CN114716528A

CN114716528A - Deacetylation modified Septin4 protein and pharmaceutical application thereof

Info

Publication number: CN114716528A
Application number: CN202210278988.2A
Authority: CN
Inventors: 孙英贤; 张莹; 张乃今
Original assignee: Individual
Current assignee: Individual
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-07-08
Also published as: WO2023174194A1

Abstract

The invention provides deacetylated modified Septin4 protein and pharmaceutical application thereof. The invention provides deacetylated modified Septin4 protein or an active fragment thereof, compared with wild Septin4 protein, the deacetylated modified Septin4 protein or the active fragment thereof comprises an amino acid sequence shown as SEQ ID NO:1, wherein lysine is deacetylated. The invention also provides application of the deacetylated modified Septin4 protein or the active fragment thereof or the pharmaceutical composition in preparation of a medicine for preventing or treating hypertensive renal injury. The applicant combines the post-translational modification of acetylated proteins with hypertensive renal injury for the first time, and provides a new thought and a new research direction for designing hypertensive renal treatment schemes and targeted drugs.

Description

Deacetylated modified Septin4 protein and pharmaceutical application thereof

Technical Field

The present application relates to the field of medicaments for the prevention or treatment of hypertensive renal injury. In particular, the application relates to deacetylated modified Septin4 protein or an active fragment thereof, a pharmaceutical composition containing deacetylated modified Septin4 protein or an active fragment thereof, and application of deacetylated modified Septin4 protein or an active fragment thereof in preventing or treating hypertensive renal injury.

Background

Hypertension is one of the most common cardiovascular diseases and is an important public health problem worldwide. Structural and functional changes in arteries can occur during aging, possibly due to hypertension, and lead to cardiovascular events and end-stage renal disease. Hypertension is a major risk factor for rapid decline in Glomerular Filtration Rate (GFR) and the development of Chronic Kidney Disease (CKD) in renal patients. Untreated hypertension can damage the kidneys by causing glomerulosclerosis and renal arteriosclerotic events.

Currently, the major drugs of 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, there is a need to further study the molecular mechanism of hypertensive nephropathy to develop new targeted drugs and clinical therapies.

Septin4 belongs to the Septins GTP-binding protein family and is involved in cell division, apoptosis, vesicle transport and other cellular processes. Septin4_ vi2 is used as a pro-apoptotic protein and participates in various apoptotic processes. Fas, etoposide, staurosporine and arabinoside can have the ability to induce apoptosis by binding Septin4/XIAP (X-linked apoptosis protein inhibitor). These stimuli may increase the expression level of Septin4, thereby promoting apoptosis. Septin4 can be involved in various diseases by inducing apoptosis, such as regulating stem cell survival critical to gut homeostasis and regeneration. Therefore, Septin4 is currently considered to be an important marker protein for organ damage.

However, it is not clear whether Septin4 plays a role in hypertensive renal injury. The prior art does not relate to the research of the relevance of Septin4 and hypertensive renal injury.

Disclosure of Invention

The technical scheme of the application is provided on the basis of the following research:

the applicant found that a new substrate of SIRT2 is apoptosis-related factor Septin 4. Applicants first demonstrated that the acetyltransferase/deacetylase activity of the CREB-binding protein (CBP)/SIRT2, respectively, modulates the acetylation of Septin4-Lys 174. Deacetylation of Septin 4K 174 may rescue kidney podocyte injury in Septin4 knockdown cells. These findings indicate that a novel SIRT 2-regulated deacetylation pathway mediates the function of Septin4 in the development and progression of hypertensive renal damage. In addition, applicants have also found that deacetylation of Septin 4K 174 by SIRT2 plays an important role in hypertensive renal injury. The discovery of the application provides a new research direction for the treatment and prevention of hypertensive nephropathy diseases.

Therefore, the purpose of the application is realized by the following technical scheme:

the first aspect of the invention provides deacetylated modified Septin4 protein or active fragment thereof, compared with wild-type Septin4 protein, the deacetylated modified Septin4 protein or active fragment thereof comprises an amino acid sequence shown as SEQ ID NO:1, wherein lysine is deacetylated.

SEQ ID NO:1

MDRSLGWQGNSVPEDRTEAGIKRFLEDTTDDGELSKFVKDFSGNASCHP PEAKTWASRPQVPEPRPQAPDLYDDDLEFRPPSRPQSSDNQQYFCAPAPLSPSA RPRSPWGKLDPYDSSEDDKEYVGFATLPNQVHRKSVKKGFDFTLMVAGESGL GKSTLVNSLFLTDLYRDRKLLGAEERIMQTVEITKHAVDIEEKGVRLRLTIVDT PGFGDAVNNTECWKPVAEYIDQQFEQYFRDESGLNRKNIQDNRVHCCLYFISP FGHGLRPLDVEFMKALHQRVNIVPILAKADTLTPPEVDHKKRKIREEIEHFGIK IYQFPDCDSDEDEDFKLQDQALKESIPFAVIGSNTVVEARGRRVRGRLYPWGIV EVENPGHCDFVKLRTMLVRTHMQDLKDVTRETHYENYRAQCIQSMTRLVVK ERNRNKLTRESGTDFPIPAVPPGTDPETEKLIREKDEELRRMQEMLHKIQKQM KENY

The deacetylated modified Septin4 protein or the active fragment thereof according to the invention, wherein lysine deacetylation is realized by lysine deacetylases (KDACs) deacetylation modification.

A second aspect of the invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises a deacetylated modified Septin4 protein or an active fragment thereof.

The pharmaceutical composition according to the present invention, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable diluent, excipient and/or carrier.

The third aspect of the invention provides a preparation, wherein the preparation deacetylates Septin4 protein, and the deacetylated Septin4 protein or active fragment thereof comprises an amino acid sequence shown as SEQ ID NO. 1, wherein lysine is deacetylated.

The formulation according to the invention, wherein the formulation comprises lysine deacetylase.

The fourth aspect of the invention provides an application of the deacetylated modified Septin4 protein or the active fragment thereof in preparing a medicament for preventing or treating hypertensive renal injury.

The use according to the present invention, wherein the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.

The fifth aspect of the present invention provides a use of the pharmaceutical composition or formulation for the manufacture of a medicament for preventing or treating hypertensive renal injury.

Compared with the prior art, the method has the following beneficial effects: the applicant combines the post-translational modification of acetylated proteins with hypertensive renal injury for the first time, and provides a new thought and a new research direction for designing hypertensive renal treatment schemes and targeted drugs.

Drawings

Embodiments of the present application are described in detail below with reference to the attached drawing figures, wherein:

figure 1 shows that SIRT2 is involved in angiotensin ii (angii) -induced renal podocyte injury;

wherein (A) at day 14, clusters of Sirtuin protein expression profiles in the heart of mice injected with saline or Ang II. (B, D) the expression level of SIRT2 in the kidney podocytes of mice was measured 48 days after stimulation with different AngII concentrations. (C, E) quantitative data as mean ± SD, # P <0.05, # P < 0.01; p < 0.001. (F) The interacting protein of SIRT2 was identified by mass spectrometry. (G) Cell lysates were immunoprecipitated with an anti-SIRT 2 antibody and then immunoblotted with a Septin4 antibody. (H) The labeled Septin4 plasmid was transfected and total lysates were Immunoprecipitated (IP) with anti-Flag antibody and detected with SIRT2 antibody. (I) PLA traversal was performed to determine the interaction between SIRT2 and Septin 4. The presence of an interaction is indicated at the arrow.

FIG. 2 shows that SIRT2 binds to the GTPase domain of Septin4, whereas Septin4 is the target for deacetylation by lysine 174-dependent SIRT2

Wherein, (A) transfects full-length Flag-tagged-Septin4 or four truncated Flag-Septin4 plasmids. Total lysates were IP with anti-Flag antibody and Western blotted with SIRT2 antibody. (B) The Septin4 contains three functional domains, including N-terminal, C-terminal and GTPase domain. (C) Cells were immunoprecipitated with acetylated antibody TSA (0.5. mu.M, 16h) and NAM (5mM, 4h) and detected with Septin4 antibody. (D) Endogenous Septin4 interacts with endogenous CBP. E respectively overexpresses labeled CBP, P300, P300/CBP related factor (PCAF) or Myc labeled GCN5, and immunoprecipitates Septin4 with anti-acetylated lysine antibody (Ac-K) for acetylation and detection with Septin4 antibody. (F) Either SIRT2 WT (wild type) or H187YQ167A (Mut, mutant), labeled with a marker, was overexpressed. Immunoprecipitation of Septin4 with anti-acetylated lysine antibody (Ac-K) acetylation, and detection with Septin4 antibody. (G) Normal controls and shSIRT2 cells treated with or without AGK2 (20. mu.M, 24 hours) were immunoprecipitated with acetylated antibody and detected with Septin4 antibody. (H) The tagged CBP was co-transfected with tagged Septin4 WT or K174R (Mut). 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 with an anti-acetylated lysine antibody (Ac-K).

Figure 3 shows that SIRT2 reduced angi-induced renal podocyte injury by deacetylating modified Septin 4.

Wherein (A) a Myc-tagged SIRT2 plasmid is transfected with or without AngII. The total lysate was IP with anti-Myc antibody and detected with Septin4 antibody. (B) The Myc-tagged SIRT2 plasmid was transfected into renal podocytes-shSIRT 2 cells with or without AngII. The total lysate was IP treated with acetylated antibody and detected with Septin4 antibody. (C) The SIRT2 plasmid labeled with the tag was transfected into renal podocyte-shSIRT 2 cells. Cells were treated with or without 10-5mol/LangII for 48 hours. clean-PARP 1 was evaluated using a western bolt. (D) Mean ± SD, # P <0.05, # P <0.01 were quantified. (E) Viability of renal podocytes was measured by the CCK8 assay. Data are expressed as mean ± SD, # P <0.05, # P < 0.01. (G) Renal podocytes were stained with anti-phalloidin-FITC antibody. Nuclei were stained with DAPI. Scale bar, 50 μm. (F) Data are expressed as mean ± SD,. P < 0.01; p < 0.001.

Fig. 4 shows that Septin4, which is involved in AngII-induced renal podocyte injury, is dependent on Septin4-K174, which is regulated by SIRT 2.

(A) Septin4 WT or K174R plasmid with a marker tag was transfected into renal podocyte-shSeptin 4 cells. Kidney podocytes were treated with NaCl or 10-5mol/LangII for 48 hours. Cleaved-PARP1 and Caspase3 were evaluated by western felt. (B) Data were quantified as mean ± SD,. P < 0.001. (C) staining renal podocytes with anti-phalloidin-FITC antibody. Nuclei were stained with DAPI. Scale bar, 50 μm. (E) Data represent mean ± SD,. P < 0.05. (D) Viability of renal podocytes was measured by the CCK8 assay. Data are expressed as mean ± SD, # P <0.05, # P < 0.01.

Figure 5 shows that mice knocked down for SIRT2 showed high acetylation levels of Septin4 and significantly exacerbated hypertensive renal injury caused by AngII.

(A) Total protein was obtained from SIRT2-WT and SIRT 2-/-mouse kidney tissues 14 days after AngII (1.5mg/kg/min) infusion. (A, D) Western blotting was performed to assess the expression levels of SIRT2, cleared-PARP 1 and cleared-Caspase 3. Quantification of (E-F) Western blot data is shown as mean ± SD (# #### # P <0.001, mice per group, n ═ 7). (B) Total lysates of kidney tissues were IP-stained with Septin4 antibody and Western-blotted with SIRT2 antibody. (C) Total lysates of kidney tissues were IP with acetylated antibody and western blotted with Septin4 antibody. (G) HE staining was performed to assess the degree of glomerular edema. Arrow, renal tubular edema. Scale bar, 20 μm. (I) Data are presented as mean ± SD, (. x.p <0.001, mice per group, n ═ 7). (H) AZAN staining was performed to assess the level of extracellular matrix secretion in the glomeruli. Arrow, extracellular matrix of glomeruli (blue). Scale bar, 20 μm. (J) Data are expressed as mean ± SD, (x #### P <0.001, mice per group, n ═ 7). (K) PAS staining was performed to assess glomerulosclerosis, arrowheads, segmental glomerulosclerosis. Scale bar, 20 μm. Data are expressed as mean ± SD, (# P <0.001, mice per group, n ═ 7). (L) lump staining was performed to assess the degree of glomerular fibrosis. Arrow, glomerular fibrosis. Scale bar, 20 μm. Data are expressed as mean ± SD, (# P <0.001, mice per group, N ═ 7).

Figure 6 shows SIRT2 transgenic (super) mice showing low acetylation levels of Septin4 and significantly reduced hypertensive renal injury caused by AngII.

Total protein was obtained from kidney tissue of WT and SIRT2 transgenic mice 14 days after (A, C) AngII (1.5mg/kg/min) infusion. Western-blot was performed to assess the levels of Flag-tagged SIRT2, cleared-PARP 1 and cleared-Caspase 3 expression. (D) Quantification of the Western blot data is shown as mean ± SD (# #### # P <0.001, mice per group, n ═ 7). (B) Total lysates of kidney tissues were IP with acetylated antibody and western blotted with Septin4 antibody. (E) HE staining was performed to assess the degree of glomerular edema. Arrow, renal tubular edema. Scale bar, 20 μm. (G) Data are presented as mean ± SD, (. x.p <0.001, mice per group, n ═ 7). (F) AZAN staining was performed to assess the level of extracellular matrix secretion in the glomeruli. Arrow, extracellular matrix of glomeruli (blue). Scale bar, 20 μm. (H) Data are expressed as mean ± SD, (x #### P <0.001, mice per group, n ═ 7). (I) PAS staining was performed to assess glomerular sclerosis arrow, segmental sclerosis of the glomerulus (pale purple). Scale bar, 20 μm. (K) Data are expressed as mean ± SD, (x #### P <0.001, mice per group, n ═ 7). (J) tumor staining was performed to assess the degree of glomerular fibrosis. Arrow, glomerular fibrosis (blue). Scale bar, 20 μm. Data are expressed as mean ± SD, (# P <0.001, mice per group, n ═ 7).

Detailed Description

The present application is further described below in conjunction with the following figures and examples, which should be understood to be illustrative only and not limiting.

The Flag-P300, Flag-CBP and Myc-GCN5 plasmids were obtained from Shanghai university of Compound Dan;

the Flag-PCAF plasmid is obtained from Shenzhen university;

SIRT2 wild-type and SIRT2 gene knockdown (SIRT2-/-) mice with exon 5-8 deletions were obtained from Shanghai Biomodel bioscience and technology development companies;

SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice were purchased from Shanghai Biomodel bioscience and technology development companies;

example 1 Septin4-K174R reduced AngII-induced vascular endothelial cell damage, apoptosis, and ROS accumulation.

Materials and methods

1.1SIRT2 Gene knockdown and transgenic mice

SIRT2 wild-type and SIRT2 gene knockdown (SIRT2-/-) mice with exon 5-8 deletions were obtained from professor Duncai university (Australian university) as a gift. Shanghai biological model bioscience and technology development companies established SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice.

All animals were kept pathogen free. All experiments were performed using 8-10 week old male mice. In a NaCl and AngII infusion model (a9525, sigma, usa), SIRT2 wild-type and SIRT2 gene knockdown (SIRT2-/-) mice (per group, N ═ 7), and SIRT2 wild-type and SIRT2 transgenic (super) mice (per group, N ═ 7) were implanted into osmotic minipumps according to the manufacturer's instructions (AlZET osmotic pump, DURECT Corporation, Cupertino, CA). An incision was made in the middle of the shoulder and an osmotic micropump was implanted. Mice were infused with NaCl or AngII (1.5 mg/kg/day) via a micropump for 14 days (Alzet, 2002 model). Blood pressure was measured daily by the tail sleeve method. SIRT2 gene knockdown and SIRT2 transgene efficiency were measured by western blot at the study endpoint.

All animal treatments were in compliance with the animal welfare regulations of the university of medical science, china. Animal study protocol was approved by the animal science committee of chinese medical university.

1.2 Immunohistochemistry (IHC) analysis

Mouse kidney tissue was immersed in 4% (V/V) paraformaldehyde for 4h and then transferred to 70% (V/V) ethanol. The individual tissues were placed in a processing cassette, dehydrated by a continuous alcohol gradient, and then embedded in paraffin blocks. Kidney tissue sections of 5 μm thickness were cut out, deparaffinized with xylene, rehydrated by immersion in reduced concentration of ethanol, and washed in PBS. Sections were then stained according to the protocol manual, according to Hematoxylin and Eosin (HE), Azan Trichrome kit (AZT-K-250, U.S. Biognost, usa), PAS (G1285, Solarbio, china) or version's Trichrome staining kit (G1340, Solarbio, china). After staining, sections were dehydrated in increasing concentrations of ethanol and xylene.

1.3 cell culture

Human podocytes were purchased from Bena Culture Collection (Beijing, China) and cultured with L-glutamine (Biological Industries) in serum-free McCoy's 5A medium (modified). HEK293T cells were purchased from Chinese sciencesShanghai cell institute, and cultured in high glucose Dulbecco's modified Eagle Medium (Israel Biochemical industry, 01-052-1). Cells were incubated with 10% Fetal Bovine Serum (FBS) (CLARK, Australia), penicillin (100U) and streptomycin (100. mu.g/ml) in 5% CO₂In a humidified atmosphere at 37 ℃.

1.4 antibodies and reagents

Antibodies used in this application include polyclonal rabbit anti-SIRT 2(S8447, Sigma), polyclonal goat anti-Septin 4(ab166788, abcam), monoclonal mouse anti-Flag (GNI4110-FG, GNI, Japan), monoclonal mouse anti-Myc (immunoprecipitation: 2276S, cell signaling; immunoblot: 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).

AngII (a9525) was purchased from Sigma, AGK2(B7323) from Apexbio, nicotinamide (NAM, S1899) and trichostatin a (TSA, S1045) from Selleck. PI (propidium iodide, ST511) is from Beyotime. Cell counting kit 8(CCK8, B34304, Bimake, USA) was from seleck. Phallodin-FITC (AAT-23102) was from Bioquest.

1.5 plasmid construction and transfection

The full-length human Septin4(Gene ID:5414) and Septin4 carrying the K174R mutation (GeneChem, China) were cloned into the 3Flag GV141 vector, creating four truncated Septin4 plasmids containing different domains: a Septin 4N-terminal domain with a tag label; a Septin 4C-terminal domain with a tag label; septin 4C-terminal with a tag label and catalytic GTPase domain. Full-length human SIRT2 (Gene ID:22933) was cloned into pCMV-Myc-N (Japanese TAKARA) and pcDNA3.1-flag/HA. SIRT 2H 187Y, Q167A mutant plasmids were generated using a rapid exchange site-directed mutagenesis kit (Stratagene, CA, usa). Flag-P300, Flag-CBP and Myc-GCN5 were obtained from quinying 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.

1.6 plasmid construction, antibodies and reagents

SIRT2 and Septin4 shRNA lentiviruses were purchased from GeneChem. Construction of stable gene knockdown cell lines was performed. Briefly, lentiviruses were harvested from HEK293T cells according to the manufacturer's instructions. Lentiviral particles were mixed with 5 XPEG-it solutions (System Biosciences, USA). Fresh plated cells in 6-well plates were infected with lentivirus. Stable cell lines were selected with puromycin (10. mu.g/ml) for 7 days. Finally, the infection efficiency of the target cells was confirmed by western blotting.

2shSirt2 target sequence 22296: TGCTCATCAACAAGGAGAA

3shSirt2 target sequence 22297: TAAGCTGGATGAAAAAAGAGAA

4shSirt2 target sequence 22298: CAACCATCTGTCACTACTT

5shSeptin4 target sequence 72648: ccTAAAGGAAAGCATCCCATT

6shSeptin4 target sequence 72649: ccTAAAGGAAAGCATCCCATT

7shSeptin4 target sequence 72650: ccTAAAGGAAAGCATCCCATT

1.7 immunoprecipitation and immunoblotting

For acetylation immunoprecipitation, cells were washed 3 times with Phosphate Buffer (PBS) and labeled lysis buffer (137mM NaCl, 10mM NaF, 50mM Tris-HCl (pH 7.6), 1mM EDTA, 0.1mM Na₃VO₄10% glycerol, 1% Nonidet P-40(NP-40) and 1mM PMSF (protease inhibitor)). In addition, 5. mu.MTSA and 20mM NAM were added to the cell lysis buffer. The cell lysates were incubated with anti-flag affinity Gel (B23102, biotool, USA) for 12 hours at 4 ℃ or with the appropriate antibodies for 3 hours at 4 ℃ followed by incubation with protein A/G immunoprecipitated magnetic beads (B23202) and biotool for 12 hours at 4 ℃. The protein-antibody complex was then lysed with cold labeled lysis buffer at 4 deg.CWashed 3 times and eluted with SDS load.

1.8 acetylation assay

Cells were treated with TSA (5. mu.M, 16h) and NAM (5mM, 4h), then harvested and lysed for immunoprecipitation and Western blot analysis. In addition, to further investigate SIRT 2-induced deacetylation of Septin4, cells were incubated with the SIRT 2-specific inhibitor AGK2(10 μ M) for 24 hours.

1.9 cell proliferation assay

Cells were cultured at 3X 10³The density of individual cells/well was seeded in triplicate in 96-well plates. 5A medium (90. mu.l) from Basic McCoy and a staining solution (10. mu.l) of CCK8 were added to the cells for 2 hours at 37 ℃. The absorbance at 450nm was measured using an absorbance reader (TECAN, switzerland).

1.10 FITC-phallodin assay

Cells were transiently transfected with plasmid for 24 hours. The following day, cells were plated at 3X 10⁴The density of cells/well was seeded into 24-well plates. After 24 hours, cells were induced by appropriate concentrations of AngII for 48 hours. The medium was then removed and the cells were washed twice with 37 ℃ pre-warmed PBS according to Bioquest's instructions and assayed using phalloidin-FITC (AAT-23102). Cells were imaged using a fluorescence microscope (Olympus).

1.11 PLA analysis

According to

The Insitu-Fluorescence handbook (DUO9210-1-1KT, sigma-Aldrich) was used for the PLA analysis. Cells on the slides were fixed with 4% PFA for 15 min. Subsequently, the slides were permeabilized with Triton X-100 for 15 minutes. Blocking solution was added to each slide and the slides were incubated in a pre-heated humidity chamber at 37 ℃ for 30 minutes. Slides with diluted primary antibody were incubated overnight at 4 ℃. The primary antibody solution was drawn off the slide and washed in 1x Wash buffer. Add PLA Probe solution and incubate at 37 ℃ in a preheated humidity cabinetFor 1 hour. The PLA probe solution was removed from the slide and washed with 1x wash buffer a. The ligation solution with ligase was added and incubated at 37 ℃ for 30 minutes in a pre-warmed humidity cabinet. The ligation solution was knocked out of the slide and washed in 1 × wash buffer a. The amplification-polymerase solution was added to each sample and incubated in a pre-heated humidity chamber at 37 ℃ for 100 minutes. Finally, the amplification-polymerase solution was knocked out of the slide and washed in 1x wash buffer B, followed by 0.01x wash buffer B. The Duolink Insitu sounding Medium with DAPI was mounted on a glass slide using a coverslip. Cells were imaged using a fluorescence microscope (Olympus).

1.12 statistical analysis

Data are presented as mean ± Standard Deviation (SD). Homogeneity of variance was assessed by F-test (panel). The Shapiro-Wilk test was performed to assess normality of the data. Differences between groups were assessed for continuous variables (expressed as mean ± SD) using a two-tailed student t-test. A one-way anova, an anova with both methods and an indicative nonparametric test were performed to compare the differences between the groups. If applicable, multiple comparison adjustments may be made to the P value. All statistical analyses were statistically significant using software version SPSS 22.0 (SPSS Inc, illinois, chicago, usa) with P < 0.05.

Second, results and analysis

2.1 SIRT2 participates in AngII-induced renal podocytes by interacting with the injury-associated protein Septin4 And (4) damaging.

To determine the sirtuin subunit expression profile in the damaged kidney, applicants have mitigated hypertensive kidney injury in Wild Type (WT) mice by Ang II infusion and have verified the sirtuin subunit expression levels obtained by iTRAQ/TMT/label free analysis, and LC-PRMMS analysis performed in shanghai using protein technology limited further quantitated the expression levels of sirtuin subunit proteins. Applicants found that SIRT2 was most upregulated in the injured kidney 14 days after Ang II infusion among the 7 sirtuin subunit proteins (fig. 1A). The results indicate that SIRT2 plays an important role in hypertensive renal injury.

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

To further explore the mechanism of SIRT2 in hypertensive renal injury, applicants identified potential protein-interacting molecules using mass spectrometry (fig. 1F). In addition to the known target proteins that rely on SIRT2 deacetylation, applicants also focused on the Septin4 protein associated with apoptosis. First, applicants investigated protein interactions between endogenous SIRT2 and Septin4 by Co-immunoprecipitation (Co-IP) (fig. 1G). In addition, the SIRT2-Septin4 interaction was also demonstrated by exogenous overexpression of Septin4 with Flag tag (FIG. 1H). Next, Flag-tagged-Septin4 was transfected into podocytes and the interaction between Flag-tagged-Septin4 and SIRT2 was confirmed by PLA analysis (FIG. 1I).

Thus, the above results demonstrate that Septin4 is a novel interacting protein of SIRT2, and that SIRT2 may be involved in AngII-induced renal podocyte injury by interacting with Septin 4.

2.2 SIRT2 binds to the GTPase domain of Septin4, whereas the GTPase domain of Septin4 passes through Lysine 174 serves as a target for SIRT 2-dependent deacetylation.

According to the UniProt database, Septin4 contains three functional domains, including N-terminal, C-terminal and GTPase domain (fig. 2B). Applicants demonstrated that SIRT2 binds to the GTPase domain of Septin4 using endogenous SIRT2 and full-length Flag-tagged-Septin4 or various truncated Flag-Septin4 plasmids (fig. 2A). These data indicate that SIRT2 interacts with the GTPase domain of Septin4, while AngII enhances binding. Next, the applicant verified whether SIRT2 could modulate the acetylation activity of Septin 4. Following treatment with trichostatin a (tsa) and Nicotinamide (NAM), the acetylation level of Septin4 increased, and these two commonly used deacetylase inhibitors inhibited histone deacetylases HDAC I and III and the Sirtuins family of deacetylases (fig. 2C). Next, to identify the acetyltransferase of Septin4, four acetyltransferases were transfected, including p300(E1A binding protein, 300kDa), CBP, PCAF (p300/CBP related factors) or GCN5(KAT2A), respectively. Applicants found that overexpression of CBP, but not of other acetyltransferases, significantly enhanced the acetylation level of Septin4 (fig. 2E). In addition, endogenous CBP bound to Septin4 (fig. 2D). Thus, CBP was demonstrated to be the acetyltransferase of Septin 4. Next, to confirm that SIRT2 could deacetylate Septin4, applicants constructed a stable SIRT2 knockdown cell line using three shRNA fragments. Applicants found that the 22297 fragment produced the best knockdown efficiency (not shown), and therefore, applicants used normal control and shSIRT2 cells with or without 20 μmol/lag 2 (the SIRT2 specific inhibitor commonly used). In agreement with previous results, acetylation levels of Septin4 were higher in shSIRT2 cells or cells treated with AGK2 compared to normal control cells (fig. 2G). Next, overexpression of wild-type (WT) SIRT2 reduced acetylation of endogenous Septin4, whereas transfection of a catalytically inactive mutant of SIRT2 (H187YQ167A) had no effect (fig. 2F). To investigate the specific site on Septin4 that was deacetylated by SIRT2, applicants subsequently mutated the acetylation site of lysine (K)174 to arginine (R, a non-acetylable mutant) using site-directed mutagenesis. Either wild-type (WT) -Septin4 or K174R mutant plasmids were transfected, along with Flag control or Flag-CBP plasmids. With or without CBP, the arginine substitution of K174 resulted in the disappearance of the acetylation of Septin4, while CBP increased the deacetylation level of WT-Septin4 (fig. 2H). Also, in contrast to WT-Septin4, arginine substitution of K174 resulted in the disappearance of Septin4 acetylation with or without SIRT2 overexpression (fig. 2I). These findings indicate that CBP is the acetyltransferase for Septin 4K 174 and that Septin 4K 174 is the target for SIRT 2-dependent deacetylation.

2.3 SIRT2 reduces AngII-induced renal podocyte injury by deacetylating modified Septin 4.

To fully understand the SIRT2-Septin4Role of signal transduction in hypertensive renal injury, applicants demonstrated that AngII caused increased binding between SIRT2 and Septin4 in renal podocytes (fig. 3A). Furthermore, AngII induced down-regulation of acetylation levels of Septin4, but increased in shSIRT2 kidney podocytes, but re-expression of SIRT2 in shSIRT2 kidney podocytes, with restoration of acetylation levels of Septin4 (fig. 3B). Subsequently, applicants used normal control and shSIRT2 renal podocytes, with or without 10^-5mol/LangII induced renal podocyte injury. ShSIRT2 cells showed a response to renal podocyte injury with elevated levels of cleared-PARP 1, while transient re-expression of WT-SIRT2 in SIRT 2-deficient renal podocytes rescued the injury (FIGS. 3C-D). Consistent with these findings, the breakdown of cytoskeleton in shSIRT2 kidney podocytes was more extensive, while transient re-expression of WT-SIRT2 in SIRT2 depleted kidney podocytes rescued the breakdown (fig. 3F, G). Similar results were obtained using CCK8 analysis (fig. 3E). In conclusion, SIRT2 could alleviate AngII-induced renal podocyte injury, and Septin4 may be involved in the response.

2.4 Septin4 involved in AngII-induced renal podocyte injury was dependent on Septin4-K174 regulated by SIRT 2.

Applicants' findings indicate that SIRT2 regulates the Septin4 by deacetylation of K174. However, the role of deacetylation of Septin4 by SIRT2 in hypertensive renal injury remains unclear. Therefore, the applicant constructed stable Septin4 knock-down (shSeptin4) renal podocytes using three shRNA fragments, and confirmed that the knock-down efficiency of 72650 fragment was the highest among them; therefore, subsequent experiments used stable knockdown cell lines. In addition, applicants have tested whether 10 is present or absent^- ⁵In mol/LangII knock-down renal podocyte injury is induced by transient re-expression of clear-PARP 1 and clear-Caspase 3 in shSeptin4 and WT-Septin4 and K174R-Septin4 (in the form of simulated SIRT2 deacetylated Septin 4). As shown in FIGS. 4A-B, levels of clear-PARP 1 and clear-Caspase 3 were higher than shSeptin4 after WT-Septin4 re-expression, whereas there was no significant difference between transient re-expression of K174R in Septin 4-deficient renal podocytes and shSeptin4 renal podocytesAnd (3) distinguishing. Consistent with previous results, cytoskeletal disintegration after WT-Septin4 re-expression in shSeptin4 kidney podocytes was greater than cytoskeletal disintegration in shSeptin4 kidney podocytes, while transient re-expression of K174R-Septin4 in shSeptin4 cells was not different from shSeptin4 kidney podocytes (fig. 4C, E). Similar results were obtained using CCK8 analysis (fig. 4D). Taken together, SIRT2 alleviated AngII-induced renal podocyte injury by deacetylating Lys174 Septin 4.

2.5 SIRT2 knock-out mice show high acetylation levels of Septin4 and significantly exacerbate AngII induction Hypertensive renal injury.

Hypertension can lead to progressive damage to the kidneys; in the early stages, renal volume and tubular epithelial cell swelling and mesangial matrix deposition increase. To investigate the role of SIRT2 in hypertensive renal injury. AngII was infused with an osmotic minipump for 2 weeks to establish a hypertensive renal injury model in vivo in SIRT2-WT and SIRT2-/-C57BL/6 mice. Applicants found that expression of SIRT2 in SIRT2-WT kidney tissue was significantly increased following ang ii-induced hypertension injury (fig. 5A, E), while SIRT 2-/-mice did not express SIRT 2.

In addition, the interaction between SIRT2 and Septin4 (fig. 5B) and the acetylation level of Septin4 (fig. 5C) were detected in hypertensive kidney-injured mice by co-immunoprecipitation. As shown by the results, consistent with the podocyte results, AngII induced an increase in its interaction, while the acetylation level of Septin4 was increased in SIRT2 knock-out mice.

Then, the applicant evaluated whether hypertensive renal injury was accompanied by changes in the expression of injury-associated proteins. The levels of clear-PARP 1 and clear-Caspase 3 were significantly increased in the SIRT 2-/-group compared to the SIRT2-WT group (FIG. 5D, F). Therefore, SIRT2 knock-out mice potentiate apoptosis in hypertensive renal injury. Thus, the applicant believes that SIRT2 may be associated with hypertensive renal injury in vivo. Next, applicants evaluated the role of SIRT2 in tubular epithelial edema and mesangial hyperstroma by H & E staining and AZAN trichrome staining at an early stage of hypertension injury. As expected, H & E and Azan trichrome staining showed that SIRT2 knockdown after AngII induction significantly aggravated the degree of tubular edema and increased the mesangial matrix area compared to SIRT2-WT mice (fig. 5G-H). Subsequently, glomerulosclerosis and renal fibrosis may occur in the late stages of renal injury. PAS and Massion staining were performed to assess the degree of glomerulosclerosis and renal fibrosis in SIRT2-WT and SIRT 2-/-mice. As shown in FIGS. 5K-L, both the segmental sclerosis and fibrosis regions in SIRT 2-/-mice were greater than those in SIRT2-WT mice (P <0.001) after hypertensive renal injury (FIGS. 5M-N). These results indicate that SIRT2 gene knockdown significantly aggravates glomerulosclerosis and fibrosis in advanced hypertensive renal injury.

In summary, SIRT2 gene knockdown exacerbated hypertensive renal injury caused by AngII by deacetylation modification of Septin 4.

2.6 SIRT2 transgenic (super) mice showed low acetylation level of Septin4 and could significantly alleviate AngII The induced hypertensive renal injury.

To further investigate the role of SIRT2 in hypertensive renal injury, SIRT2 transgenic mice were used to validate the above experiments. As shown in fig. 6A, SIRT2 transgenic (super) mice were successfully constructed. Acetylation levels of Septin4 were detected by co-immunoprecipitation in hypertensive kidney-injured mice (fig. 6B). Acetylation level of Septin4 was reduced in SIRT2 transgenic (super) mice. Furthermore, the SIRT2 transgenic (super) group significantly reduced the amount of cleared-PARP 1 and cleared-Caspase 3 compared to the WT group (FIGS. 6C-D). Thus, SIRT2 transgenic (super) mice showed reduced apoptosis in hypertensive kidney injury. Subsequently, H & E and Azan trichrome staining showed that transfection of SIRT2 (super) significantly reduced the degree of renovascular edema following AngII induction and increased the area of mesangial matrix compared to WT mice (fig. 6E-H). Following hypertensive renal injury, both the segmental sclerosis and fibrotic regions of SIRT2 transgenic (super) mice were smaller than wild-type mice (P <0.001) (fig. 6I-L). Thus, the SIRT2 transgene (super) reduced hypertensive kidney injury caused by AngII. This further demonstrates Septin4 dependent deacetylation regulation of SIRT2 can reduce hypertensive renal injury.

Discussion of the related Art

Discussion and conclusions

Applicants' findings indicate that deacetylation of Septin4-K174 can rescue renal podocyte injury in Septin4 knock-out renal podocytes. In addition, SIRT2 knock-out mice showed high acetylation levels of Septin4 and significantly exacerbated hypertensive renal injury caused by AngII. However, SIRT2 transgenic (super) mice had lower levels of Septin4 acetylation and had the opposite effect in hypertensive renal injury caused by AngII. These observations reveal a novel SIRT 2-regulated deacetylation pathway mediating the role of Septin4 in hypertensive renal injury. In addition, the deacetylation of Septin4 at K174 provides a theoretical basis for designing therapeutic regimens and targeted drugs.

SIRT2 is an NAD + -dependent class III histone deacetylase, playing an important role in endothelial cell and heart related diseases. Specific inhibitors SIRT2, AGK2, reduced H₂O₂Induced endothelial cytotoxicity. In addition, activated SIRT2 signaling reduces DOX-induced cardiotoxicity. SIRT2 deficient mice experience spontaneous heart failure and exhibit cardiac hypertrophy, remodeling, fibrosis and dysfunction at increasing age. SIRT2 activation can protect the heart from the effects of aging-related and isoproterenol-induced pathological myocardial hypertrophy by inhibiting NFAT transcription factors. However, there is no evidence that SIRT2 plays a role in hypertensive renal injury.

Using the iTRAQ/TMT/label free analysis and the LC-PRMMS analysis, the applicants found that SIRT2 was first implicated in hypertensive renal injury. Here, the applicant reported that SIRT2 knockout mice exhibited markedly increased tubular edema with excessive secretion of glomerular extracellular matrix at an early stage of hypertensive renal injury. However, SIRT2 transgenic (super) mice can reduce hypertensive kidney injury. In addition, glomerulosclerosis and renal fibrosis are markedly aggravated at an advanced stage. These results demonstrate that SIRT2 plays a protective role in hypertensive renal injury. Upregulation of SIRT2 plays an important role in adipocytes and HUVEC cells under oxidant stimulation. Also, in applicants' studies, SIRT2 has a role in a renal podocyte injury model. Re-expression of SIRT2 rescued cytoskeletal disassembly in SIRT2 knockdown cells. In addition, SIRT2 regulates many common substrates that depend on NAD + deacetylation activity, including FoxO1, FoxO3, and NF-. kappa.B. SIRT2 promotes AMPK activity by deacetylating LKB132, an AMPK upstream kinase, thereby protecting the heart from AngII-induced hypertrophic stimulation. The applicant found a new apoptosis-related protein downstream of SIRT2 Septin 4. In addition, AngII significantly increased the expression of Septin4 after deletion of SIRT 2. These results indicate that Septin4 may be involved in hypertensive renal injury responses.

Septin4 is currently considered to be an important marker protein for organ damage. ARTs (Septin4 isform2) can be involved in various diseases by inducing apoptosis, for example by modulating stem cell survival in ISC niches. In addition, Septin4 plays a crucial role in apoptosis and can reduce liver fibrosis by promoting apoptosis. However, the role of Septin4 and signal transduction SIRT2-Septin4 in hypertensive nephropathy is still unknown. Applicants have demonstrated that acetyltransferase/deacetylase activity of the respective CBP/SIRT2 modulates the acetylation of Septin4-Lys 174. In addition, applicants found that deacetylation of Septin 4K 174 could rescue kidney podocyte injury in Septin4 knockdown cells. .

In summary, applicants have for the first time identified an acetylation dependent regulatory mechanism controlling the function of Septin4 in hypertension. Septin4 deacetylation can prevent hypertensive nephropathy. Applicants' findings indicate that Septin4 may be critical in SIRT 2-mediated hypertension-related diseases, providing a potential mechanism for SIRT2 to act as a protective factor in hypertensive nephropathy. These observations further demonstrate the potential utility of targeting Septin 4K 174 deacetylation for treatment of hypertensive nephropathy.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the present application has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

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Zhang Ying

Zhang Jing jin

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Claims

1. Deacetylated modified Septin4 protein or active fragment thereof, wherein compared with wild-type Septin4 protein, the deacetylated modified Septin4 protein or active fragment thereof comprises an amino acid sequence shown as SEQ ID NO:1, and lysine is deacetylated.

2. The deacetylated modified Septin4 protein or active fragment thereof according to claim 1, wherein lysine deacetylation is achieved by lysine deacetylase (KDACs) deacetylation modification.

3. A pharmaceutical composition comprising the deacetylated modified Septin4 protein or active fragment thereof according to claim 1.

4. The pharmaceutical composition of claim 3, further comprising a pharmaceutically acceptable diluent, excipient and/or carrier.

5. A preparation, characterized in that the preparation deacetylates Septin4 protein, the deacetylated Septin4 protein or active fragment thereof comprises an amino acid sequence as shown in SEQ ID NO:1, wherein lysine is deacetylated.

6. A formulation as claimed in claim 5 wherein the formulation comprises lysine deacetylase.

7. Use of a deacetylated modified Septin4 protein or an active fragment thereof according to claim 1 or 2, in the preparation of a medicament for preventing or treating hypertensive renal injury.

8. The use according to claim 7, wherein the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.

9. Use of the pharmaceutical composition of claim 3 or 4, the formulation of claim 5 or 6, in the manufacture of a medicament for preventing or treating hypertensive renal injury.

10. The use according to claim 9, wherein the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.