WO2023174194A1 - Deacetylation-modified septin4 protein and pharmaceutical use thereof - Google Patents

Deacetylation-modified septin4 protein and pharmaceutical use thereof Download PDF

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WO2023174194A1
WO2023174194A1 PCT/CN2023/080991 CN2023080991W WO2023174194A1 WO 2023174194 A1 WO2023174194 A1 WO 2023174194A1 CN 2023080991 W CN2023080991 W CN 2023080991W WO 2023174194 A1 WO2023174194 A1 WO 2023174194A1
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septin4
sirt2
protein
deacetylation
modified
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PCT/CN2023/080991
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French (fr)
Chinese (zh)
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孙英贤
张莹
张乃今
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孙英贤
张莹
张乃今
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Publication of WO2023174194A1 publication Critical patent/WO2023174194A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
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    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This application relates to the field of drugs for preventing or treating hypertensive renal injury.
  • the present application relates to a deacetylation-modified Septin4 protein or an active fragment thereof.
  • the present application also relates to a pharmaceutical composition comprising a deacetylation-modified Septin4 protein or an active fragment thereof, as well as a deacetylation-modified Septin4 protein or its activity. Use of the fragments to prevent or treat hypertensive renal injury.
  • Hypertension is one of the most common cardiovascular diseases and an important public health problem worldwide. Structural and functional changes in arteries occur during aging, may be caused by hypertension, and lead to cardiovascular events as well as end-stage renal disease. Hypertension is a major risk factor for the rapid decline of 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 arteriosclerosis.
  • GFR glomerular filtration rate
  • CKD chronic kidney disease
  • RAAS inhibitors angiotensin-converting enzyme (ACE) inhibitors and angiotensin II receptor blockers (ARBs), which mainly affect the control of blood pressure (BP).
  • ACE angiotensin-converting enzyme
  • ARBs angiotensin II receptor blockers
  • Septin4 belongs to the Septins GTP-binding protein family and is involved in cell division, apoptosis, vesicle trafficking 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 apoptosis.
  • Septin4 can be involved in various diseases by inducing apoptosis, such as regulating stem cell survival that is critical for homeostasis and regeneration of the intestine. Therefore, Septin4 is currently considered an important marker protein for organ damage.
  • SIRT2 apoptosis-related factor
  • Applicants first demonstrated that the respective acetyltransferase/deacetylase activities of CREB binding protein (CBP)/SIRT2 regulate the acetylation of Septin4-Lys174.
  • CBP CREB binding protein
  • SIRT2 CREB binding protein
  • Deacetylation of Septin4 K174 rescues renal podocyte damage in Septin4 knockdown cells.
  • CBP CREB binding protein
  • SIRT2 CREB binding protein
  • a novel SIRT2-regulated deacetylation pathway mediates the function of Septin4 in the occurrence and development of hypertensive renal damage.
  • Applicants also found that deacetylation of Septin4 K174 by SIRT2 plays an important role in hypertensive kidney injury.
  • the findings of this application provide new research directions for the treatment and prevention of hypertensive nephropathy.
  • the first aspect of the present invention provides a deacetylation-modified Septin4 protein or an active fragment thereof.
  • the deacetylation-modified Septin4 protein or an active fragment thereof includes such as SEQ ID NO: 1
  • the amino acid sequence shown is wherein the lysine is deacetylated.
  • the lysine deacetylation is achieved by deacetylation modification by lysine deacetylase (KDACs).
  • a second aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition contains deacetylation-modified Septin4 protein or an active fragment thereof.
  • composition according to the present invention, wherein the pharmaceutical composition further contains pharmaceutically acceptable diluents, excipients and/or carriers.
  • a third aspect of the present invention provides a preparation, wherein the preparation deacetylates the Septin4 protein, and the deacetylated Septin4 protein or its active fragment comprises the amino acid sequence shown in SEQ ID NO:1 , wherein the lysine is deacetylated.
  • the preparation according to the present invention wherein the preparation comprises lysine deacetylase.
  • the fourth aspect of the present invention provides the use of the deacetylation-modified Septin4 protein or active fragment thereof in preparing a medicament for preventing or treating hypertensive renal injury.
  • the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.
  • a fifth aspect of the present invention provides the use of the pharmaceutical composition or preparation in the preparation of a medicament for preventing or treating hypertensive renal injury.
  • the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.
  • the applicant combines post-translational modification of acetylated proteins with hypertensive kidney damage for the first time, providing new ideas and methods for designing hypertensive kidney treatment programs and targeted drugs. research direction.
  • FIG 1 shows that SIRT2 is involved in angiotensin II (AngII)-induced renal podocyte injury
  • Figure 2 shows that SIRT2 binds the GTPase domain of Septin4, which is the target of SIRT2-dependent deacetylation via lysine 174.
  • Figure 3 shows that SIRT2 alleviates AngII-induced renal podocyte injury by deacetylating Septin4.
  • FIG. 4 shows that Septin4 involved in AngII-induced renal podocyte injury is dependent on Septin4-K174, which is regulated by SIRT2.
  • Figure 5 shows that SIRT2 knockdown mice display high acetylation levels of Septin4 and significantly aggravate AngII-induced hypertensive renal injury.
  • A Total protein obtained from kidney tissue of SIRT2-WT and SIRT2-/- mice 14 days after AngII (1.5 mg/kg/min) infusion.
  • A,D Western blotting was performed to assess SIRT2, Cleaved-PARP1 and Cleaved-Caspase3 expression levels.
  • B Total lysates of kidney tissue were IP with Septin4 antibody and Western blotted with SIRT2 antibody.
  • C Total lysates of kidney tissue were IP with an acetylation antibody and Western blotted with a Septin4 antibody.
  • (G) HE staining was performed to evaluate the degree of glomerular edema. Arrow, tubular edema. Scale bar, 20 ⁇ m.
  • (I) Data are expressed as mean ⁇ SD, (***P ⁇ 0.001, mice per group, n 7).
  • (H) AZAN staining was performed to assess the content of extracellular matrix secretion in glomeruli. Arrow, glomerular extracellular matrix (blue). Scale bar, 20 ⁇ m.
  • (J) Data are expressed as mean ⁇ SD, (***###P ⁇ 0.001, mice in each group, n 7).
  • PAS staining was performed to assess glomerulosclerosis, arrowhead, and segmental glomerulosclerosis. Scale bar, 20 ⁇ m.
  • Figure 6 shows that SIRT2 transgenic (super) mice display low acetylation levels of Septin4 and significantly alleviate AngII-induced hypertensive renal injury.
  • A, C Total protein obtained from kidney tissue of WT and SIRT2 transgenic mice 14 days after AngII (1.5 mg/kg/min) infusion. Western-blot was performed to evaluate Flag-tagged SIRT2, Cleaved-PARP1 and Cleaved-Caspase3 expression levels.
  • B Total lysates of kidney tissue were IP with an acetylation antibody and Western blotted with a Septin4 antibody.
  • E HE staining was performed to evaluate the degree of glomerular edema. Arrow, tubular edema. Scale bar, 20 ⁇ m.
  • Flag-P300, Flag-CBP and Myc-GCN5 plasmids were obtained from Fudan University in Shanghai;
  • Flag-PCAF plasmid was obtained from Shenzhen University;
  • SIRT2 wild-type and SIRT2 knockout mice with deletion of exons 5-8 were obtained from Shanghai Biological Model Bioscience and Technology Development Company;
  • SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice were purchased from Shanghai Biological Model Bioscience and Technology Development Company;
  • Example 1 Septin4-K174R reduces AngII-induced vascular endothelial cell damage, apoptosis and ROS accumulation.
  • SIRT2 wild-type and SIRT2 knockout mice with deletion of exons 5-8 were obtained as gifts from Professor Deng Chuxia (University of Macau). Shanghai Biological Model Bioscience and Technology Development Corporation established SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice.
  • 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 tail cuff method. SIRT2 knockdown and SIRT2 transgene efficiency were measured by Western blotting of study endpoints.
  • Mouse kidney tissue was immersed in 4% (V/V) paraformaldehyde for 4 h and then transferred to 70% (V/V) ethanol.
  • Individual tissues were placed in a processing cassette, dehydrated through a sequential alcohol gradient, and embedded in paraffin blocks. Renal tissue sections with a thickness of 5 ⁇ m were cut, deparaffinized with xylene, and rehydrated by immersion in ethanol of reduced concentration, followed by washing in PBS.
  • HE hematoxylin and eosin
  • Azan Trichrome kit Azan Trichrome kit
  • PAS PAS
  • Massion's Trichrome staining kit G1340, Solarbio, China
  • 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 the Shanghai Institute of Cell Research, Chinese Academy of Sciences, and cultured in high glucose Dulbecco's modified Eagle medium (Israel BioIndustry, 01-052-1). Cells were cultured with 10% fetal bovine serum (FBS) (CLARK, Australia), penicillin (100 U) and streptomycin (100 ⁇ g/ml) in a humidified atmosphere of 5% CO2 at 37°C.
  • FBS fetal bovine serum
  • penicillin 100 U
  • streptomycin 100 ⁇ g/ml
  • 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; Western blot: GNI4110-MC, GNI, Japan), monoclonal mouse anti-GAPDH (10494-1-AP, Proteintech), polyclonal rabbit anti-CBP (7389S, Cell Signaling) ), anti-acetylation-lysine (9441S, Cell Signaling), polyclonal rabbit anti-cleavage PARP (5625S, Cell Signaling), polyclonal rabbit anti-cleavage Caspase3 (19677-1-AP, Proteintech) .
  • AngII (A9525) was purchased from Sigma, AGK2 (B7323) from Apexbio, nicotinamide (NAM, S1899) and tricugustatin A (TSA, S1045) from Selleck.
  • PI propidium iodide, ST511) was from Beyotime.
  • Cell counting kit 8 (CCK8, B34304, Bimake, USA) was from Selleck.
  • Phalloidin-FITC (AAT-23102) is from Bioquest.
  • 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.
  • SIRT2 and Septin4 shRNA lentivirus were purchased from GeneChem. Construct stable gene knockdown cell lines. Briefly, lentivirus was collected from HEK293T cells according to the manufacturer's instructions. Lentiviral particles were mixed with 5 ⁇ PEG-itTM Solution (System Biosciences, USA). Infect freshly plated cells in 6-well culture plates with lentivirus. Stable cell lines were selected with puromycin (10 ⁇ g/ml) for 7 days. Finally, the infection efficiency of target cells was confirmed by Western blotting.
  • shSirt2 target sequence 22296 TGCTCATCAACAAGGAGAA
  • shSirt2 target sequence 22297 TAAGCTGGATGAAAAAAGAGAA
  • shSirt2 target sequence 22298 CAACCATCTGTCACTACTT
  • shSeptin4 target sequence 72648 ccTAAAGGAAAGCATCCCATT
  • acetylation immunoprecipitation cells were washed 3 times with phosphate buffered saline (PBS) and lysed with Flag lysis buffer (137mM NaCl, 10mM NaF, 50mM Tris-HCl (pH 7.6), 1mM EDTA, 0.1mM Na 3 VO 4. 10% glycerol, 1% NonidetP-40 (NP-40) and 1mM PMSF (protease inhibitor)). Additionally, add 5 ⁇ M TSA and 20 mM NAM to the cell lysis buffer.
  • PBS phosphate buffered saline
  • Flag lysis buffer 137mM NaCl, 10mM NaF, 50mM Tris-HCl (pH 7.6), 1mM EDTA, 0.1mM Na 3 VO 4. 10% glycerol, 1% NonidetP-40 (NP-40) and 1mM PMSF (protease inhibitor)
  • Cells were transiently transfected with plasmid for 24 hours. The next day, cells were seeded into 24-well plates at a density of 3 ⁇ 10 cells/well. After 24 h, cells were induced by appropriate concentrations of AngII for 48 h. The medium was then removed and the cells were washed twice with 37°C pre-warmed PBS according to Bioquest's instructions and assayed using phalloidin-FITC (AAT-23102). Cells were imaged using a fluorescence microscope (Olympus).
  • ligation solution with ligase and incubate at 37 °C for 30 min in a preheated humidity chamber. Tap the ligation solution from the slides and wash in 1x Wash Buffer A. Amplification-polymerase solution was added to each sample and incubated in a preheated humidity chamber at 37°C for 100 minutes. Finally, the amplification-polymerase solution was knocked out of the slides and washed in 1x Wash Buffer B followed by 0.01x Wash Buffer B. Duolink Insitu Mounting Medium with DAPI is mounted on the slide with a coverslip. Cells were imaged using a fluorescence microscope (Olympus).
  • SD mean ⁇ standard deviation
  • F test group pairs
  • Shapiro-Wilk test was performed to assess the normality of the data. Differences between groups were assessed using a two-tailed Student's t test for continuous variables (expressed as mean ⁇ SD).
  • One-way ANOVA, two-method ANOVA and indicative non-parametric tests were performed to compare differences between multiple groups. If applicable, P values can be adjusted for multiple comparisons. All statistical analyzes were performed using SPSS version 22.0 software (SPSS Inc, Chicago, IL, USA), and P ⁇ 0.05 indicated statistical significance.
  • SIRT2 participates in AngII-induced renal podocyte injury by interacting with the injury-related protein-Septin4.
  • SIRT2 binds to the GTPase domain of Septin4, and the GTPase domain of Septin4 serves as the target of SIRT2-dependent deacetylation through lysine 174.
  • Septin4 contains three functional domains, including N-terminal, C-terminal and GTPase domains ( Figure 2B).
  • Figure 2B Using endogenous SIRT2 and full-length Flag-tagged-Septin4 or various truncated Flag-Septin4 plasmids, Applicants demonstrated that SIRT2 binds to the GTPase domain of Septin4 ( Figure 2A). These data indicate that SIRT2 interacts with the GTPase domain of Septin4 and that AngII enhances binding. Next, Applicants verified whether SIRT2 could modulate the acetylation activity of Septin4.
  • Septin4 acetylation levels are increased after treatment with trichostatin A (TSA) and nicotinamide (NAM), two commonly used deacetylase inhibitors that inhibit the histone deacetylase HDAC I and III and Deacetylase of the Sirtuins family (Fig. 2C).
  • TSA trichostatin A
  • NAM nicotinamide
  • acetyltransferases were transfected separately, including p300 (E1A binding protein, 300 kDa), CBP, PCAF (p300/CBP-related factor) or GCN5 (KAT2A).
  • acetylation level of Septin4 was higher in shSIRT2 cells or cells treated with AGK2 compared with normal control cells (Fig. 2G).
  • overexpression of wild-type (WT) SIRT2 reduced endogenous Septin4 acetylation, whereas transfection of a catalytically inactive mutant of SIRT2 (H187YQ167A) had no effect (Fig. 2F).
  • WT wild-type
  • H187YQ167A catalytically inactive mutant of SIRT2
  • Applicants 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 loss of Septin4 acetylation in the presence or absence of CBP, while CBP increased the deacetylation level of WT-Septin4 (Fig. 2H).
  • arginine substitution of K174 resulted in loss of Septin4 acetylation with or without SIRT2 overexpression compared with WT-Septin4 (Fig. 2I).
  • SIRT2 alleviates AngII-induced renal podocyte injury by deacetylating Septin4.
  • AngII caused increased binding between SIRT2 and Septin4 in renal podocytes (Figure 3A).
  • AngII induced the down-regulation of Septin4 acetylation levels, which was increased in shSIRT2 renal podocytes, but was restored after re-expression of SIRT2 in shSIRT2 renal podocytes (Fig. 3B).
  • Fig. 3B Applicants subsequently used normal control and shSIRT2 renal podocytes with or without renal podocyte injury induced by 10 ⁇ 5 mol/LAngII.
  • ShSIRT2 cells showed increased levels of Cleaved-PARP1 in response to renal podocyte injury, whereas transient reexpression of WT-SIRT2 in SIRT2-depleted renal podocytes rescued the injury (Fig. 3C-D). Consistent with these findings, cytoskeletal disassembly was more extensive in shSIRT2 renal podocytes, whereas transient reexpression of WT-SIRT2 in SIRT2-depleted renal podocytes rescued this disassembly (Fig. 3F,G). Similar results were obtained using CCK8 analysis (Fig. 3E). In summary, SIRT2 can alleviate AngII-induced renal podocyte injury, and Septin4 may be involved in the response.
  • Transient reexpression of deacetylated Septin4 induces renal podocyte injury.
  • the levels of Cleaved-PARP1 and Cleaved-Caspase3 after re-expression of WT-Septin4 were higher than those of shSeptin4, while there was no difference between transient re-expression of K174R in Septin4-depleted renal podocytes and shSeptin4 renal podocytes. significant difference.
  • AngII was infused for 2 weeks using an osmotic minipump to establish hypertensive renal injury models in SIRT2-WT and SIRT2-/-C57BL/6 mice in vivo. Applicants found that the expression of SIRT2 in SIRT2-WT kidney tissue increased significantly after AngII-induced hypertensive injury (Fig. 5A, E), while SIRT2-/- mice did not express SIRT2.
  • SIRT2 knockdown exacerbates AngII-induced hypertensive renal injury by deacetylating Septin4.
  • SIRT2 transgenic mice were used to verify the above experiments. As shown in Figure 6A, SIRT2 transgenic (super) mice were successfully constructed. The acetylation level of Septin4 was detected in hypertensive renal injury mice by co-immunoprecipitation (Fig. 6B). Septin4 acetylation levels are reduced in SIRT2 transgenic (super) mice. In addition, compared with the WT group, the SIRT2 transgenic (super) group significantly alleviated the amounts of Cleaved-PARP1 and Cleaved-Caspase3 (Fig. 6C-D).
  • SIRT2 transgenic (super) mice display attenuated apoptosis in hypertensive renal injury. Subsequently, H&E and Azan trichrome staining showed that compared with WT mice, transfection of SIRT2 (super) could significantly alleviate the degree of tubular edema induced by AngII and increase the area of glomerular mesangial matrix (Figure 6E-H) . After hypertensive renal injury, SIRT2 transgenic (super) mice had smaller segmental sclerosis and fibrosis areas than wild-type mice (P ⁇ 0.001) ( Figure 6I-L). Therefore, SIRT2 transgene (super) attenuated AngII-induced hypertensive renal injury. This further demonstrates that Septin4-dependent deacetylation regulation of SIRT2 alleviates hypertensive renal injury.
  • SIRT2 is an NAD+-dependent class III histone deacetylase that plays an important role in endothelial cells and heart-related diseases.
  • a specific inhibitor of SIRT2, AGK2 reduces H 2 O 2 -induced endothelial cell toxicity.
  • activated SIRT2 signaling alleviated DOX-induced cardiotoxicity.
  • SIRT2-deficient mice undergo spontaneous heart failure and exhibit cardiac hypertrophy, remodeling, fibrosis, and dysfunction with increasing age. SIRT2 activation protects the heart from aging-related and isoproterenol-induced pathological cardiac hypertrophy by inhibiting the NFAT transcription factor.
  • SIRT2 plays a role in hypertensive kidney injury.
  • SIRT2 is implicated in hypertensive renal injury for the first time.
  • SIRT2 transgenic (super) mice can attenuate hypertensive kidney damage.
  • glomerulosclerosis and renal fibrosis are significantly aggravated in the late stages.
  • SIRT2 had a role in a model of renal podocyte injury. Reexpression of SIRT2 rescued cytoskeletal disorganization in SIRT2 knockdown cells. Furthermore, SIRT2 regulates many common substrates that depend on NAD+ deacetylation activity, including FoxO1, FoxO3, and NF- ⁇ B. SIRT2 promotes AMPK activity by deacetylating AMPK's upstream kinase LKB132, thereby protecting the heart from AngII-induced hypertrophic stimulation. The applicant discovered a new apoptosis-related protein downstream of SIRT2Septin4. In addition, AngII significantly increased the expression of Septin4 after deletion of SIRT2. These results indicate that Septin4 may be involved in the response to hypertensive renal injury.
  • Septin4 is currently considered an important marker protein for organ damage. ARTs (Septin4isform2) can be involved in various diseases by inducing apoptosis, such as by regulating stem cell survival in the ISC niche. Furthermore, Septin4 plays a crucial 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. Applicants demonstrate that the respective acetyltransferase/deacetylase activities of CBP/SIRT2 regulate the acetylation of Septin4-Lys174. In addition, Applicants found that deacetylation of Septin4 K174 can rescue renal podocyte injury in Septin4 knockdown cells. .
  • Applicants have identified for the first time an acetylation-dependent regulatory mechanism controlling Septin4 function in hypertension.
  • Septin4 deacetylation protects against hypertensive nephropathy.
  • Applicants' findings indicate that Septin4 may be critical in SIRT2-mediated hypertension-related diseases, providing a potential mechanism by which SIRT2 functions as a protective factor in hypertensive nephropathy.

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Abstract

Provided are a deacetylation-modified Septin4 protein and the pharmaceutical use thereof. Also provided is a deacetylation-modified Septin4 protein or an active fragment thereof. Compared with a wild-type Septin4 protein, the deacetylation-modified Septin4 protein or the active fragment thereof contains an amino acid sequence as shown in SEQ ID NO: 1, wherein the lysine is deacetylated. Further provided is the use of the deacetylation-modified Septin4 protein or the active fragment thereof or the pharmaceutical composition in the preparation of a drug for preventing or treating hypertensive kidney injuries. For the first time, post-translational modification of an acetylated protein is combined with hypertensive kidney injuries, providing new ideas and research directions for the design of treatment regimens and targeted drugs for hypertensive kidney injuries.

Description

[根据细则26改正 21.03.2023]脱乙酰化修饰的Septin4蛋白及其制药用途[Amended in accordance with Rule 26 21.03.2023] Deacetylation-modified Septin4 protein and its pharmaceutical use 技术领域Technical field
本申请涉及预防或治疗高血压性肾损伤的药物领域。具体地，本申请涉及脱乙酰化修饰的Septin4蛋白或其活性片段，本申请还涉及包含脱乙酰化修饰的Septin4蛋白或其活性片段的药物组合物，以及脱乙酰化修饰的Septin4蛋白或其活性片段用于预防或治疗高血压性肾损伤的用途。This application relates to the field of drugs for preventing or treating hypertensive renal injury. Specifically, the present application relates to a deacetylation-modified Septin4 protein or an active fragment thereof. The present application also relates to a pharmaceutical composition comprising a deacetylation-modified Septin4 protein or an active fragment thereof, as well as a deacetylation-modified Septin4 protein or its activity. Use of the fragments to prevent or treat hypertensive renal injury.
背景技术Background technique
高血压是最常见的心血管疾病之一，并且是全世界范围内的重要公共卫生问题。动脉的结构和功能变化会在衰老过程中发生，可能是由高血压引起的，并导致心血管事件以及终末期肾脏疾病。高血压是肾病患者肾小球滤过率(GFR)快速下降和慢性肾脏病(CKD)发生的主要危险因素。未经治疗的高血压可通过引起肾小球硬化和肾小动脉硬化损害肾脏。Hypertension is one of the most common cardiovascular diseases and an important public health problem worldwide. Structural and functional changes in arteries occur during aging, may be caused by hypertension, and lead to cardiovascular events as well as end-stage renal disease. Hypertension is a major risk factor for the rapid decline of 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 arteriosclerosis.
目前，高血压肾病的主要药物包括RAAS抑制剂，血管紧张素转化酶(ACE)抑制剂和血管紧张素II受体阻滞剂(ARBs)，它们主要影响血压(BP)的控制。但是，严格的BP控制不会延迟终末期肾脏疾病(ESRD)的发作和肾脏功能的显著恶化。因此，有必要进一步研究高血压肾病的分子机制，以开发新型靶向药物和临床治疗方法。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 mechanisms of hypertensive nephropathy to develop novel targeted drugs and clinical treatments.
Septin4属于Septins GTP结合蛋白家族，与细胞分裂，凋亡，囊泡运输和其他细胞过程有关。Septin4_vi2作为促凋亡蛋白，参与各种凋亡过程。Fas、依托泊苷、星形孢菌素和阿拉伯糖苷可通过结合Septin4/XIAP(X连锁凋亡蛋白抑制剂)而具有诱导凋亡的能力。这些刺激可以增加Septin4的表达水平，从而促进细胞凋亡。Septin4可以通过诱导细胞凋亡来参与各种疾病，例如调节对肠道的稳态和再生至关重要的干细胞存活。因此，Septin4目前被认为是器官损伤的重要标志蛋白。Septin4 belongs to the Septins GTP-binding protein family and is involved in cell division, apoptosis, vesicle trafficking 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 apoptosis. Septin4 can be involved in various diseases by inducing apoptosis, such as regulating stem cell survival that is critical for homeostasis and regeneration of the intestine. Therefore, Septin4 is currently considered an important marker protein for organ damage.
然而，Septin4是否在高血压肾损伤中起作用尚不清楚。现有技术中并没有涉及Septin4与高血压肾损伤的相关性的研究。However, whether Septin4 plays a role in hypertensive kidney injury is unclear. There are no studies involving the correlation between Septin4 and hypertensive renal damage in the prior art.
发明内容Contents of the invention
本申请的技术方案是基于以下研究的基础上提出的：The technical solution of this application is proposed based on the following research:
本申请人发现，SIRT2的新底物为凋亡相关因子Septin4。申请人首先证实，CREB结合蛋白(CBP)/SIRT2各自的乙酰转移酶/脱乙酰化酶活性调节Septin4-Lys174的乙酰化作用。Septin4 K174的脱乙酰基可以挽救Septin4敲减细胞中的肾足细胞损伤。这些发现表明，新型的SIRT2调节的脱乙酰途径介导了Septin4在高血压肾脏损害的发生和发展中的功能。此外，申请人还发现，通过SIRT2使Septin4 K174脱乙酰化在高血压肾损伤中起重要作用。本申请的发现为高血压肾病疾病的治疗和预防提供了新的研究方向。The applicant discovered that a new substrate of SIRT2 is the apoptosis-related factor Septin4. Applicants first demonstrated that the respective acetyltransferase/deacetylase activities of CREB binding protein (CBP)/SIRT2 regulate the acetylation of Septin4-Lys174. Deacetylation of Septin4 K174 rescues renal podocyte damage in Septin4 knockdown cells. These findings indicate that a novel SIRT2-regulated deacetylation pathway mediates the function of Septin4 in the occurrence and development of hypertensive renal damage. In addition, Applicants also found that deacetylation of Septin4 K174 by SIRT2 plays an important role in hypertensive kidney injury. The findings of this application provide new research directions for the treatment and prevention of hypertensive nephropathy.
因此，本申请的目的是通过以下技术方案实现的：Therefore, the purpose of this application is achieved through the following technical solutions:
本发明的第一方面提供了一种脱乙酰化修饰的Septin4蛋白或其活性片段，与野生型Septin4蛋白相比，所述脱乙酰化修饰的Septin4蛋白或其活性片段包含如SEQ ID NO:1所示的氨基酸序列，其中所述赖氨酸脱乙酰化。The first aspect of the present invention provides a deacetylation-modified Septin4 protein or an active fragment thereof. Compared with the wild-type Septin4 protein, the deacetylation-modified Septin4 protein or an active fragment thereof includes such as SEQ ID NO: 1 The amino acid sequence shown is wherein the lysine is deacetylated.
SEQ ID NO:1 SEQ ID NO:1
根据本发明所述的脱乙酰化修饰的Septin4蛋白或其活性片段，其中，所述赖氨酸脱乙酰化通过赖氨酸脱乙酰化酶(KDACs)脱乙酰化修饰实现。According to the deacetylation-modified Septin4 protein or active fragment thereof according to the present invention, the lysine deacetylation is achieved by deacetylation modification by lysine deacetylase (KDACs).
本发明的第二方面提供了一种药物组合物，其中，所述药物组合物包含脱乙酰化修饰的Septin4蛋白或其活性片段。A second aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition contains deacetylation-modified Septin4 protein or an active fragment thereof.
根据本发明所述的药物组合物，其中，所述药物组合物还包含药学上可接受的稀释剂、赋形剂和/或载体。The pharmaceutical composition according to the present invention, wherein the pharmaceutical composition further contains pharmaceutically acceptable diluents, excipients and/or carriers.
本发明的第三方面提供了一种制剂，其中，所述制剂使Septin4蛋白脱乙酰化修饰，所述脱乙酰化修饰的Septin4蛋白或其活性片段包含如SEQ ID NO:1所示的氨基酸序列，其中所述赖氨酸脱乙酰化。A third aspect of the present invention provides a preparation, wherein the preparation deacetylates the Septin4 protein, and the deacetylated Septin4 protein or its active fragment comprises the amino acid sequence shown in SEQ ID NO:1 , wherein the lysine is deacetylated.
根据本发明所述的制剂，其中所述制剂包含赖氨酸脱乙酰化酶。The preparation according to the present invention, wherein the preparation comprises lysine deacetylase.
本发明的第四方面提供了所述脱乙酰化修饰的Septin4蛋白或其活性片段在制备用于预防或治疗高血压性肾损伤的药物中的用途。The fourth aspect of the present invention provides the use of the deacetylation-modified Septin4 protein or active fragment thereof in preparing a medicament for preventing or treating hypertensive renal injury.
根据本发明所述的用途，其中所述高血压性肾损伤为血管紧张素II诱导的高血压性肾损伤。According to the use of the present invention, the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.
本发明的第五方面提供了所述药物组合物或制剂在制备用于预防或治疗高血压性肾损伤的药物中的用途。A fifth aspect of the present invention provides the use of the pharmaceutical composition or preparation in the preparation of a medicament for preventing or treating hypertensive renal injury.
根据本发明所述的用途，其中，所述高血压性肾损伤为血管紧张素II诱导的高血压性肾损伤。According to the use of the present invention, the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.
与现有技术相比，本申请具有以下有益效果：申请人首次将乙酰化蛋白的翻译后修饰与高血压肾脏损伤相结合，为设计高血压肾脏治疗方案和靶向药物提供了新的思路和研究方向。Compared with the existing technology, this application has the following beneficial effects: The applicant combines post-translational modification of acetylated proteins with hypertensive kidney damage for the first time, providing new ideas and methods for designing hypertensive kidney treatment programs and targeted drugs. research direction.
附图说明Description of the drawings
以下，结合附图来详细说明本申请的实施方案，其中：Below, the embodiments of the present application are described in detail with reference to the accompanying drawings, wherein:
图1示出为SIRT2参与了血管紧张素II(AngII)诱导的肾足细胞损伤；Figure 1 shows that SIRT2 is involved in angiotensin II (AngII)-induced renal podocyte injury;
其中，(A)在第14天，注入盐水或Ang II的小鼠心脏中Sirtuin蛋白表达谱的簇。(B，D)在用不同的AngII浓度刺激48天后，测量了在小鼠的肾脏足细胞中SIRT2的表达水平。(C，E)定量数据为平均值±SD，*P<0.05，**P<0.01；***P＜0.001。(F)通过质谱鉴定SIRT2的相互作用蛋白。(G)用抗SIRT2抗体免疫沉淀细胞裂解物，然后用Septin4抗体免疫印迹。(H)转染带有标志的Septin4质粒，并用抗Flag抗体对总裂解物进行免疫沉淀(IP)处理，并用SIRT2抗体进行检测。(I)执行PLA遍历以确定SIRT2和Septin4之间的相互作用。箭头处表示存在相互作用。Among them, (A) Clusters of Sirtuin protein expression profiles in the hearts of mice injected with saline or Ang II at day 14. (B, D) SIRT2 expression levels were measured in mouse kidney podocytes after stimulation with different AngII concentrations for 48 days. (C, E) Quantitative data are mean ± SD, *P<0.05, **P<0.01; ***P<0.001. (F) Identification of SIRT2-interacting proteins by mass spectrometry. (G) Cell lysates were immunoprecipitated with anti-SIRT2 antibody and then immunoblotted with Septin4 antibody. (H) Flag-bearing Septin4 plasmid was transfected, and total lysates were immunoprecipitated (IP) with anti-Flag antibody and detected with SIRT2 antibody. (I) Perform a PLA traversal to determine the interaction between SIRT2 and Septin4. Arrows indicate the presence of interactions.
图2示出为SIRT2结合Septin4的GTPase结构域，而Septin4则是通过赖氨酸174依赖SIRT2的脱乙酰基作用的靶标Figure 2 shows that SIRT2 binds the GTPase domain of Septin4, which is the target of SIRT2-dependent deacetylation via lysine 174.
其中，(A)转染全长Flag-tagged-Septin4或四个截短的Flag-Septin4质粒。用抗-Flag抗体对总裂解物进行IP，并用SIRT2抗体进行蛋白质印迹。(B)Septin4包含三个功能域，包括N端，C端和GTPase域。(C)用乙酰化抗体免疫沉淀TSA(0.5μM，16h)和NAM(5mM，4h)处理细胞并用Septin4抗体检测。(D)内源性Septin4与内源性CBP相互作用。E分别将标记标记的CBP、P300、p300/CBP相关因子(PCAF)或Myc标记的GCN5过表达，并用抗乙酰化的赖氨酸抗体(Ac-K)免疫沉淀Septin4乙酰化并用Septin4抗体检测。(F)标记有标志的SIRT2WT(野生型)或H187YQ167A(Mut，突变型)过表达。用抗乙酰化的赖氨酸抗体(Ac-K)免疫沉淀Septin4乙酰化，并用Septin4抗体检测。(G)用或不用AGK2(20μM，24小时)处理的正常对照和shSIRT2细胞用乙酰化抗体免疫沉淀并用Septin4抗体检测。(H)标记了标签的CBP与标记了标签的Septin4 WT或K174R(Mut)共转染。使用抗乙酰化的赖氨酸抗体(Ac-K)通过IP检测Septin4的乙酰化。(I)Myc标签的SIRT2与Flag标签的Septin4 WT或K174R(Mut)共转染。用抗乙酰化的赖氨酸抗体(Ac-K)检测Septin4的乙酰化。Among them, (A) transfected full-length Flag-tagged-Septin4 or four truncated Flag-Septin4 plasmids. Total lysates were subjected to IP with anti-Flag antibody and Western blotted with SIRT2 antibody. (B) Septin4 contains three functional domains, including N-terminal, C-terminal and GTPase domains. (C) Cells were treated with acetylated antibodies to immunoprecipitate TSA (0.5 μM, 16 h) and NAM (5 mM, 4 h) and detected with Septin4 antibodies. (D) Endogenous Septin4 interacts with endogenous CBP. E. Tag-tagged CBP, P300, p300/CBP-associated factor (PCAF) or Myc-tagged GCN5 were overexpressed respectively, and Septin4 acetylation was immunoprecipitated with anti-acetylated lysine antibody (Ac-K) and detected with Septin4 antibody. (F) Overexpression of Flag-tagged SIRT2WT (wild type) or H187YQ167A (Mut, mutant). Septin4 acetylation was immunoprecipitated with anti-acetylated lysine antibody (Ac-K) and detected with Septin4 antibody. (G) Normal control and shSIRT2 cells treated with or without AGK2 (20 μM, 24 h) were immunoprecipitated with acetylated antibodies and probed with Septin4 antibodies. (H) 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 using anti-acetylated lysine antibody (Ac-K).
图3示出为SIRT2通过脱乙酰基修饰Septin4减轻了AngII诱导的肾足细胞损伤。Figure 3 shows that SIRT2 alleviates AngII-induced renal podocyte injury by deacetylating Septin4.
其中，(A)用或不用AngII转染Myc标签的SIRT2质粒。用抗Myc抗体对总裂解物进行IP，并用Septin4抗体检测。(B)将Myc标记的SIRT2质粒转染到具有或不具有AngII的肾足细胞-shSIRT2细胞中。将总裂解物用乙酰化抗体进行IP处理，并用Septin4抗体进行检测。(C)将标记了标签的SIRT2质粒转染到肾足细胞-shSIRT2细胞中。将细胞在有或没有10-5mol/L AngII的情况下处理48小时。Cleaved-PARP1用western bolt评估。(D)定量数据平均值±SD，*P<0.05，**P<0.01。(E)通过CCK8测定法测量肾足细胞的生存力。数据表示为平均值±SD，*P<0.05，**P<0.01。(G)用抗phalloidin-FITC抗体对肾足细胞进行染色。细胞核用DAPI染色。比例尺，50μm。(F)数据表示为平均值±SD，**P<0.01；***P＜0.001。Among them, (A) Myc-tagged SIRT2 plasmid was transfected with or without AngII. Total lysates were IPed with anti-Myc antibody and probed with Septin4 antibody. (B) Myc-tagged SIRT2 plasmid was transfected into renal podocyte-shSIRT2 cells with or without AngII. Total lysates were IP treated with acetylated antibodies and detected with Septin4 antibodies. (C) The tagged SIRT2 plasmid was transfected into renal podocyte-shSIRT2 cells. Cells were treated with or without 10-5 mol/L AngII for 48 hours. Cleaved-PARP1 was evaluated using western bolt. (D) Quantitative data mean±SD, *P<0.05, **P<0.01. (E) Measurement of renal podocyte viability 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. Cell nuclei were stained with DAPI. Scale bar, 50 μm. (F) Data are expressed as mean±SD, **P<0.01; ***P<0.001.
图4示出为参与AngII诱导的肾足细胞损伤的Septin4依赖于Septin4-K174，其受SIRT2调控。Figure 4 shows that Septin4 involved in AngII-induced renal podocyte injury is dependent on Septin4-K174, which is regulated by SIRT2.
(A)将带有标志标签的Septin4WT或K174R质粒转染到肾足细胞-shSeptin4细胞中。肾足细胞用NaCl或10-5mol/LAngII处理48小时。Cleaved-PARP1和Caspase3通过westernbolt评估。(B)定量数据，平均值±SD，***P＜0.001。(C)用抗鬼笔环肽-FITC抗体对肾足细胞进行染色。细胞核用DAPI染色。比例尺，50μm。(E)数据表示平均值±SD，*P＜0.05。(D)通过CCK8测定法测量肾足细胞的生存力。数据表示为平均值±SD，*P<0.05，**P<0.01。(A) Septin4WT or K174R plasmid with Flag tag was transfected into renal podocyte-shSeptin4 cells. Renal podocytes were treated with NaCl or 10-5 mol/LAngII for 48 hours. Cleaved-PARP1 and Caspase3 assessed by westernbolt. (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) Renal podocyte viability measured by CCK8 assay. Data are expressed as mean±SD, *P<0.05, **P<0.01.
图5示出为敲减SIRT2的小鼠显示出Septin4的乙酰化水平高，并且明显加重了AngII引起的高血压性肾损伤。Figure 5 shows that SIRT2 knockdown mice display high acetylation levels of Septin4 and significantly aggravate AngII-induced hypertensive renal injury.
(A)为AngII(1.5mg/kg/min)输注14天后，从SIRT2-WT和SIRT2-/-小鼠肾脏组织中获得总蛋白。(A，D)进行蛋白质印迹以评估SIRT2、Cleaved-PARP1和Cleaved-Caspase3表达水平。(E-F)Western印迹数据的定量显示为平均值±SD(***###P＜0.001，每组小鼠，n＝7)。(B)用Septin4抗体对肾组织的总裂解物进行IP，并用SIRT2抗体进行蛋白质印迹。(C)用乙酰化抗体对肾组织的总裂解物进行IP，并用Septin4抗体进行蛋白质印迹。(G)进行HE染色以评估肾小球水肿程度。箭头，肾小管水肿。比例尺，20μm。(I)数据表示为平均值±SD，(***P＜0.001，每组小鼠，n＝7)。(H)进行AZAN染色以评估肾小球中细胞外基质分泌的含量。箭头，肾小球的细胞外基质(蓝色)。比例尺，20μm。(J)数据表示为平均值±SD，(***###P＜0.001，每组小鼠，n＝7)。(K)进行PAS染色以评估肾小球硬化症、箭头、肾小球节段性硬化症。比例尺，20μm。(M)数据表示为平均值±SD，(***###P＜0.001，每组小鼠，n＝7)。(L)进行肿块染色以评估肾小球纤维化程度。箭头，肾小球纤维化。比例尺，20μm。(N)数据表示为平均值±SD，(***###P＜0.001，每组小鼠，n＝7)。(A) Total protein obtained from kidney tissue of SIRT2-WT and SIRT2-/- mice 14 days after AngII (1.5 mg/kg/min) infusion. (A,D) Western blotting was performed to assess SIRT2, Cleaved-PARP1 and Cleaved-Caspase3 expression levels. (E-F) Quantification of Western blot data shown as mean±SD (***###P<0.001, mice per group, n=7). (B) Total lysates of kidney tissue were IP with Septin4 antibody and Western blotted with SIRT2 antibody. (C) Total lysates of kidney tissue were IP with an acetylation antibody and Western blotted with a 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, mice per group, n=7). (H) AZAN staining was performed to assess the content of extracellular matrix secretion in glomeruli. Arrow, glomerular extracellular matrix (blue). Scale bar, 20 μm. (J) Data are expressed as mean±SD, (***###P<0.001, mice in each group, n=7). (K) PAS staining was performed to assess glomerulosclerosis, arrowhead, and segmental glomerulosclerosis. Scale bar, 20 μm. (M) Data are expressed as mean±SD, (***###P<0.001, mice in each group, n=7). (L) Mass staining was performed to assess the extent of glomerular fibrosis. Arrow, glomerular fibrosis. Scale bar, 20 μm. (N) Data are expressed as mean±SD, (***###P<0.001, mice in each group, n=7).
图6示出为SIRT2转基因(超级)小鼠显示出Septin4的乙酰化水平低，并显著缓解了AngII引起的高血压性肾损伤。Figure 6 shows that SIRT2 transgenic (super) mice display low acetylation levels of Septin4 and significantly alleviate AngII-induced hypertensive renal injury.
(A，C)AngII(1.5mg/kg/min)输注14天后，从WT和SIRT2转基因小鼠肾脏组织获得总蛋白。进行了Western-blot评估Flag标签的SIRT2，Cleaved-PARP1和Cleaved-Caspase3表达水平。(D)Western印迹数据的定量显示为平均值±SD(***###P＜0.001，每组小鼠，n＝7)。(B)用乙酰化抗体对肾组织的总裂解物进行IP，并用Septin4抗体进行蛋白质印迹。(E)进行HE染色以评估肾小球水肿程度。箭头，肾小管水肿。比例尺，20μm。(G)数据表示为平均值±SD，(***P＜0.001，每组小鼠，n＝7)。(F)进行AZAN染色以评估肾小球中细胞外基质分泌的含量。箭头，肾小球的细胞外基质(蓝色)。比例尺，20μm。(H)数据表示为平均值±SD，(***###P＜0.001，每组小鼠，n＝7)。(I)进行PAS染色以评估肾小球硬化箭头，肾小球节段性硬化(淡紫色)。比例尺，20μm。(K)数据表示为平均值±SD，(***###P＜0.001，每组小鼠，n＝7)。(J)进行肿块染色以评估肾小球纤维化程度。箭头，肾小球纤维化(蓝色)。比例尺，20μm。(L)数据表示为平均值±SD，(***###P＜0.001，每组小鼠，n＝7)。(A, C) Total protein obtained from kidney tissue of WT and SIRT2 transgenic mice 14 days after AngII (1.5 mg/kg/min) infusion. Western-blot was performed to evaluate Flag-tagged SIRT2, Cleaved-PARP1 and Cleaved-Caspase3 expression levels. (D) Quantification of Western blot data shown as mean±SD (***###P<0.001, mice per group, n=7). (B) Total lysates of kidney tissue were IP with an acetylation antibody and Western blotted with a Septin4 antibody. (E) HE staining was performed to evaluate the degree of glomerular edema. Arrow, tubular edema. Scale bar, 20 μm. (G) Data are expressed as mean±SD, (***P<0.001, mice per group, n=7). (F) AZAN staining was performed to assess the content of extracellular matrix secretion in glomeruli. Arrow, glomerular extracellular matrix (blue). Scale bar, 20 μm. (H) Data are expressed as mean±SD, (***###P<0.001, mice in each group, n=7). (I) PAS staining was performed to assess glomerulosclerosis arrowhead, segmental sclerosis of the glomerulus (lavender). Scale bar, 20 μm. (K) Data are expressed as mean±SD, (***###P<0.001, mice in each group, n=7). (J) Mass staining was performed to assess the extent of glomerular fibrosis. Arrow, glomerular fibrosis (blue). Scale bar, 20 μm. (L) Data are expressed as mean±SD, (***###P<0.001, mice in each group, n=7).
具体实施方式Detailed ways
下面结合附图和实施例进一步说明本申请，应当理解，实施例仅用于进一步说明和阐释本申请，并非用于限制本申请。The present application will be further described below with reference to the accompanying drawings and examples. It should be understood that the examples are only used to further illustrate and illustrate the present application, and are not intended to limit the present application.
Flag-P300，Flag-CBP和Myc-GCN5质粒获得自上海复旦大学；Flag-P300, Flag-CBP and Myc-GCN5 plasmids were obtained from Fudan University in Shanghai;
Flag-PCAF质粒获得自深圳大学；Flag-PCAF plasmid was obtained from Shenzhen University;
外显子5-8缺失的SIRT2野生型和SIRT2基因敲减(SIRT2-/-)小鼠获得自上海生物模型生物科学与技术发展公司；SIRT2 wild-type and SIRT2 knockout (SIRT2-/-) mice with deletion of exons 5-8 were obtained from Shanghai Biological Model Bioscience and Technology Development Company;
SIRT2野生型和Flag-SIRT2转基因(超级)小鼠购自上海生物模型生物科学与技术发展公司；SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice were purchased from Shanghai Biological Model Bioscience and Technology Development Company;
实施例1 Septin4-K174R减少AngII诱导的血管内皮细胞损伤，凋亡以及ROS累积。Example 1 Septin4-K174R reduces AngII-induced vascular endothelial cell damage, apoptosis and ROS accumulation.
一、材料和方法1. Materials and methods
1.1SIRT2基因敲减和转基因小鼠1.1SIRT2 knockout and transgenic mice
外显子5-8缺失的SIRT2野生型和SIRT2基因敲减(SIRT2-/-)小鼠获得自邓初夏教授(澳门大学)馈赠。上海生物模型生物科学与技术发展公司建立了SIRT2野生型和Flag-SIRT2转基因(超级)小鼠。SIRT2 wild-type and SIRT2 knockout (SIRT2-/-) mice with deletion of exons 5-8 were obtained as gifts from Professor Deng Chuxia (University of Macau). Shanghai Biological Model Bioscience and Technology Development Corporation established SIRT2 wild-type and Flag-SIRT2 transgenic (super) mice.
所有动物均在无病原体的条件下饲养。所有实验均使用8-10周大的雄性小鼠进行。在NaCl和AngII输注模型(A9525，sigma，美国)中，SIRT2野生型和SIRT2基因敲减(SIRT2-/-)小鼠(每组，N＝7)，以及SIRT2野生型和SIRT2转基因(超级)小鼠(每组，N＝7)根据制造商的说明(AlZET渗透泵，DURECT Corporation，Cupertino，CA)植入渗透微型泵。在肩中部切开一个切口，并植入一个渗透性微型泵。通过微型泵向小鼠注入NaCl或AngII(1.5mg/kg/天)，持续14天(Alzet，2002年模型)。每天通过尾袖法测量血压。SIRT2基因敲减和SIRT2转基因效率是通过研究终点的蛋白质印迹法测量的。All animals were maintained 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 (N=7 per group) were implanted with osmotic minipumps according to the manufacturer's instructions (AlZET Osmotic Pump, DURECT Corporation, Cupertino, CA). An incision is made in the mid-shoulder and an osmotic minipump is 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 tail cuff method. SIRT2 knockdown and SIRT2 transgene efficiency were measured by Western blotting of study endpoints.
所有动物处理均符合中国医科大学动物福利规定。中国医科大学动物学科委员会批准了动物研究方案。All animal handling complied with the animal welfare regulations of China Medical University. The animal study protocol was approved by the Animal Science Committee of China Medical University.
1.2免疫组织化学(IHC)分析1.2 Immunohistochemistry (IHC) analysis
将小鼠肾脏组织浸入4％(V/V)多聚甲醛中4h，然后转移至70％(V/V)乙醇中。将各个组织放入处理盒中，通过连续的酒精梯度进行脱水，然后包埋在石蜡块中。切下厚度为5μm的肾组织切片，用二甲苯脱蜡，并通过浸入浓度降低的乙醇中重新水化，然后在PBS中洗涤。然后根据苏木精和曙红(HE)，Azan Trichrome试剂盒(AZT-K-250，美国Biognost，美国)，PAS(G1285，Solarbio，中国)或Massion’s Trichrome染色试剂盒(G1340，Solarbio，中国)根据操作手册对切片进行染色。染色后，将切片在浓度越来越高的乙醇和二甲苯中脱水。Mouse kidney tissue was immersed in 4% (V/V) paraformaldehyde for 4 h and then transferred to 70% (V/V) ethanol. Individual tissues were placed in a processing cassette, dehydrated through a sequential alcohol gradient, and embedded in paraffin blocks. Renal tissue sections with a thickness of 5 μm were cut, deparaffinized with xylene, and rehydrated by immersion in ethanol of reduced concentration, followed by washing in PBS. Then stain according to hematoxylin and eosin (HE), Azan Trichrome kit (AZT-K-250, Biognost, USA), PAS (G1285, Solarbio, China) or Massion's Trichrome staining kit (G1340, Solarbio, China) Stain sections according to the operating manual. After staining, sections were dehydrated in increasing concentrations of ethanol and xylene.
1.3细胞培养1.3 Cell culture
人足细胞购自Bena Culture Collection(中国北京)，并在无血清的McCoy's 5A培养基(改良)中与L-谷氨酰胺(Biological Industries)一起培养。HEK293T细胞购自中国科学院上海细胞研究所，并在高葡萄糖Dulbecco改良的Eagle培养基(以色列生物产业，01-052-1)中进行培养。将细胞与10％胎牛血清(FBS)(CLARK，澳大利亚)，青霉素(100U)和链霉素(100μg/ml)在5％CO2的湿润气氛中于37℃培养。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 the Shanghai Institute of Cell Research, Chinese Academy of Sciences, and cultured in high glucose Dulbecco's modified Eagle medium (Israel BioIndustry, 01-052-1). Cells were cultured with 10% fetal bovine serum (FBS) (CLARK, Australia), penicillin (100 U) and streptomycin (100 μg/ml) in a humidified atmosphere of 5% CO2 at 37°C.
1.4抗体和试剂1.4 Antibodies and reagents
本申请中使用的抗体包括多克隆兔抗SIRT2(S8447，Sigma)，多克隆山羊抗Septin4(ab166788，abcam)，单克隆小鼠抗Flag(GNI4110-FG，GNI，Japan)，单克隆小鼠抗Myc(免疫沉淀：2276S，细胞信号传导；免疫印迹：GNI4110-MC，GNI，日本)，单克隆小鼠抗GAPDH(10494-1-AP，Proteintech)，多克隆兔抗CBP(7389S，细胞信号传导)，抗乙酰化-赖氨酸(9441S，细胞信号转导)，多克隆兔抗切割的PARP(5625S，细胞信号转导)，多克隆兔抗切割的Caspase3(19677-1-AP，Proteintech)。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; Western blot: GNI4110-MC, GNI, Japan), monoclonal mouse anti-GAPDH (10494-1-AP, Proteintech), polyclonal rabbit anti-CBP (7389S, Cell Signaling) ), anti-acetylation-lysine (9441S, Cell Signaling), polyclonal rabbit anti-cleavage PARP (5625S, Cell Signaling), polyclonal rabbit anti-cleavage Caspase3 (19677-1-AP, Proteintech) .
AngII(A9525)购自Sigma、AGK2(B7323)购自Apexbio、烟酰胺(NAM，S1899)和曲古他汀A(TSA，S1045)购自Selleck。PI(碘化丙啶，ST511)来自Beyotime。细胞计数试剂盒8(CCK8，B34304，Bimake，USA)来自Selleck。Phalloidin-FITC(AAT-23102)来自Bioquest。AngII (A9525) was purchased from Sigma, AGK2 (B7323) from Apexbio, nicotinamide (NAM, S1899) and tricugustatin A (TSA, S1045) from Selleck. PI (propidium iodide, ST511) was from Beyotime. Cell counting kit 8 (CCK8, B34304, Bimake, USA) was from Selleck. Phalloidin-FITC (AAT-23102) is from Bioquest.
1.5质粒构建和转染1.5 Plasmid construction and transfection
将人类全长Septin4(Gene ID:5414)和携带K174R突变的Septin4(GeneChem，中国)克隆到3Flag GV141载体中，构建了四个截短的Septin4质粒，它们包含不同的域：具有标志标签的Septin4 N末端结构域；具有标志标签的Septin4C末端结构域；具有标志标签的Septin4C末端和催化GTP酶结构域。将全长人SIRT2(Gene ID:22933)克隆到pCMV-Myc-N(日本TAKARA)和pcDNA3.1-flag/HA。使用快速更换定点诱变试剂盒(Stratagene，CA，美国)产生SIRT2 H187Y，Q167A突变质粒。Flag-P300，Flag-CBP和Myc-GCN5获得自Qunying Lei(中国上海医科大学)。Flag-PCAF获得自Weiguo Zhu(深圳大学，中国深圳)。根据制造商的说明，使用Lipofectamine 3000(Invitrogen，California，USA)进行质粒转染。转染后36-48小时收集细胞。Human full-length Septin4 (Gene ID: 5414) and Septin4 carrying K174R mutation (GeneChem, China) were cloned into the 3Flag GV141 vector, and four truncated Septin4 plasmids were constructed, which contain different domains: Septin4 with flag tag N-terminal domain; 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.
1.6质粒构建，抗体和试剂1.6 Plasmid construction, antibodies and reagents
SIRT2和Septin4shRNA慢病毒购自GeneChem。进行稳定基因敲减细胞系的构建。简而言之，根据制造商的说明从HEK293T细胞中收集慢病毒。将慢病毒颗粒与5×PEG-itTM Solution(System Biosciences，美国)混合。用慢病毒感染6孔培养板中的新鲜铺板细胞。用嘌呤霉素(10μg/ml)选择稳定的细胞系7天。最后，通过蛋白质印迹证实了靶细胞的感染效率。SIRT2 and Septin4 shRNA lentivirus were purchased from GeneChem. Construct stable gene knockdown cell lines. Briefly, lentivirus was collected from HEK293T cells according to the manufacturer's instructions. Lentiviral particles were mixed with 5×PEG-itTM Solution (System Biosciences, USA). Infect freshly plated cells in 6-well culture plates with lentivirus. 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.
SEQ ID NO:2 shSirt2靶序列22296：TGCTCATCAACAAGGAGAA SEQ ID NO:3shSirt2靶序列22297：TAAGCTGGATGAAAAAAGAGAASEQ ID NO: 2 shSirt2 target sequence 22296: TGCTCATCAACAAGGAGAA SEQ ID NO: 3 shSirt2 target sequence 22297: TAAGCTGGATGAAAAAAGAGAA
SEQ ID NO:4 shSirt2靶序列22298：CAACCATCTGTCACTACTT SEQ ID NO:5shSeptin4靶序列72648：ccTAAAGGAAAGCATCCCATTSEQ ID NO:4 shSirt2 target sequence 22298: CAACCATCTGTCACTACTT SEQ ID NO:5 shSeptin4 target sequence 72648: ccTAAAGGAAAGCATCCCATT
SEQ ID NO:6shSeptin4靶序列72649：ccTAAAGGAAAGCATCCCATTSEQ ID NO:6shSeptin4 target sequence 72649: ccTAAAGGAAAGCATCCCATT
SEQ ID NO:7shSeptin4靶序列72650：ccTAAAGGAAAGCATCCCATTSEQ ID NO:7shSeptin4 target sequence 72650:ccTAAAGGAAAGCATCCCATT
1.7免疫沉淀和免疫印迹1.7 Immunoprecipitation and Western Blotting
为了进行乙酰化免疫沉淀，将细胞用磷酸盐缓冲液(PBS)洗涤3次，并用标志裂解缓冲液(137mM NaCl，10mM NaF，50mM Tris-HCl(pH 7.6)，1mM EDTA，0.1mM Na3VO4、10％甘油，1％NonidetP-40(NP-40)和1mM PMSF(蛋白酶抑制剂))裂解。另外，将5μMTSA和20mM NAM添加到细胞裂解缓冲液中。将细胞裂解物与抗FlagAffinity Gel(B23102，biotool，USA)在4℃孵育12小时，或与适当的抗体在4℃孵育3小时，然后与ProteinA/G免疫沉淀磁珠(B23202)孵育，biotool)在4℃下放置12小时。然后将蛋白质-抗体复合物用冷标记裂解缓冲液在4℃洗涤3次，并用SDS上样洗脱。For acetylation immunoprecipitation, cells were washed 3 times with phosphate buffered saline (PBS) and lysed with Flag lysis buffer (137mM NaCl, 10mM NaF, 50mM Tris-HCl (pH 7.6), 1mM EDTA, 0.1mM Na 3 VO 4. 10% glycerol, 1% NonidetP-40 (NP-40) and 1mM PMSF (protease inhibitor)). Additionally, add 5 μM TSA and 20 mM NAM to the cell lysis buffer. Cell lysates were incubated with anti-FlagAffinity Gel (B23102, biotool, USA) for 12 h at 4°C or with appropriate antibodies for 3 h at 4°C and then incubated with ProteinA/G immunoprecipitation magnetic beads (B23202, biotool) Leave at 4°C for 12 hours. The protein-antibody complexes were then washed three times with cold labeling lysis buffer at 4°C and eluted with SDS loading.
1.8乙酰化测定1.8 Acetylation determination
用TSA(5μM，16h)和NAM(5mM，4h)处理细胞，然后收获并裂解以进行免疫沉淀和蛋白质印迹分析。此外，为了进一步研究SIRT2诱导的Septin4脱乙酰化，将细胞与SIRT2特异性抑制剂AGK2(10μM)孵育24小时。Cells were treated with TSA (5 μM, 16 h) and NAM (5 mM, 4 h), then harvested and lysed for immunoprecipitation and Western blot analysis. In addition, to further study SIRT2-induced Septin4 deacetylation, cells were incubated with SIRT2-specific inhibitor AGK2 (10 μM) for 24 h.
1.9细胞增殖分析1.9 Cell proliferation analysis
将细胞以3×103个细胞/孔的密度一式三份地接种在96孔板中。在37℃下将Basic McCoy的5A培养基(90μl)和CCK8染色溶液(10μl)添加到细胞中2小时。使用吸光度读数器(瑞士TECAN)测量450nm处的吸光度。Cells were seeded in triplicate in 96-well plates at a density of 3 × 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).
1.10 FITC-phalloidin测定法1.10 FITC-phalloidin assay
用质粒瞬时转染细胞24小时。第二天，将细胞以3×104细胞/孔的密度接种到24孔板中。24小时后，通过适当浓度的AngII诱导细胞48小时。然后除去培养基，并根据Bioquest的指示用37℃预热的PBS洗涤细胞两次，并使用phalloidin-FITC(AAT-23102)测定。使用荧光显微镜(Olympus)使细胞成像。Cells were transiently transfected with plasmid for 24 hours. The next day, cells were seeded into 24-well plates at a density of 3 × 10 cells/well. After 24 h, cells were induced by appropriate concentrations of AngII for 48 h. The medium was then removed and the cells were washed twice with 37°C 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分析1.11 PLA analysis
按照Fluorescence手册(DUO9210-1-1KT，sigma-Aldrich)进行PLA分析。载玻片上的细胞用4％PFA固定15分钟。随后，用Triton X-100将载玻片透化15分钟。将封闭溶液添加到每个载玻片中，并将载玻片在预热的湿度室中于37℃孵育30分钟。将带有稀释的一抗的载玻片在4℃孵育过夜。从载玻片上抽出一级抗体溶液，并在1xWash BufferA中洗涤。加入PLA探针溶液，并在预热的湿度箱中于37℃孵育1小时。从载玻片上取下PLA探针溶液，并用1x洗涤缓冲液A洗涤。加入具有连接酶的连接溶液，并在预热的湿度箱中于37℃孵育30分钟。从载玻片上敲出连接溶液，并在1x洗涤缓冲液A中洗涤。将扩增-聚合酶溶液添加至每个样品中，并在预热的湿度室中于37℃温育100分钟。最终，从载玻片上敲出扩增-聚合酶溶液，并在1x洗涤缓冲液B中洗涤，随后在0.01x洗涤缓冲液B中洗涤。带有DAPI的Duolink Insitu Mounting Medium用盖玻片固定在载玻片上。使用荧光显微镜(Olympus)使细胞成像。according to Fluorescence manual (DUO9210-1-1KT, sigma-Aldrich) for PLA analysis. Cells on the slides were fixed with 4% PFA for 15 minutes. Subsequently, the slides were permeabilized with Triton X-100 for 15 minutes. Add blocking solution to each slide and incubate the slides in a preheated humidity chamber at 37 °C for 30 min. Incubate slides with diluted primary antibodies overnight at 4°C. Aspirate the primary antibody solution from the slide and wash in 1xWash BufferA. Add PLA probe solution and incubate for 1 hour at 37 °C in a preheated humidity chamber. Remove the PLA probe solution from the slide and wash with 1x Wash Buffer A. Add ligation solution with ligase and incubate at 37 °C for 30 min in a preheated humidity chamber. Tap the ligation solution from the slides and wash in 1x Wash Buffer A. Amplification-polymerase solution was added to each sample and incubated in a preheated humidity chamber at 37°C for 100 minutes. Finally, the amplification-polymerase solution was knocked out of the slides and washed in 1x Wash Buffer B followed by 0.01x Wash Buffer B. Duolink Insitu Mounting Medium with DAPI is mounted on the slide with a coverslip. Cells were imaged using a fluorescence microscope (Olympus).
1.12统计分析1.12 Statistical analysis
数据表示为平均值±标准偏差(SD)。方差的同质性通过F检验(组对)进行评估。进行了Shapiro-Wilk测试以评估数据的正常性。使用两尾学生t检验对连续变量(表示为平均值±SD)评估组之间的差异。进行了一种单因素方差分析，两种方法的方差分析和指示性非参数检验以比较多个组之间的差异。如果适用，可以对P值进行多次比较调整。所有统计分析均使用SPSS 22.0版软件(SPSS Inc，美国芝加哥伊利诺伊州)，P<0.05表示具有统计学意义。Data are expressed as mean ± standard deviation (SD). Homogeneity of variances was assessed by F test (group pairs). Shapiro-Wilk test was performed to assess the normality of the data. Differences between groups were assessed using a two-tailed Student's t test for continuous variables (expressed as mean±SD). One-way ANOVA, two-method ANOVA and indicative non-parametric tests were performed to compare differences between multiple groups. If applicable, P values can be adjusted for multiple comparisons. All statistical analyzes were performed using SPSS version 22.0 software (SPSS Inc, Chicago, IL, USA), and P<0.05 indicated statistical significance.
二、结果与分析2. Results and analysis
2.1 SIRT2通过与损伤相关蛋白-Septin4相互作用而参与AngII诱导的肾足细胞损伤。2.1 SIRT2 participates in AngII-induced renal podocyte injury by interacting with the injury-related protein-Septin4.
为了确定受损肾脏中Sirtuins亚基表达谱，申请人通过Ang II输注减轻了野生型(WT)小鼠的高血压肾损伤，并验证了通过iTRAQ/TMT/label free分析获得的Sirtuins亚基表达水平，在上海应用蛋白技术有限公司进行的LC-PRMMS分析进一步定量了Sirtuins亚基蛋白的表达水平。申请人发现，在7种Sirtuins亚基蛋白中，Ang II输注14天后，SIRT2在受伤的肾脏中上调最高(图1A)。结果表明SIRT2在高血压肾损伤中起重要作用。To determine the Sirtuins subunit expression profile in damaged kidneys, Applicants attenuated hypertensive renal injury in wild-type (WT) mice via Ang II infusion and validated the Sirtuins subunits obtained by iTRAQ/TMT/label-free analysis Expression levels, LC-PRMMS analysis performed at Shanghai Applied Protein Technology Co., Ltd. further quantified the expression levels of Sirtuins subunit proteins. Applicants found that among the seven Sirtuins subunit proteins, SIRT2 was the most upregulated in injured kidneys 14 days after Ang II infusion (Figure 1A). The results indicate that SIRT2 plays an important role in hypertensive kidney injury.
为了进一步证实SIRT2在高血压性肾损伤中的作用，使用不同浓度的AngII体外诱导人足细胞的肾足细胞损伤(图1B)。申请人发现，SIRT2的表达在此浓度梯度中逐渐增加(图1B，D)。与以前的结果一致，SIRT2在Ang II诱导的小鼠中也高度表达(图1C，E)。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 1B). Applicants found that the expression of SIRT2 gradually increased in this concentration gradient (Figure 1B, D). Consistent with previous results, SIRT2 was also highly expressed in Ang II-induced mice (Fig. 1C,E).
为了进一步探讨SIRT2在高血压肾损伤中的机制，申请人使用质谱法(图1F)鉴定了潜在的蛋白质相互作用分子。除了已知的依赖SIRT2的脱乙酰基作用的靶蛋白外，申请人还将重点放在与细胞凋亡相关的Septin4蛋白上。首先，申请人通过共免疫沉淀(Co-IP)研究了内源性SIRT2和Septin4之间的蛋白质相互作用(图1G)。此外，SIRT2-Septin4相互作用还通过外源过度表达带有Flag标签的Septin4(图1H)来证明。接下来，将Flag-tagged-Septin4转染到足细胞中，并通过PLA分析确认Flag-tagged-Septin4和SIRT2之间的相互作用(图1I)。To further explore the mechanism of SIRT2 in hypertensive kidney injury, Applicants identified potential protein-interacting molecules using mass spectrometry (Figure 1F). In addition to known target proteins for SIRT2-dependent deacetylation, the applicants will focus on the Septin4 protein, which is associated with apoptosis. First, Applicants studied the protein interaction between endogenous SIRT2 and Septin4 by co-immunoprecipitation (Co-IP) (Figure 1G). In addition, SIRT2-Septin4 interaction was also demonstrated by exogenous overexpression of Flag-tagged Septin4 (Fig. 1H). Next, Flag-tagged-Septin4 was transfected into podocytes, and the interaction between Flag-tagged-Septin4 and SIRT2 was confirmed by PLA analysis (Figure 1I).
因此，以上结果证实Septin4是SIRT2的新的相互作用蛋白，SIRT2可能通过与Septin4相互作用而参与AngII诱导的肾足细胞损伤。Therefore, the above results confirm that Septin4 is a new interacting protein of SIRT2, and SIRT2 may participate in AngII-induced renal podocyte injury by interacting with Septin4.
2.2SIRT2与Septin4的GTPase结构域结合，而Septin4的GTPase结构域则是通过赖氨酸174作为SIRT2依赖的脱乙酰基作用的靶标。2.2 SIRT2 binds to the GTPase domain of Septin4, and the GTPase domain of Septin4 serves as the target of SIRT2-dependent deacetylation through lysine 174.
根据UniProt数据库，Septin4包含三个功能域，包括N端，C端和GTPase域(图2B)。使用内源性SIRT2和全长Flag-tagged-Septin4或各种截短的Flag-Septin4质粒，申请人证明了SIRT2与Septin4的GTPase结构域结合(图2A)。这些数据表明SIRT2与Septin4的GTPase结构域相互作用，而AngII增强了结合。接下来，申请人验证SIRT2是否可以调节Septin4的乙酰化活性。用曲古抑菌素A(TSA)和烟酰胺(NAM)治疗后，Septin4的乙酰化水平增加，这两种常用的脱乙酰基酶抑制剂可抑制组蛋白脱乙酰基酶HDAC I和III和Sirtuins家族的脱乙酰基酶(图2C)。接下来，为了鉴定Septin4的乙酰基转移酶，分别转染了四个乙酰基转移酶，包括p300(E1A结合蛋白，300kDa)，CBP，PCAF(p300/CBP相关因子)或GCN5(KAT2A)。申请人发现CBP的过表达，而不是其他乙酰转移酶的过表达，显著增强了Septin4的乙酰化水平(图2E)。此外，内源性CBP与Septin4结合(图2D)。因此，CBP被证明是Septin4的乙酰基转移酶。接下来，为确认SIRT2可以使Septin4脱乙酰，申请人使用三个shRNA片段构建了稳定的SIRT2敲减细胞系。申请人发现22297片段产生了最佳的敲减效率(未示出)，因此，申请人使用有或没有20μmol/LAGK2(通常使用的SIRT2特异性抑制剂)的正常对照和shSIRT2细胞。与先前的结果一致，与正常对照细胞相比，shSIRT2细胞或用AGK2处理的细胞中Septin4的乙酰化水平更高(图2G)。接下来，野生型(WT)SIRT2的过表达减少了内源性Septin4乙酰化，而SIRT2的催化失活突变体(H187YQ167A)的转染则没有效果(图2F)。为了研究Septin4上被SIRT2脱乙酰化的特定位点，申请人随后使用定点诱变将赖氨酸(K)174乙酰化位点突变为精氨酸(R，不可乙酰化突变体)。转染野生型(WT)-Septin4或K174R突变质粒，同时与Flag对照或Flag-CBP质粒一起转染。有或没有CBP时，K174的精氨酸取代会导致Septin4乙酰化消失，而CBP会增加WT-Septin4的脱乙酰基水平(图2H)。同样，与WT-Septin4相比，K174的精氨酸取代会导致Septin4乙酰化在有或没有SIRT2过表达的情况下消失(图2I)。这些发现表明，CBP是Septin4 K174的乙酰基转移酶，Septin4 K174是SIRT2依赖性脱乙酰基作用的靶标。According to the UniProt database, Septin4 contains three functional domains, including N-terminal, C-terminal and GTPase domains (Figure 2B). Using endogenous SIRT2 and full-length Flag-tagged-Septin4 or various truncated Flag-Septin4 plasmids, Applicants demonstrated that SIRT2 binds to the GTPase domain of Septin4 (Figure 2A). These data indicate that SIRT2 interacts with the GTPase domain of Septin4 and that AngII enhances binding. Next, Applicants verified whether SIRT2 could modulate the acetylation activity of Septin4. Septin4 acetylation levels are increased after treatment with trichostatin A (TSA) and nicotinamide (NAM), two commonly used deacetylase inhibitors that inhibit the histone deacetylase HDAC I and III and Deacetylase of the Sirtuins family (Fig. 2C). Next, to identify the acetyltransferase of Septin4, four acetyltransferases were transfected separately, including p300 (E1A binding protein, 300 kDa), CBP, PCAF (p300/CBP-related factor) or GCN5 (KAT2A). Applicants found that overexpression of CBP, but not other acetyltransferases, significantly enhanced the acetylation level of Septin4 (Figure 2E). Furthermore, endogenous CBP bound to Septin4 (Fig. 2D). Therefore, CBP was shown to be an acetyltransferase of Septin4. Next, to confirm that SIRT2 can deacetylate Septin4, the Applicants used three shRNA fragments to construct a stable SIRT2 knockdown cell line. Applicants found that the 22297 fragment produced the best knockdown efficiency (not shown), so Applicants used normal control and shSIRT2 cells with or without 20 μmol/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 (Fig. 2G). Next, overexpression of wild-type (WT) SIRT2 reduced endogenous Septin4 acetylation, whereas transfection of a catalytically inactive mutant of SIRT2 (H187YQ167A) had no effect (Fig. 2F). To study the specific site on Septin4 that is deacetylated by SIRT2, Applicants 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 loss of Septin4 acetylation in the presence or absence of CBP, while CBP increased the deacetylation level of WT-Septin4 (Fig. 2H). Likewise, arginine substitution of K174 resulted in loss of Septin4 acetylation with or without SIRT2 overexpression compared with WT-Septin4 (Fig. 2I). These findings indicate that CBP is an acetyltransferase of Septin4 K174, which is the target of SIRT2-dependent deacetylation.
2.3 SIRT2通过脱乙酰基修饰Septin4减轻了AngII诱导的肾足细胞损伤。2.3 SIRT2 alleviates AngII-induced renal podocyte injury by deacetylating Septin4.
为了充分理解SIRT2-Septin4信号转导在高血压肾损伤中的作用，申请人证实AngII引起了肾足细胞中SIRT2和Septin4之间的结合增加(图3A)。此外，AngII诱导了Septin4的乙酰化水平下调，而在shSIRT2肾足细胞中则升高了，但是在shSIRT2肾足细胞中SIRT2的重新表达后，Septin4的乙酰化水平得以恢复(图3B)。随后，申请人使用正常对照和shSIRT2肾足细胞，伴或不伴有10-5mol/LAngII诱导的肾足细胞损伤。ShSIRT2细胞显示出对肾脏足细胞损伤的应答，其Cleaved-PARP1水平升高，而在SIRT2缺失的肾足细胞中WT-SIRT2的瞬时重新表达挽救了该损伤(图3C-D)。与这些发现一致，shSIRT2肾足细胞中细胞骨架的分解更为广泛，而SIRT2耗尽的肾足细胞中WT-SIRT2的瞬时再表达挽救了该分解作用(图3F，G)。使用CCK8分析获得了相似的结果(图3E)。总之，SIRT2可以缓解AngII诱导的肾足细胞的损伤，Septin4可能参与了反应。To fully understand the role of SIRT2-Septin4 signaling in hypertensive renal injury, Applicants demonstrated that AngII caused increased binding between SIRT2 and Septin4 in renal podocytes (Figure 3A). In addition, AngII induced the down-regulation of Septin4 acetylation levels, which was increased in shSIRT2 renal podocytes, but was restored after re-expression of SIRT2 in shSIRT2 renal podocytes (Fig. 3B). Applicants subsequently used normal control and shSIRT2 renal podocytes with or without renal podocyte injury induced by 10 −5 mol/LAngII. ShSIRT2 cells showed increased levels of Cleaved-PARP1 in response to renal podocyte injury, whereas transient reexpression of WT-SIRT2 in SIRT2-depleted renal podocytes rescued the injury (Fig. 3C-D). Consistent with these findings, cytoskeletal disassembly was more extensive in shSIRT2 renal podocytes, whereas transient reexpression of WT-SIRT2 in SIRT2-depleted renal podocytes rescued this disassembly (Fig. 3F,G). Similar results were obtained using CCK8 analysis (Fig. 3E). In summary, SIRT2 can alleviate AngII-induced renal podocyte injury, and Septin4 may be involved in the response.
2.4参与AngII诱导的肾足细胞损伤的Septin4依赖于SIRT2调控的Septin4-K174。2.4 Septin4 involved in AngII-induced renal podocyte injury depends on Septin4-K174 regulated by SIRT2.
申请人的发现表明，SIRT2通过K174的脱乙酰作用来调节Septin4。然而，经由SIRT2使Septin4脱乙酰化在高血压肾损伤中的作用仍不清楚。因此，申请人使用三个shRNA片段构建了稳定的Septin4敲减(shSeptin4)肾足细胞，并确认了其中72650片段的敲减效率最高；因此，随后的实验使用了稳定的敲减细胞系。此外，申请人检测了在有或没有10-5mol/L AngII的敲减肾足细胞中，shSeptin4中Cleaved-PARP1和Cleaved-Caspase3的表达，WT-Septin4和K174R-Septin4(形式为模拟的SIRT2脱乙酰化Septin4)的瞬时重新表达诱导肾脏足细胞损伤。如图4A-B所示，WT-Septin4重新表达后Cleaved-PARP1和Cleaved-Caspase3的水平高于shSeptin4，而在Septin4缺失的肾足细胞和shSeptin4肾足细胞中，K174R的瞬时重新表达之间没有显著差异。与先前的结果一致，在shSeptin4肾足细胞中WT-Septin4重新表达后的细胞骨架崩解大于shSeptin4肾足细胞中的细胞骨架崩解，而在shSeptin4细胞中K174R-Septin4的瞬时再表达与shSeptin4肾脏足细胞相比没有差异(图4C，E)。使用CCK8分析获得了相似的结果(图4D)。综上所述，SIRT2通过使Lys174Septin4脱乙酰化，缓解了AngII诱导的肾足细胞的损伤。Applicants' findings indicate that SIRT2 regulates Septin4 through deacetylation of K174. However, the role of Septin4 deacetylation via SIRT2 in hypertensive renal injury remains unclear. Therefore, the applicant used three shRNA fragments to construct stable Septin4 knockdown (shSeptin4) renal podocytes, and confirmed that the 72650 fragment had the highest knockdown efficiency; therefore, subsequent experiments used stable knockdown cell lines. In addition, Applicants examined the expression of Cleaved-PARP1 and Cleaved-Caspase3 in shSeptin4, WT-Septin4 and K174R-Septin4 (formed as simulated SIRT2) in knockdown renal podocytes with or without 10 -5 mol/L AngII. Transient reexpression of deacetylated Septin4 induces renal podocyte injury. As shown in Figure 4A-B, the levels of Cleaved-PARP1 and Cleaved-Caspase3 after re-expression of WT-Septin4 were higher than those of shSeptin4, while there was no difference between transient re-expression of K174R in Septin4-depleted renal podocytes and shSeptin4 renal podocytes. significant difference. Consistent with previous results, cytoskeletal collapse upon reexpression of WT-Septin4 in shSeptin4 renal podocytes was greater than that in shSeptin4 renal podocytes, whereas transient reexpression of K174R-Septin4 in shSeptin4 cells was associated with greater There was no difference compared with podocytes (Figure 4C,E). Similar results were obtained using CCK8 analysis (Fig. 4D). In summary, SIRT2 alleviates AngII-induced renal podocyte injury by deacetylating Lys174Septin4.
2.5 SIRT2基因敲除小鼠显示Septin4的高乙酰化水平，并显著加重了AngII诱导的高血压肾损伤。2.5 SIRT2 knockout mice display high acetylation levels of Septin4 and significantly aggravate AngII-induced hypertensive renal injury.
高血压会导致肾脏进行性损害；在早期，肾体积和肾小管上皮细胞肿胀和肾小球系膜基质沉积增加。为了调查SIRT2在高血压肾损伤中的作用。用渗透微型泵输注2周AngII，用于在体内建立SIRT2-WT和SIRT2-/-C57BL/6小鼠的高血压肾损伤模型。申请人发现AngII引起的高血压损伤后，SIRT2-WT肾脏组织中SIRT2的表达显著增加(图5A，E)，而SIRT2-/-小鼠不表达SIRT2。Hypertension causes progressive renal damage; 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 for 2 weeks using an osmotic minipump to establish hypertensive renal injury models in SIRT2-WT and SIRT2-/-C57BL/6 mice in vivo. Applicants found that the expression of SIRT2 in SIRT2-WT kidney tissue increased significantly after AngII-induced hypertensive injury (Fig. 5A, E), while SIRT2-/- mice did not express SIRT2.
另外，通过免疫共沉淀在高血压肾损伤小鼠中检测到SIRT2和Septin4之间的相互作用(图5B)和Septin4的乙酰化水平(图5C)。如结果所示，与足细胞结果一致，AngII诱导其相互作用增加，而SIRT2基因敲除小鼠中Septin4的乙酰化水平增加。In addition, the interaction between SIRT2 and Septin4 (Fig. 5B) and the acetylation level of Septin4 (Fig. 5C) were detected in hypertensive renal injury mice by co-immunoprecipitation. As shown in the results, consistent with the podocyte results, AngII induced an increase in its interaction, and the acetylation level of Septin4 increased in SIRT2 knockout mice.
然后，申请人评估了高血压肾损伤是否伴有损伤相关蛋白的表达变化。与SIRT2-WT组相比，SIRT2-/-组的Cleaved-PARP1和Cleaved-Caspase3含量显著升高(图5D，F)。因此，SIRT2基因敲除小鼠在高血压肾损伤中加剧细胞凋亡。因此，申请人认为SIRT2可能与体内高血压性肾损伤有关。接下来，申请人在高血压损伤早期通过H&E染色和AZAN三色染色，评估了SIRT2在肾小管上皮细胞水肿和肾小球系膜基质过多中的作用。如预期，H&E和Azan三色染色显示，与SIRT2-WT小鼠相比，AngII诱导后SIRT2敲减显著加重了肾小管水肿程度并增加了肾小球系膜基质面积(图5G-H)。随后，肾损伤的晚期可能发生肾小球硬化和肾纤维化。进行PAS和Massion染色以评估SIRT2-WT和SIRT2-/-小鼠的肾小球硬化和肾纤维化程度。如图5K-L所示，高血压肾脏损伤后，SIRT2-/-小鼠的节段性硬化和纤维化区域均大于SIRT2-WT小鼠中的节段性硬化和纤维化区域(P<0.001)(图5M-N)。这些结果表明，SIRT2基因敲减在晚期高血压肾损伤中显著加重了肾小球硬化和纤维化。Applicants then assessed whether hypertensive renal injury is accompanied by changes in the expression of injury-associated proteins. Compared with the SIRT2-WT group, the levels of Cleaved-PARP1 and Cleaved-Caspase3 in the SIRT2-/- group were significantly increased (Figure 5D, F). Therefore, SIRT2 knockout mice have exacerbated apoptosis in hypertensive renal injury. Therefore, the applicants believe that SIRT2 may be associated with hypertensive renal injury in vivo. Next, the Applicants evaluated the role of SIRT2 in tubular epithelial cell edema and glomerular mesangial matrix excess in the early stages of hypertensive injury by H&E staining and AZAN trichrome staining. As expected, H&E and Azan trichrome staining showed that SIRT2 knockdown after AngII induction significantly aggravated tubular edema and increased mesangial matrix area compared with SIRT2-WT mice (Figure 5G-H). Subsequently, glomerulosclerosis and renal fibrosis may occur in late stages of renal injury. PAS and Massion staining were performed to evaluate the degree of glomerulosclerosis and renal fibrosis in SIRT2-WT and SIRT2-/- mice. As shown in Figure 5K-L, 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 5M-N). These results indicate that SIRT2 knockdown significantly aggravates glomerulosclerosis and fibrosis in advanced hypertensive renal injury.
总而言之，SIRT2基因敲减通过脱乙酰基修饰Septin4加剧了AngII引起的高血压性肾损伤。In summary, SIRT2 knockdown exacerbates AngII-induced hypertensive renal injury by deacetylating Septin4.
2.6 SIRT2转基因(超级)小鼠显示Septin4的低乙酰化水平，并能明显缓解AngII引起的高血压肾损伤。2.6 SIRT2 transgenic (super) mice show low acetylation levels of Septin4 and can significantly alleviate AngII-induced hypertensive renal damage.
为了进一步探讨SIRT2在高血压肾损伤中的作用，使用SIRT2转基因小鼠来验证上述实验。如图6A所示，成功构建了SIRT2转基因(超级)小鼠。通过免疫共沉淀法在高血压肾损伤小鼠中检测到Septin4的乙酰化水平(图6B)。SIRT2转基因(超级)小鼠中Septin4的乙酰化水平降低。此外，与WT组相比，SIRT2转基因(超级)组显著减轻了Cleaved-PARP1和Cleaved-Caspase3的量(图6C-D)。因此，SIRT2转基因(超级)小鼠在高血压肾损伤中显示出减弱的凋亡。随后，H&E和Azan三色染色显示与WT小鼠相比，转染SIRT2(超级)可显著缓解AngII诱导后的肾小管水肿程度，并增加肾小球系膜基质的面积(图6E-H)。高血压肾损伤后，SIRT2转基因(超级)小鼠的节段性硬化和纤维化区域均比野生型小鼠小(P<0.001)(图6I-L)。因此，SIRT2转基因(超级)减轻了AngII引起的高血压性肾损伤。这进一步证明SIRT2的Septin4依赖的脱乙酰化调节可减轻高血压性肾损伤。In order to further explore the role of SIRT2 in hypertensive renal injury, SIRT2 transgenic mice were used to verify the above experiments. As shown in Figure 6A, SIRT2 transgenic (super) mice were successfully constructed. The acetylation level of Septin4 was detected in hypertensive renal injury mice by co-immunoprecipitation (Fig. 6B). Septin4 acetylation levels are reduced in SIRT2 transgenic (super) mice. In addition, compared with the WT group, the SIRT2 transgenic (super) group significantly alleviated the amounts of Cleaved-PARP1 and Cleaved-Caspase3 (Fig. 6C-D). Accordingly, SIRT2 transgenic (super) mice display attenuated apoptosis in hypertensive renal injury. Subsequently, H&E and Azan trichrome staining showed that compared with WT mice, transfection of SIRT2 (super) could significantly alleviate the degree of tubular edema induced by AngII and increase the area of glomerular mesangial matrix (Figure 6E-H) . After hypertensive renal injury, SIRT2 transgenic (super) mice had smaller segmental sclerosis and fibrosis areas than wild-type mice (P<0.001) (Figure 6I-L). Therefore, SIRT2 transgene (super) attenuated AngII-induced hypertensive renal injury. This further demonstrates that Septin4-dependent deacetylation regulation of SIRT2 alleviates hypertensive renal injury.
讨论discuss
讨论和结论Discussion and conclusion
申请人的发现表明，Septin4-K174的脱乙酰基可以挽救Septin4敲除的肾足细胞中的肾足细胞损伤。此外，SIRT2基因敲除小鼠显示高乙酰化水平的Septin4，并显著加重了AngII引起的高血压性肾损伤。但是，SIRT2转基因(超级)小鼠的Septin4乙酰化水平较低，并且在AngII引起的高血压性肾损伤中具有相反的作用。这些观察结果揭示了介导Septin4在高血压肾脏损伤中起作用的新型SIRT2调节的脱乙酰途径。此外，K174处的Septin4脱乙酰基为设计治疗方案和靶向药物提供了理论基础。Applicants' findings demonstrate that deacetylation of Septin4-K174 can rescue renal podocyte injury in Septin4 knockout renal podocytes. Furthermore, SIRT2 knockout mice displayed high acetylation levels of Septin4 and significantly aggravated AngII-induced hypertensive renal injury. However, SIRT2 transgenic (super) mice have lower levels of Septin4 acetylation and have opposite effects in AngII-induced hypertensive renal injury. These observations reveal a novel SIRT2-regulated deacetylation pathway that mediates Septin4's role in hypertensive kidney injury. In addition, the deacetylation of Septin4 at K174 provides a theoretical basis for designing therapeutic regimens and targeted drugs.
SIRT2是NAD+依赖的III类组蛋白脱乙酰基酶，在内皮细胞和心脏相关疾病中起重要作用。特定的抑制剂SIRT2，AGK2，降低的H2O2诱导的内皮细胞毒性。此外，激活的SIRT2信号传导减轻了DOX诱导的心脏毒性。SIRT2缺陷型小鼠经历自发性心力衰竭，并在年龄增大时表现出心脏肥大，重塑，纤维化和功能障碍。SIRT2激活可通过抑制NFAT转录因子保护心脏免受衰老相关和异丙肾上腺素引起的病理性心肌肥大的影响。然而，没有证据表明SIRT2在高血压性肾损伤中起作用。SIRT2 is an NAD+-dependent class III histone deacetylase that plays an important role in endothelial cells and heart-related diseases. A specific inhibitor of SIRT2, AGK2, reduces H 2 O 2 -induced endothelial cell toxicity. Furthermore, activated SIRT2 signaling alleviated DOX-induced cardiotoxicity. SIRT2-deficient mice undergo spontaneous heart failure and exhibit cardiac hypertrophy, remodeling, fibrosis, and dysfunction with increasing age. SIRT2 activation protects the heart from aging-related and isoproterenol-induced pathological cardiac hypertrophy by inhibiting the NFAT transcription factor. However, there is no evidence that SIRT2 plays a role in hypertensive kidney injury.
使用iTRAQ/TMT/label free分析和LC-PRMMS分析，申请人发现SIRT2首次涉及高血压肾损伤。在这里，申请人报道在高血压肾损伤的早期阶段，SIRT2基因敲除小鼠表现出明显加重的肾小管水肿，并伴有肾小球细胞外基质的过度分泌。但是，SIRT2转基因(超级)小鼠可以减轻高血压性肾损伤。此外，肾小球硬化和肾纤维化在晚期明显加重。这些结果证实SIRT2在高血压性肾损伤中起保护作用。SIRT2的上调在氧化剂刺激下在脂肪细胞和HUVEC细胞中起重要作用。同样，在申请人的研究中，SIRT2在肾足细胞损伤模型中有作用。SIRT2的重新表达挽救了SIRT2敲减细胞中的细胞骨架解体。此外，SIRT2调节许多取决于NAD+脱乙酰基活性的常见底物，包括FoxO1，FoxO3和NF-κB。SIRT2通过使AMPK的上游激酶LKB132脱乙酰基来促进AMPK的活性，从而保护心脏免受AngII诱导的肥大性刺激。申请人在SIRT2Septin4的下游发现了一个新的凋亡相关蛋白。另外，缺失SIRT2后AngII显著增加了Septin4的表达。这些结果表明Septin4可能参与了高血压肾损伤反应。Using iTRAQ/TMT/label free assays and LC-PRMMS assays, Applicants discovered that SIRT2 is implicated in hypertensive renal injury for the first time. Here, Applicants report that in the early stages of hypertensive renal injury, SIRT2 knockout mice exhibit significantly increased tubular edema accompanied by excessive secretion of glomerular extracellular matrix. However, SIRT2 transgenic (super) mice can attenuate hypertensive kidney damage. In addition, glomerulosclerosis and renal fibrosis are significantly aggravated in the late stages. These results confirm that SIRT2 plays a protective role in hypertensive renal injury. Upregulation of SIRT2 plays an important role in adipocytes and HUVEC cells upon oxidant stimulation. Likewise, in Applicants' studies, SIRT2 had a role in a model of renal podocyte injury. Reexpression of SIRT2 rescued cytoskeletal disorganization in SIRT2 knockdown cells. Furthermore, SIRT2 regulates many common substrates that depend on NAD+ deacetylation activity, including FoxO1, FoxO3, and NF-κB. SIRT2 promotes AMPK activity by deacetylating AMPK's upstream kinase LKB132, thereby protecting the heart from AngII-induced hypertrophic stimulation. The applicant discovered a new apoptosis-related protein downstream of SIRT2Septin4. In addition, AngII significantly increased the expression of Septin4 after deletion of SIRT2. These results indicate that Septin4 may be involved in the response to hypertensive renal injury.
Septin4目前被认为是器官损伤的重要标志蛋白。ARTs(Septin4isform2)可以通过诱导细胞凋亡来参与各种疾病，例如通过调节ISC生态位中的干细胞存活。此外，Septin4在细胞凋亡中起着至关重要的作用，并且可以通过促进细胞凋亡来减轻肝脏纤维化。然而，Septin4和信号转导SIRT2-Septin4在高血压肾病中的作用仍然未知。申请人证实，CBP/SIRT2各自的乙酰转移酶/脱乙酰化酶活性调节Septin4-Lys174的乙酰化作用。此外，申请人发现Septin4 K174的脱乙酰基可以挽救Septin4敲减细胞中的肾足细胞损伤。。Septin4 is currently considered an important marker protein for organ damage. ARTs (Septin4isform2) can be involved in various diseases by inducing apoptosis, such as by regulating stem cell survival in the ISC niche. Furthermore, Septin4 plays a crucial 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. Applicants demonstrate that the respective acetyltransferase/deacetylase activities of CBP/SIRT2 regulate the acetylation of Septin4-Lys174. In addition, Applicants found that deacetylation of Septin4 K174 can rescue renal podocyte injury in Septin4 knockdown cells. .
总而言之，申请人首次确定了高血压病中控制Septin4功能的乙酰化依赖性调节机制。Septin4脱乙酰基可预防高血压肾病。申请人的发现表明，Septin4在SIRT2介导的高血压相关疾病中可能至关重要，提供了SIRT2在高血压肾病中起保护因子作用的潜在机制。这些观察结果进一步表明靶向Septin4 K174脱乙酰基治疗高血压肾病的潜在实用性。In summary, Applicants have identified for the first time an acetylation-dependent regulatory mechanism controlling Septin4 function in hypertension. Septin4 deacetylation protects against hypertensive nephropathy. Applicants' findings indicate that Septin4 may be critical in SIRT2-mediated hypertension-related diseases, providing a potential mechanism by which SIRT2 functions as a protective factor in hypertensive nephropathy. These observations further demonstrate the potential utility of targeting Septin4 K174 deacetylation for the treatment of hypertensive nephropathy.
最后说明的是，以上优选实施例仅用以说明本申请的技术方案而非限制，尽管通过上述优选实施例已经对本申请进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本申请权利要求书所限定的范围。Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present application and are not limiting. Although the present application has been described in detail through the above preferred embodiments, those skilled in the art should understand that the technical solutions can be modified in form and Various changes can be made to the details without departing from the scope defined by the claims of this application.

Claims (10)

  1. 脱乙酰化修饰的Septin4蛋白或其活性片段，其特征在于，与野生型Septin4蛋白相比，所述脱乙酰化修饰的Septin4蛋白或其活性片段包含如SEQ ID NO:1所示的氨基酸序列，其中所述赖氨酸脱乙酰化。Deacetylation-modified Septin4 protein or active fragment thereof, characterized in that, compared with wild-type Septin4 protein, the deacetylation-modified Septin4 protein or active fragment thereof includes the amino acid sequence shown in SEQ ID NO: 1, wherein said lysine is deacetylated.
  2. 根据权利要求1所述的脱乙酰化修饰的Septin4蛋白或其活性片段，其特征在于，所述赖氨酸脱乙酰化通过赖氨酸脱乙酰化酶(KDACs)脱乙酰化修饰实现。The deacetylation-modified Septin4 protein or active fragment thereof according to claim 1, wherein the lysine deacetylation is achieved by deacetylation modification by lysine deacetylase (KDACs).
  3. 药物组合物，其特征在于，所述药物组合物包含如权利要求1所述的脱乙酰化修饰的Septin4蛋白或其活性片段。A pharmaceutical composition, characterized in that the pharmaceutical composition contains the deacetylation-modified Septin4 protein or active fragment thereof according to claim 1.
  4. 根据权利要求3所述的药物组合物，其特征在于，所述药物组合物还包含药学上可接受的稀释剂、赋形剂和/或载体。The pharmaceutical composition according to claim 3, characterized in that the pharmaceutical composition further comprises a pharmaceutically acceptable diluent, excipient and/or carrier.
  5. 制剂，其特征在于，所述制剂使Septin4蛋白脱乙酰化修饰，所述脱乙酰化修饰的Septin4蛋白或其活性片段包含如SEQ ID NO:1所示的氨基酸序列，其中所述赖氨酸脱乙酰化。Preparation, characterized in that the preparation deacetylates the Septin4 protein, and the deacetylated Septin4 protein or its active fragment includes the amino acid sequence shown in SEQ ID NO: 1, wherein the lysine deacetylation Acetylation.
  6. 如权利要求5所示的制剂，其中所述制剂包含赖氨酸脱乙酰化酶。The formulation of claim 5, wherein said formulation comprises lysine deacetylase.
  7. 如权利要求1或2所述的脱乙酰化修饰的Septin4蛋白或其活性片段在制备用于预防或治疗高血压性肾损伤的药物中的用途。The use of the deacetylation-modified Septin4 protein or active fragment thereof according to claim 1 or 2 in the preparation of a medicament for preventing or treating hypertensive renal injury.
  8. 根据权利要求7所述的用途，其特征在于，所述高血压性肾损伤为血管紧张素II诱导的高血压性肾损伤。The use according to claim 7, wherein the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.
  9. 如权利要求3或4所述的药物组合物、权利要求5或6所述的制剂在制备用于预防或治疗高血压性肾损伤的药物中的用途。The use of the pharmaceutical composition according to claim 3 or 4 and the preparation according to claim 5 or 6 in the preparation of medicaments for preventing or treating hypertensive renal injury.
  10. 根据权利要求9所述的用途，其特征在于，所述高血压性肾损伤为血管紧张素II诱导的高血压性肾损伤。 The use according to claim 9, wherein the hypertensive renal injury is angiotensin II-induced hypertensive renal injury.
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ZHANG NAIJIN, ZHANG YING, WU BOQUAN, WU SHAOJUN, YOU SHILONG, LU SAIEN, LIU JINGWEI, HUANG XINYUE, XU JIAQI, CAO LIU, SUN YINGXIAN: "Deacetylation-dependent regulation of PARP1 by SIRT2 dictates ubiquitination of PARP1 in oxidative stress-induced vascular injury", REDOX BIOLOGY, ELSEVIER, NL, vol. 47, 1 November 2021 (2021-11-01), NL , pages 102141, XP093091362, ISSN: 2213-2317, DOI: 10.1016/j.redox.2021.102141 *
ZHANG NAIJIN, ZHANG YING, YOU SHILONG, TIAN YICHEN, LU SAIEN, CAO LIU, SUN YINGXIAN: "Septin4 Prevents PDGF-BB-induced HAVSMC Phenotypic Transformation, Proliferation and Migration by Promoting SIRT1-STAT3 Deacetylation and Dephosphorylation", INTERNATIONAL JOURNAL OF BIOLOGICAL SCIENCES, vol. 16, no. 4, 1 January 2020 (2020-01-01), pages 708 - 718, XP093091352, ISSN: 1449-2288, DOI: 10.7150/ijbs.39843 *

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