val 340 345 350 Val Glu Ala Arg Gly Arg Arg Val Arg Gly Arg Leu Tyr Pro Trp Gly 355 360 365 Ile Val Glu Val Glu Asn Pro Gly His Cys Asp Phe Val Lys Leu Arg 370 375 380 Thr Met Leu Val Arg Thr His Met Gln Asp Leu Lys Asp Val Thr Arg 385 390 395 400 Glu Thr His Tyr Glu Asn Tyr Arg Ala Gln Cys Ile Gln Ser Met Thr 405 410 415 Arg Leu Val Val Lys Glu Arg Asn Arg Asn Lys Leu Thr Arg Glu Ser 420 425 430 Gly Thr Asp Phe Pro Ile Pro Ala Val Pro Pro Gly Thr Asp Pro Glu 435 440 445 Thr Glu Lys Leu Ile Arg Glu Lys Asp Glu Glu Leu Arg Arg Met Gln 450 455 460 Glu Met Leu His Lys Ile Gln Lys Gln Met Lys Glu Asn Tyr 465 470 475 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <400> 2 tgctcatcaa caaggagaa 19 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 taagctggat gaaaaaagag aa 22 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <400> 4 caaccatctg tcactactt 19 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 cctaaaggaa agcatcccat t 21 <210> 6 <211> twenty one <212> DNA <213> Artificial Sequence <400> 6 cctaaaggaa agcatcccat t 21 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 cctaaaggaa agcatcccat t 21

Claims

1. A deacetylated Septin4 protein, characterized in that: Compared with the wild-type Septin 4 protein, the amino acid sequence of the deacetylated modified Septin 4 protein is shown in SEQ ID NO: 1, wherein the lysine at position 174 is deacetylated.

2. The deacetylated Septin4 protein according to claim 1, characterized in that The lysine deacetylation is achieved by deacetylation modification of lysine deacetylases (KDACs).

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