WO2023236863A1 - Deacetylation modified baf155 protein and pharmaceutical use thereof - Google Patents
Deacetylation modified baf155 protein and pharmaceutical use thereof Download PDFInfo
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- WO2023236863A1 WO2023236863A1 PCT/CN2023/098023 CN2023098023W WO2023236863A1 WO 2023236863 A1 WO2023236863 A1 WO 2023236863A1 CN 2023098023 W CN2023098023 W CN 2023098023W WO 2023236863 A1 WO2023236863 A1 WO 2023236863A1
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- baf155
- parp1
- sirt2
- protein
- deacetylation
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Classifications
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
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- 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
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- 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
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
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- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
- C12Y305/01017—Acyl-lysine deacylase (3.5.1.17)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- Non-communicable diseases which account for nearly half of cardiovascular diseases (CVD) have surpassed infectious diseases to become a major global pathology. At the same time, cardiovascular disease remains the most lethal pathology globally. With the rapid improvement of living standards and dramatic changes in lifestyle in China, the prevalence and mortality of cardiovascular diseases have increased significantly. With the advent of an aging society, heart disease has become one of the most important health problems worldwide. Ventricular remodeling, including myocardial hypertrophy and fibrosis, forms the etiological basis of heart failure. Poly(ADP-ribose) polymerase 1 (PARP1) is an important damage factor in CVD, especially cardiac remodeling caused by various factors. PARP1 upregulation and enhanced PARP1 activity occur during cardiac remodeling, resulting in extremely high energy consumption by damaged cardiomyocytes. However, it is currently unclear how PARP1 is regulated in cardiac remodeling.
- PARP1 Poly(ADP-ribose) polymerase 1
- SWI/SNF switching type switching/sucrose non-fermenting
- BAF155 also known as SMARCC1
- SMARCC1 As a helicase and ATPase, BAF15 regulates gene transcription by changing the chromatin structure around genes.
- BAF155 has been reported to contribute to a variety of physiological and pathological events, including cancer, development, and more.
- AngII-induced cardiac remodeling was significantly attenuated in BAF155 cardiac muscle-specific knockout mice.
- overexpression of BAF155 in mice significantly exacerbated cardiac remodeling.
- BAF155 binds PARP1 in the PARP1-BRCT domain and inhibits PARP1 ubiquitination at K249 by interfering with WWP2, an important E3 ubiquitin ligase.
- BAF155 was identified as a novel substrate for acetyltransferase CBP and deacetylase SIRT2 under physiological conditions.
- CBP/SIRT2 interacts with BAF155 and acetylates/deacetylates BAF155 at K948.
- the same lysine site of BAF155 is ubiquitinated by WWP2, thereby inducing the downstream degradation of BAF155 through the proteasome.
- Crosstalk between acetylation and ubiquitination of BAF155 dynamically regulates the stability of the BAF155-PARP1 complex in a competitive manner.
- Applicants' studies identify a novel role for BAF155 and its upstream and downstream regulatory mechanisms in cardiac remodeling. Specifically, BAF155 interacts with PARP1, reducing PARP1 degradation and exacerbating cardiac remodeling by inhibiting WWP2.
- BAF155 is regulated by SIRT2-mediated deacetylation, which helps mobilize WWP2 to degrade BAF155 and dissociate the BAF155-PARP1 cardiac remodeling damage complex.
- the findings of this application provide new research directions for the treatment and prevention of cardiac remodeling injury.
- a first aspect of the present invention provides a deacetylation-modified BAF155 protein or an active fragment thereof, wherein, compared with a wild-type BAF155 protein, the deacetylation-modified BAF155 protein or an active fragment thereof comprises SEQ ID NO: 1
- the amino acid sequence shown is in which lysine at position 948 is deacetylated.
- the deacetylation of the lysine at position 948 is achieved by deacetylation modification by lysine deacetylase (KDACs).
- 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 BAF155 protein, and the deacetylated BAF155 protein or active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1 , wherein the lysine at position 948 is deacetylated.
- the cardiac remodeling is AngII-induced cardiac remodeling
- the cardiac remodeling is selected from one or more of cardiac hypertrophy, cardiac fibrosis and/or heart failure.
- the cardiac remodeling is selected from one or more of cardiac hypertrophy, cardiac fibrosis and/or heart failure.
- BAF155 interacts with PARP1, reducing PARP1 degradation and exacerbating cardiac remodeling by inhibiting WWP2.
- BAF155 is regulated by SIRT2-mediated deacetylation, which helps mobilize WWP2 to degrade BAF155 and dissociate the BAF155-PARP1 cardiac remodeling damage complex to prevent cardiac remodeling. This provides new ideas and research directions for the treatment and prevention of cardiac remodeling injury.
- Figure 1A shows BAF155-cWT mice and myocardial-specific BAF155 knockout (BAF155-cKO) mice exposed to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks;
- Figure 1B-C show the results of immunofluorescence and Western blotting respectively, showing that BAF155 is specifically knocked out in cardiac tissue;
- Figures 1E and 1F show increases in left ventricular ejection fraction (EF) % and fractional shortening (FS) %, respectively, in mice;
- Figure 1G shows H&E and WGA staining data, showing that in BAF155-cWT mice, AngII increased the size of cardiomyocytes and the cross-sectional area of cardiomyocytes, while this change in BAF155-cKO mice given AngII was not significant;
- Figure 1J shows that compared with BAF155-cWT mice, BAF155-cKO significantly reduced the AngII-induced expression of mouse ANP, BNP, cleavedcaspase-3 and PARP1;
- Figure 2 shows that BAF155 overexpression significantly exacerbates cardiac hypertrophy and heart failure in a mouse model, where:
- Figure 2A shows the exposure of BAF155-WT and BAF155 transgenic mice (BAF155-TG) to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks;
- Figure 2B shows a Western blot showing that BAF155 is specifically overexpressed in cardiac tissue
- Figure 2C shows that compared with BAF155-WT mice, BAF155-TG significantly aggravated AngII-induced cardiac dysfunction
- Figures 2D and 2E show increases in left ventricular ejection fraction (EF) % and fractional shortening (FS) %, respectively, in mice;
- Figure 2G-H shows that compared with BAF155-WT mice, BAF155-TG significantly exacerbated AngII-induced increases in HW/BW and HW/TL ratios, indicating that overexpression of BAF155 causes cardiac hypertrophy in mice;
- Figure 2I- Figure 2K respectively show that compared with BAF155-WT mice, AngII-induced expression of ANP, BNP, cleavedcaspase-3 and PARP1 were all increased in BAF155-TG mice ( Figure 2I). Similarly, compared with BAF155 - Compared with WT mice, BAF155-TG significantly aggravated AngII-induced myocardial fibrosis in mice ( Figure 2J), and upregulated ⁇ -SMA and collagen I ( Figure 2K);
- FIG. 3 shows that PARP1 acts as a key BAF155-binding protein under physiological conditions and suggests that the BAF155-PARP1 axis may regulate cardiac remodeling, where:
- Figure 3D shows the interaction of BAF155 with the 476-779 amino acid domain of PARP1, that is, the PARP-A-helical domain;
- Figure 4 shows the regulatory mechanism of PARP1 by BAF155, where:
- Figure 4A shows that increased expression of BAF155 leads to synchronized expression of PARP1
- Figure 4B shows that BAF155 silenced with 56438shBAF155 RNA significantly downregulated PARP1 expression
- Figure 4C shows that the abundance of PARP1 gradually decreased in the Flag control group, while BAF155 overexpression maintained PARP1 expression in the presence of increased CHX, a transcription inhibitor used to inhibit PARP1 protein synthesis;
- Figure 4G shows that BAF155 reduced the ubiquitination level in the PARP1-WT group, but not in PARP1-K249R cells, indicating that BAF155 inhibited PARP1 ubiquitination on K249;
- Figures 4H-4K respectively show that under the treatment of MG132, the degradation of PARP1 and BAF155 was blocked by exogenous immunoassay, resulting in enhanced interaction of BAF155 and PARP1 with WWP2 (4H); in addition, after BAF155 knockdown , the binding of PARP1 to WWP2 increased ( Figure 4I), while the overexpression of BAF155 led to the reduced binding of PARP1 to WWP2 ( Figure 4J); when WWP2 was overexpressed, compared with the Flag control group, the overexpression of Flag-BAF155 increased the binding of PARP1 to WWP2 ( Figure 4J). Ubiquitination levels decreased (Fig. 4K);
- Figures 4L to 4O show that the binding of PARP1 to WWP2 and SIRT2, respectively, was enhanced after treatment with AngII and significantly increased in the heart tissue of BAF155-cKO mice compared with WT with or without AngII treatment ( Figure 4L);
- Figure 4M In terms of ubiquitination levels of PARP1, Applicants' data show that AngII induces ubiquitination in heart tissue of WT mice ( Figure 4M); furthermore, ubiquitination of PARP1 increased in BAF155-cKO compared with WT mice significantly increased in mouse heart tissue (Fig.
- Figure 5A-C demonstrates that endogenous and exogenous co-immunoprecipitation confirms that SIRT2 interacts with BAF155;
- FIG. 5G shows that the acetylation level of BAF155 increased after treatment with trichostatin A (TSA) and nicotinamide (NAM);
- Figure 5H shows that overexpression of CBP significantly increased the acetylation level of BAF155
- FIGS. 5I and 5J show that CBP interacts with BAF155 under both endogenous and exogenous conditions
- Figure 5K shows that the acetylation level of BAF155 is upregulated in shSIRT2 cells and cells treated with AGK2 compared with normal control cells;
- Figure 5L shows that BAF155 levels of BAF55 were significantly enhanced in heart tissue samples from SIRT2 knockout animals compared with WT animals;
- FIG. 6A shows SIRT2-regulated mouse cardiac lysine acetylation changes in SIRT2 knockout mice (SIRT2-KO), SIRT2 overexpression mice (SIRT2-TG) and wild-type animals (SIRT2-WT);
- FIG. 7 shows that SIRT2 promotes BAF155-K948 and PARP1-K294 ubiquitination via WWP2, where:
- Figure 7A shows that BAF155 abundance increases after SIRT2 downregulation; Applicants observed similar trends using three fragments of shSIRT2 (61965, 61966, and 61967), and used the 61966-shSIRT2 fragment for subsequent experiments;
- Figure 7B shows that the rate and extent of BAF155 upregulation in the heart tissue of SIRT2-WT mice was increased compared with SIRT2-KO animals administered MG132;
- FIG. 7C shows that BAF155 expression in SIRT2-WT mouse heart tissue significantly decreased after CHX administration, while the SIRT2-KO mouse group maintained very high BAF155 expression over time;
- Figure 7D shows that consistent with degradation through the ubiquitin-proteasome pathway, enhanced ubiquitination levels of BAF155 were detected after Myc-SIRT2 overexpression and MG132 treatment compared with the Flag-control plasmid group;
- Figure 7E shows that the ubiquitination level of BAF155 increased after overexpression of WT-SIRT2-Flag, but not H187YQ167A-SIRT2-Flag (mutated SIRT2 without deacetylation activity);
- Figure 7H shows that SIRT2 overexpression enhanced ubiquitination levels in BAF155-WT cells compared with the BAF155-K948R counterpart, indicating that BAF155 is deacetylated by SIRT2 at K948, leading to ubiquitination at the same site and promoting Degradation of BAF155;
- Figure 7I- Figure 7O demonstrates that WWP2 is involved in SIRT2-mediated deacetylation-induced BAF155 degradation.
- HA-WWP2 is expressed in NC and shSIRT2H9c2 cell lines; among them, the abundance of BAF155 gradually decreased in NC cell lines, while in shSIRT2 cells maintained at high levels in NC cells (Fig. 7I); compared with NC cells, the level of BAF155 ubiquitination mediated by WWP2 was reduced in shSIRT2 treatment (Fig.
- Figure 8 shows proteomic analysis of differential proteins in SIRT2 knockout and transgenic mice indicating that SIRT2 promotes the ubiquitination of BAF155 and PARP1 through WWP2 in vivo, where:
- Figure 8A shows that the binding of BAF155 to PARP1 was enhanced after treatment with AngII compared with SIRT2-WT mouse samples with or without AngII administration, and was significantly enhanced in the heart tissue of SIRT2-KO mice;
- Figures 8B and 8C show that in SIRT2-WT mouse heart tissue, respectively, the ubiquitination levels of BAF155 and PARP1 were reduced after AngII treatment compared with WT mouse samples, and significantly in SIRT2-KO mouse heart tissue. reduce;
- Figures 8D-8H show that compared with SIRT2-WT animals, the binding of WWP2 to BAF155 and PARP1 was reduced in the heart tissue of SIRT2-KO mice administered or not administered AngII (Fig. 8D); conversely, compared with SIRT2-WT animals, Or compared with the SIRT2-WT group without AngII administration, the binding of BAF155 to PARP1 was reduced in the heart tissue of SIRT2-TG mice ( Figure 8E); compared with SIRT2-WT mice, BAF155 in the heart tissue of SIRT2-TG mice and PARP1 ubiquitination levels were significantly increased ( Figures 8F and 8G); furthermore, compared with SIRT2-WT animals, WWP2 interacted with BAF155 and PARP1 in the heart tissue of SIRT2-TG mice administered or not administered AngII. Binding increased (Fig. 8H).
- Conditional cardiomyocyte-specific knockout (KO) mice including Myh6Cre+, BAF155Fl/Fl (BAF155-cKO) and Myh6Cre-, BAF155Fl/Fl (BAF155-cWT), BAF155-WT and BAF155-TG mice (CAG-initiated Sub) obtained from Shanghai Southern Model Biotechnology Co., Ltd.;
- SIRT2 transgenic mice (SIRT2-TG) mice (CAG promoter) were obtained from Shanghai Southern Model Biotechnology Co., Ltd.
- mice 8- to 10-week-old specific pathogen-free (SPF) male mice were included.
- Cardiac remodeling was then induced by incision and subcutaneous implantation of an osmotic minipump (Alzet) in the midscapula according to the manufacturer's instructions. Over the next 14 days, the mice were euthanized by cervical dislocation. All animal studies were approved by the Animal Science Committee of China Medical University (Protocol No. 2019026).
- Specimens were pulverized in liquid nitrogen and placed in 5-mL tubes, supplemented with four volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail, 3 ⁇ M TSA, 50 mM NAM) and sonicated on ice with a Scientz ultrasonic homogenizer. Process 3 times. Subsequently clear by centrifugation at 12,000g and 4°C for 10 minutes. The protein concentration of the resulting supernatant was measured using a BCA kit.
- the peptides were dissolved in IP buffer (100mM NaCl, 1mM EDTA, 50mM Tris-HCl, 0.5% NP-40, pH 8.0) and mixed with prewashed anti-lysine acetylation and anti-ubiquitin residue antibody resin (PTM- 104 and PTM-1104; Hangzhou Jingjie PTM-Bio) and shake gently at 4°C overnight. Wash the antibody resin with IP buffer and deionized water respectively. Finally, the enriched peptides were eluted three times with 0.1% trifluoroacetic acid and washed with C18ZipTips.
- Ionization was performed with an injected capillary ion source and a TIMS-TOFPro mass spectrometer (ion source voltage, 1.6 kV; scan range, 100-1700 mA).
- Parallel accumulated serial fragmentation (PASEF) mode is enabled for data acquisition.
- Precursors with charge states 0 to 5 were selected for fragmentation, and 10 PASEFMS/MS scans were performed per cycle.
- MS/MS scans have a dynamic exclusion time of 30 seconds to prevent multiple scans of the same precursor ion.
- Maxquant (v1.6.15.0) searches raw mass spectrometry data against the Swissprot protein sequence database (Mus_musculus_10090_SP_20201214.fasta), which contains reverse decoy entries and common contaminating proteins. Trypsin/P digestion allows up to 2 missing cleavages, requiring at least 7 amino acids per peptide. The mass error tolerance is 10 ppm for precursor ions and 20 ppm for product ions. Cysteine alkylation (carbamoylmethyl [C]) is considered a fixed modification. Variable modifications are methionine oxidation and N-terminal acetylation. Lysine acetylation and diglycine on lysine were also set as variable modifications for corresponding modification enrichment analysis. The FDR for both protein and PSM identification was 1%.
- UniProt-GOA database www.http://www.ebi.ac.uk/GOA/) was used for GO annotation.
- the obtained protein identifications are mapped to GOIDs based on their single-port IDs.
- InterProScan is used to annotate GO functions based on protein sequence alignment. Proteins were assigned to biological processes, cellular components, and molecular functions as GO terms. Within each category, a two-tailed Fisher test was used to evaluate the enrichment of differentially expressed proteins against all detected proteins; a corrected p ⁇ 0.05 indicates statistical significance.
- Myocardial tissue samples were fixed in formalin (4%) for 4 hours, embedded in paraffin, and sectioned at 5 ⁇ m. After dewaxing in xylene, they were rehydrated with graded ethanol, and then stained with H&E and Masson's trichrome (G1340; Solarbio, China).
- Frozen myocardial tissue sections were examined by immunofluorescence.
- the cross-sectional area of cardiomyocytes was evaluated in images obtained after staining with 5 ⁇ M MWGA (Thermo, USA).
- H9c2 cells were given 10-5 ⁇ M AngII or treated with Nacl for 48 hours. Then, cells were fixed with 4% formalin and treated with WGA (5 ⁇ M) for 10 min at room temperature before fluorescence microscopy analysis.
- Cardiac function measurements were based on interventricular septal dimension (IVSd), left ventricular posterior wall dimension (PWTd), systolic LV internal dimension (LVD), diastolic LV internal dimension (LVDd), and LV mass measurements.
- IVSd interventricular septal dimension
- PWTd left ventricular posterior wall dimension
- LPD systolic LV internal dimension
- RVDd diastolic LV internal dimension
- LV mass measurements were based on left ventricular ejection fraction (EF%) and fractional shortening (FS%).
- HEK293T and H9c2 cells were cultured in high glucose Dulbecco's modified Eagle medium containing 10% FBS (HyClone) at 37°C in a humidified 5% CO2 incubator.
- Lipofectamine 3000 (Invitrogen, USA) was used for transfection. Lentivirus is used for shRNA transduction. Table 2 lists all antibodies used in the study.
- MG132 (A2585), a proteasome inhibitor, and cycloheximide (CHX, A8244) were purchased from Apexbio (USA) and dissolved with DMSO.
- AngII (A9525; Sigma, USA) in DMSO was used at a concentration of 10 ⁇ M.
- AGK2 is provided by Selleck (USA).
- Mouse myocardial tissue samples were lysed using 1% SDS buffer (TrispH7.5, 0.5mMEDTA and 1mMDTT), boiled for 10 minutes, and then saturated with Tris-HCL (pH8.0).
- Cells transfected with HA-tagged (HA)-ubiquitin, full-length human Myc-PARP1 and mutant Myc-PARP1 plasmids; or full-length human Flag-BAF155 and mutant Flag-BAF155 plasmids were also cleaved as described above.
- Cell lysates were sequentially incubated with anti-PARP1 or anti-BAF155 antibodies (1 ⁇ g/mg cell lysate; 4°C) and protein A/G (B23202) or anti-Myc (B26302) or anti-Flag immunoprecipitation magnetic beads (Biotool, USA) 12 Hour.
- BAF155-cKO significantly reduced AngII-induced expression of ANP, BNP, cleavedcaspase-3, and PARP1 in mice compared with BAF155-cWT mice (Fig. 1J).
- the level of AngII-induced myocardial fibrosis in BAF155-cKO mice was significantly reduced (Figure 1K), and the expression of myocardial fibrosis-related proteins ⁇ -SMA and collagen I was reduced ( Figure 1L) .
- BAF155-WT and BAF155 transgenic mice were exposed to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks (Fig. 2A).
- Western blotting showed that BAF155 was specifically overexpressed in cardiac tissue (Fig. 2B).
- BAF155-TG significantly exacerbated AngII-induced cardiac dysfunction (Fig. 2C), as reflected by increases in EF% (Fig. 2D) and FS% (Fig. 2E) in mice compared with BAF155-WT mice.
- 2.3.PARP1 acts as a key BAF155-binding protein under physiological conditions
- 2.4.BAF155 stabilizes PARP1 and reduces PARP1-K249 by inhibiting E3 ubiquitin ligase WWP2 ubiquitination
- AngII induces ubiquitination in WT mouse heart tissue (Figure 4M).
- ubiquitination of PARP1 was significantly increased in heart tissue of BAF155-cKO mice compared with WT mice (Fig. 4M).
- PARP1 binding to WWP2 and SIRT2 was reduced in heart tissue of BAF155-TG mice compared with WT mice (Fig. 4N).
- Ubiquitination of PARP1 was significantly reduced in heart tissue of BAF155-TG mice compared with WT mice (Fig. 4O).
- BAF155 contains five functional domains, including Chromo, SWIRM, SANT, Glu-rich and Pro-rich domains.
- Applicants demonstrated that the SWIRM domain of BAF155 is the binding site for SIRT2 ( Figure 5F).
- Acetylation levels of BAF155 are increased after treatment with trichostatin A (TSA) and nicotinamide (NAM), members of the histone deacetylase HDACI and III and deacetylase families of inhibitor ( Figure 5G).
- TSA trichostatin A
- NAM nicotinamide
- Figure 5G deacetylase families of inhibitor
- K948 may represent an important acetylation site for BAF155 regulated by SIRT2. K948 is found throughout evolution, from Drosophila melanogaster to mammalian species ( Figure 5Q). To further investigate the critical role of acetylation on K948, an antibody specifically recognizing acetylated K948 of BAF155 was generated (Fig. 5R). The acetylation level of BAF155-K948 increased after administration of TSA and NAM (Fig. 5S). In addition, only CBP increased the acetylation level of BAF155-K948 after exogenous transfection of four acetyltransferases (Fig. 5T).
- SIRT2-KO SIRT2 knockout mice
- SIRT2-TG SIRT2 overexpression mice
- SIRT2-WT wild-type animals
- BAF155 SMARCC1
- SMARCC1 BAF155
- WWP2 E3 ubiquitin ligases
- low-activity SIRT2 retains higher levels of acetylation on K948 of BAF155 and K249 of PARP1, thereby allowing BAF155 and PARP1 to maintain an interactive state and prevent ubiquitination by WWP2.
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Abstract
Description
本申请涉及预防或治疗心脏重塑的药物领域。具体地,本申请涉及一种脱乙酰化修饰的BAF155蛋白或其活性片段,本申请还涉及脱乙酰化修饰的BAF155蛋白或其活性片段的制药用途。The present application relates to the field of drugs for preventing or treating cardiac remodeling. Specifically, the present application relates to a deacetylation-modified BAF155 protein or an active fragment thereof. The present application also relates to the pharmaceutical use of the deacetylation-modified BAF155 protein or an active fragment thereof.
占心血管疾病(CVD)近一半的非传染性疾病(NCDs)已超过传染病成为全球主要病理。同时,心血管疾病仍然是全球最致命的病理学。随着中国生活水平的快速提高和生活方式的巨大改变,心血管疾病的患病率和死亡率显著增加。随着老龄化社会的到来,心脏病已成为全球最重要的健康问题之一。心室重构,包括心肌肥大和纤维化,构成心力衰竭的病因基础。聚(ADP-核糖)聚合酶1(PARP1)是CVD的重要损伤因素,尤其是各种因素引起的心脏重塑。PARP1上调和增强的PARP1活性发生在心脏重塑中,导致受损心肌细胞消耗极高的能量。然而,目前尚不清楚PARP1如何在心脏重塑中受到调节。Non-communicable diseases (NCDs), which account for nearly half of cardiovascular diseases (CVD), have surpassed infectious diseases to become a major global pathology. At the same time, cardiovascular disease remains the most lethal pathology globally. With the rapid improvement of living standards and dramatic changes in lifestyle in China, the prevalence and mortality of cardiovascular diseases have increased significantly. With the advent of an aging society, heart disease has become one of the most important health problems worldwide. Ventricular remodeling, including myocardial hypertrophy and fibrosis, forms the etiological basis of heart failure. Poly(ADP-ribose) polymerase 1 (PARP1) is an important damage factor in CVD, especially cardiac remodeling caused by various factors. PARP1 upregulation and enhanced PARP1 activity occur during cardiac remodeling, resulting in extremely high energy consumption by damaged cardiomyocytes. However, it is currently unclear how PARP1 is regulated in cardiac remodeling.
SWI/SNF(交配型转换/蔗糖非发酵)是一种多亚基ATP依赖性染色质重塑复合物,是基因转录的基本表观遗传调节因子。BAF155,也称为SMARCC1,代表SWI/SNF亚基。作为解旋酶和ATP酶,BAF15通过改变基因周围的染色质结构来调节基因转录。据报道,BAF155有助于多种生理和病理事件,包括癌症、发育等。SWI/SNF (mating type switching/sucrose non-fermenting) is a multi-subunit ATP-dependent chromatin remodeling complex and a fundamental epigenetic regulator of gene transcription. BAF155, also known as SMARCC1, stands for SWI/SNF subunit. As a helicase and ATPase, BAF15 regulates gene transcription by changing the chromatin structure around genes. BAF155 has been reported to contribute to a variety of physiological and pathological events, including cancer, development, and more.
然而,虽然在心脏中,BAF155丰度很高,但其在心肌中的作用尚未明确。However, although BAF155 is highly abundant in the heart, its role in the myocardium is not yet clear.
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,在BAF155心肌特异性敲除小鼠中,AngII诱导的心脏重塑(包括心脏肥大、纤维化和衰竭)显著减轻。相反,在小鼠中过表达BAF155会显著加重心脏重塑。研究发现,BAF155在PARP1-BRCT结构域中结合PARP1,并通过干扰WWP2(一种重要的E3泛素连接酶)抑制PARP1在K249的泛素化。申请人发现,BAF155在生理条件下被鉴定为乙酰转移酶CBP和脱乙酰化酶SIRT2的新型底物。CBP/SIRT2与BAF155相互作用并在BAF155的K948处乙酰化/脱乙酰化。BAF155的相同赖氨酸位点被WWP2泛素化,从而通过蛋白酶体诱导BAF155的下游降解。BAF155的乙酰化和泛素化之间的串扰以竞争方式动态调节BAF155-PARP1复合物的稳定性。总之,申请人的研究确定了BAF155的新作用及其在心脏重塑中的上游和下游调节机制。具体而言,BAF155与PARP1相互作用,通过抑制WWP2减少PARP1的降解并加剧心脏重塑。BAF155受SIRT2介导的脱乙酰化调节,这有助于调动WWP2降解BAF155并解离BAF155-PARP1心脏重塑损伤复合物。本申请的发现为心脏重塑损伤的治疗和预防提供了新的研究方向。The Applicants found that AngII-induced cardiac remodeling, including cardiac hypertrophy, fibrosis and failure, was significantly attenuated in BAF155 cardiac muscle-specific knockout mice. In contrast, overexpression of BAF155 in mice significantly exacerbated cardiac remodeling. The study found that BAF155 binds PARP1 in the PARP1-BRCT domain and inhibits PARP1 ubiquitination at K249 by interfering with WWP2, an important E3 ubiquitin ligase. Applicants discovered that BAF155 was identified as a novel substrate for acetyltransferase CBP and deacetylase SIRT2 under physiological conditions. CBP/SIRT2 interacts with BAF155 and acetylates/deacetylates BAF155 at K948. The same lysine site of BAF155 is ubiquitinated by WWP2, thereby inducing the downstream degradation of BAF155 through the proteasome. Crosstalk between acetylation and ubiquitination of BAF155 dynamically regulates the stability of the BAF155-PARP1 complex in a competitive manner. In summary, Applicants' studies identify a novel role for BAF155 and its upstream and downstream regulatory mechanisms in cardiac remodeling. Specifically, BAF155 interacts with PARP1, reducing PARP1 degradation and exacerbating cardiac remodeling by inhibiting WWP2. BAF155 is regulated by SIRT2-mediated deacetylation, which helps mobilize WWP2 to degrade BAF155 and dissociate the BAF155-PARP1 cardiac remodeling damage complex. The findings of this application provide new research directions for the treatment and prevention of cardiac remodeling injury.
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脱乙酰化修饰的BAF155蛋白或其活性片段,其中,与野生型BAF155蛋白相比,所述脱乙酰化修饰的BAF155蛋白或其活性片段包含如SEQ ID NO:1所示的氨基酸序列,其中位于948位的赖氨酸脱乙酰化。
A first aspect of the present invention provides a deacetylation-modified BAF155 protein or an active fragment thereof, wherein, compared with a wild-type BAF155 protein, the deacetylation-modified BAF155 protein or an active fragment thereof comprises SEQ ID NO: 1 The amino acid sequence shown is in which lysine at position 948 is deacetylated.
根据本发明所述的脱乙酰化修饰的BAF155蛋白或其活性片段,其中,所述位于948位的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 BAF155 protein or active fragment thereof according to the present invention, the deacetylation of the lysine at position 948 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所述的脱乙酰化修饰的BAF155蛋白或其活性片段。A second aspect of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition contains the deacetylation-modified BAF155 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BAF155蛋白脱乙酰化修饰,所述脱乙酰化修饰的BAF155蛋白或其活性片段包含如SEQ ID NO:1所示的氨基酸序列,其中所述位于948位的赖氨酸脱乙酰化。A third aspect of the present invention provides a preparation, wherein the preparation deacetylates the BAF155 protein, and the deacetylated BAF155 protein or active fragment thereof comprises the amino acid sequence shown in SEQ ID NO: 1 , wherein the lysine at position 948 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脱乙酰化修饰的BAF155蛋白或其活性片段在制备用于预防或治疗心脏重塑的药物中的用途。A fourth aspect of the present invention provides the use of deacetylation-modified BAF155 protein or active fragments thereof in the preparation of medicaments for preventing or treating cardiac remodeling.
所述心脏重塑为AngII诱导的心脏重塑;和/或The cardiac remodeling is AngII-induced cardiac remodeling; and/or
所述心脏重塑选自心肌肥大、心脏纤维化和/或心脏衰竭中的一种或多种。 The cardiac remodeling is selected from one or more of cardiac hypertrophy, cardiac fibrosis and/or heart failure.
本发明的第五方面提供了所述的药物组合物、所述的制剂在制备用于预防或治疗心脏重塑的药物中的用途。The fifth aspect of the present invention provides the use of the pharmaceutical composition and the preparation in the preparation of medicaments for preventing or treating cardiac remodeling.
根据本发明所述的应用,所述心脏重塑为AngII诱导的心脏重塑;和/或According to the application of the present invention, the cardiac remodeling is AngII-induced cardiac remodeling; and/or
所述心脏重塑选自心肌肥大、心脏纤维化和/或心脏衰竭中的一种或多种。The cardiac remodeling is selected from one or more of cardiac hypertrophy, cardiac fibrosis and/or heart failure.
与现有技术相比,本申请具有以下有益效果:Compared with the existing technology, this application has the following beneficial effects:
BAF155与PARP1相互作用,通过抑制WWP2减少PARP1的降解并加剧心脏重塑。BAF155受SIRT2介导的去乙酰化调节,这有助于调动WWP2降解BAF155并解离BAF155-PARP1心脏重塑损伤复合物以防止心脏重塑。从而为心脏重塑损伤的治疗和预防提供了新的思路和研究方向。BAF155 interacts with PARP1, reducing PARP1 degradation and exacerbating cardiac remodeling by inhibiting WWP2. BAF155 is regulated by SIRT2-mediated deacetylation, which helps mobilize WWP2 to degrade BAF155 and dissociate the BAF155-PARP1 cardiac remodeling damage complex to prevent cardiac remodeling. This provides new ideas and research directions for the treatment and prevention of cardiac remodeling injury.
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BAF155的心脏特异性敲除减轻了小鼠的心脏重塑,其中:Figure 1 shows that cardiac-specific knockout of BAF155 alleviates cardiac remodeling in mice, where:
其中,图1A为BAF155-cWT小鼠和心肌特异性BAF155敲除(BAF155-cKO)小鼠暴露于持续的0.9%NaCl和AngII(2mg/kg/天)两周;Among them, Figure 1A shows BAF155-cWT mice and myocardial-specific BAF155 knockout (BAF155-cKO) mice exposed to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks;
图1B-C分别示出为免疫荧光和蛋白质印迹的结果,显示BAF155在心脏组织中被特异性敲除;Figure 1B-C show the results of immunofluorescence and Western blotting respectively, showing that BAF155 is specifically knocked out in cardiac tissue;
图1D示出与BAF155-cWT小鼠相比,BAF155-cKO显著减轻了AngII诱导的心功能不全;Figure 1D shows that BAF155-cKO significantly alleviated AngII-induced cardiac dysfunction compared with BAF155-cWT mice;
图1E和图1F分别示出在小鼠中左心室射血分数(EF)%和缩短分数(FS)%的增加;Figures 1E and 1F show increases in left ventricular ejection fraction (EF) % and fractional shortening (FS) %, respectively, in mice;
图1G为H&E和WGA染色数据,显示在BAF155-cWT小鼠中,AngII提高了心肌细胞的大小以及心肌细胞的横截面积,而BAF155-cKO小鼠中给予AngII的这种变化并不显著;Figure 1G shows H&E and WGA staining data, showing that in BAF155-cWT mice, AngII increased the size of cardiomyocytes and the cross-sectional area of cardiomyocytes, while this change in BAF155-cKO mice given AngII was not significant;
图1H-I分别示出与BAF155-cWT小鼠相比,BAF155-cKO显著抑制AngII诱导的HW/BW和HW/TL比值升高,这表明BAF155的心脏特异性敲除减轻了小鼠的心脏肥大;Figure 1H-I show that BAF155-cKO significantly inhibited the AngII-induced increase in HW/BW and HW/TL ratios compared with BAF155-cWT mice, respectively, indicating that cardiac-specific knockout of BAF155 alleviated the cardiac Hypertrophy;
图1J示出为与BAF155-cWT小鼠相比,BAF155-cKO显著降低了AngII诱导的小鼠ANP、BNP、cleavedcaspase-3和PARP1的表达;Figure 1J shows that compared with BAF155-cWT mice, BAF155-cKO significantly reduced the AngII-induced expression of mouse ANP, BNP, cleavedcaspase-3 and PARP1;
图1K示出与BAF155-cWT小鼠相比,BAF155-cKO小鼠的AngII诱导的心肌纤维化水平显著降低;Figure 1K shows that the level of AngII-induced myocardial fibrosis in BAF155-cKO mice was significantly reduced compared with BAF155-cWT mice;
图1L示出与BAF155-cWT小鼠相比,BAF155-cKO小鼠的心肌纤维化相关蛋白α-SMA和胶原蛋白I的表达降低;Figure 1L shows that compared with BAF155-cWT mice, BAF155-cKO mice have reduced expression of cardiac fibrosis-related proteins α-SMA and collagen I;
图2示出为BAF155过表达显著加重小鼠模型中的心肌肥大和心力衰竭,其中:Figure 2 shows that BAF155 overexpression significantly exacerbates cardiac hypertrophy and heart failure in a mouse model, where:
图2A示出为将BAF155-WT和BAF155转基因小鼠(BAF155-TG)暴露于持续的0.9%NaCl和AngII(2mg/kg/天)两周;Figure 2A shows the exposure of BAF155-WT and BAF155 transgenic mice (BAF155-TG) to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks;
图2B示出为蛋白质印迹显示BAF155在心脏组织中特异性过表达;Figure 2B shows a Western blot showing that BAF155 is specifically overexpressed in cardiac tissue;
图2C示出为与BAF155-WT小鼠相比,BAF155-TG显著加重了AngII诱导的心功能不全; Figure 2C shows that compared with BAF155-WT mice, BAF155-TG significantly aggravated AngII-induced cardiac dysfunction;
图2D和图2E分别示出在在小鼠中左心室射血分数(EF)%和缩短分数(FS)%的增加;Figures 2D and 2E show increases in left ventricular ejection fraction (EF) % and fractional shortening (FS) %, respectively, in mice;
图2F示出H&E和WGA染色数据,与BAF155-WT小鼠相比,AngII给药导致BAF155-TG小鼠心肌细胞大小和心肌细胞横截面积升高;Figure 2F shows H&E and WGA staining data. Compared with BAF155-WT mice, AngII administration resulted in an increase in cardiomyocyte size and cardiomyocyte cross-sectional area in BAF155-TG mice;
图2G-H示出与BAF155-WT小鼠相比,BAF155-TG显著加重了AngII诱导的HW/BW和HW/TL比值升高,这表明BAF155的过表达导致小鼠心脏肥大;Figure 2G-H shows that compared with BAF155-WT mice, BAF155-TG significantly exacerbated AngII-induced increases in HW/BW and HW/TL ratios, indicating that overexpression of BAF155 causes cardiac hypertrophy in mice;
图2I-图2K分别示出与BAF155-WT小鼠相比,AngII诱导的ANP、BNP、cleavedcaspase-3和PARP1的表达在BAF155-TG小鼠中均升高(图2I),同样,与BAF155-WT小鼠相比,BAF155-TG显著加重了AngII诱导的小鼠心肌纤维化(图2J),并上调了α-SMA和胶原蛋白I(图2K);Figure 2I-Figure 2K respectively show that compared with BAF155-WT mice, AngII-induced expression of ANP, BNP, cleavedcaspase-3 and PARP1 were all increased in BAF155-TG mice (Figure 2I). Similarly, compared with BAF155 - Compared with WT mice, BAF155-TG significantly aggravated AngII-induced myocardial fibrosis in mice (Figure 2J), and upregulated α-SMA and collagen I (Figure 2K);
图3示出PARP1在生理条件下充当关键的BAF155结合蛋白,并表明BAF155-PARP1轴可能调节心脏重塑,其中:Figure 3 shows that PARP1 acts as a key BAF155-binding protein under physiological conditions and suggests that the BAF155-PARP1 axis may regulate cardiac remodeling, where:
图3A和3B为通过免疫共沉淀评估内源性BAF155与PARP1的相互作用;Figures 3A and 3B evaluate the interaction between endogenous BAF155 and PARP1 by co-immunoprecipitation;
图3C示出为BAF155与PARP1的相互作用是由AngII诱导;Figure 3C shows that the interaction between BAF155 and PARP1 is induced by AngII;
图3D示出为BAF155与PARP1的476-779个氨基酸结构域相互作用,即PARP-A-螺旋结构域;Figure 3D shows the interaction of BAF155 with the 476-779 amino acid domain of PARP1, that is, the PARP-A-helical domain;
图4示出BAF155对PARP1的调节机制,其中:Figure 4 shows the regulatory mechanism of PARP1 by BAF155, where:
图4A示出BAF155的表达升高导致PARP1的同步表达;Figure 4A shows that increased expression of BAF155 leads to synchronized expression of PARP1;
图4B示出用56438shBAF155RNA沉默的BAF155显著下调PARP1表达;Figure 4B shows that BAF155 silenced with 56438shBAF155 RNA significantly downregulated PARP1 expression;
图4C示出在Flag对照组中PARP1的丰度逐渐降低,而BAF155过表达在增加CHX的情况下维持PARP1表达,CHX是用于抑制PARP1蛋白合成的转录抑制剂;Figure 4C shows that the abundance of PARP1 gradually decreased in the Flag control group, while BAF155 overexpression maintained PARP1 expression in the presence of increased CHX, a transcription inhibitor used to inhibit PARP1 protein synthesis;
图4D示出,在MG132处理后,与Flag对照细胞相比,PARP1的表达在BAF155过表达细胞中(速率和程度)增加;Figure 4D shows that after MG132 treatment, PARP1 expression increased (rate and extent) in BAF155 overexpressing cells compared with Flag control cells;
图4E示出与Flag-对照质粒组相比,用Flag-BAF155转染后PARP1的泛素化水平降低;Figure 4E shows that compared with the Flag-control plasmid group, the ubiquitination level of PARP1 was reduced after transfection with Flag-BAF155;
图4F示出PARP1的泛素化水平因BAF155敲低而增加;Figure 4F shows that the ubiquitination level of PARP1 is increased by BAF155 knockdown;
图4G示出BAF155降低了PARP1-WT组的泛素化水平,但在PARP1-K249R细胞中没有,表明BAF155抑制了K249上的PARP1泛素化;Figure 4G shows that BAF155 reduced the ubiquitination level in the PARP1-WT group, but not in PARP1-K249R cells, indicating that BAF155 inhibited PARP1 ubiquitination on K249;
图4H-4K分别示出在MG132的处理下,通过外源性免疫分析,阻断了PARP1和BAF155的降解,导致BAF155和PARP1与WWP2的相互作用增强(4H);此外,在BAF155敲低后,PARP1与WWP2的结合增加(图4I),而BAF155的过表达导致PARP1与WWP2的结合减少(图4J);当WWP2过表达时,与Flag对照组相比,过表达Flag-BAF155后PARP1的泛素化水平下降(图4K);Figures 4H-4K respectively show that under the treatment of MG132, the degradation of PARP1 and BAF155 was blocked by exogenous immunoassay, resulting in enhanced interaction of BAF155 and PARP1 with WWP2 (4H); in addition, after BAF155 knockdown , the binding of PARP1 to WWP2 increased (Figure 4I), while the overexpression of BAF155 led to the reduced binding of PARP1 to WWP2 (Figure 4J); when WWP2 was overexpressed, compared with the Flag control group, the overexpression of Flag-BAF155 increased the binding of PARP1 to WWP2 (Figure 4J). Ubiquitination levels decreased (Fig. 4K);
图4L至图4O分别显示PARP1与WWP2和SIRT2的结合在用AngII治疗后得到增强,并且在BAF155-cKO小鼠心脏组织中显著增加,与有或没有AngII治疗的WT相比(图4L);就PARP1的泛素化水平而言,申请人的数据显示AngII在WT小鼠心脏组织中诱导泛素化(图4M);此外,与WT小鼠相比,PARP1的泛素化在BAF155-cKO小鼠心脏组织中显著增加(图4M);相反,与WT小鼠相 比,在BAF155-TG小鼠心脏组织中,PARP1与WWP2和SIRT2的结合比WT降低(图4N);与WT小鼠相比,BAF155-TG小鼠心脏组织中PARP1的泛素化显著降低(图4O);Figures 4L to 4O show that the binding of PARP1 to WWP2 and SIRT2, respectively, was enhanced after treatment with AngII and significantly increased in the heart tissue of BAF155-cKO mice compared with WT with or without AngII treatment (Figure 4L); In terms of ubiquitination levels of PARP1, Applicants' data show that AngII induces ubiquitination in heart tissue of WT mice (Figure 4M); furthermore, ubiquitination of PARP1 increased in BAF155-cKO compared with WT mice significantly increased in mouse heart tissue (Fig. 4M); conversely, compared with WT mice Compared with WT mice, the binding of PARP1 to WWP2 and SIRT2 in the heart tissue of BAF155-TG mice was reduced compared with WT (Figure 4N); compared with WT mice, the ubiquitination of PARP1 in the heart tissue of BAF155-TG mice was significantly reduced ( Figure 4O);
图5示出BAF155上的K948受SIRT2特异性调控,其中:Figure 5 shows that K948 on BAF155 is specifically regulated by SIRT2, where:
图5A-C证明了内源性和外源性免疫共沉淀证实SIRT2与BAF155相互作用;Figure 5A-C demonstrates that endogenous and exogenous co-immunoprecipitation confirms that SIRT2 interacts with BAF155;
图5D和E示出在AngII的诱导下,SIRT2和BAF155之间的相互作用得到增强;Figure 5D and E show that the interaction between SIRT2 and BAF155 is enhanced under the induction of AngII;
图5F证明BAF155的SWIRM结构域是SIRT2的结合位点;Figure 5F demonstrates that the SWIRM domain of BAF155 is the binding site for SIRT2;
图5G示出用曲古抑菌素A(TSA)和烟酰胺(NAM)治疗后,BAF155的乙酰化水平增加;Figure 5G shows that the acetylation level of BAF155 increased after treatment with trichostatin A (TSA) and nicotinamide (NAM);
图5H示出CBP的过表达显著增加了BAF155的乙酰化水平;Figure 5H shows that overexpression of CBP significantly increased the acetylation level of BAF155;
图5I和5J示出CBP在内源性和外源性条件下都与BAF155相互作用;Figures 5I and 5J show that CBP interacts with BAF155 under both endogenous and exogenous conditions;
图5K示出与正常对照细胞相比,BAF155的乙酰化水平在shSIRT2细胞和用AGK2处理的细胞中上调;Figure 5K shows that the acetylation level of BAF155 is upregulated in shSIRT2 cells and cells treated with AGK2 compared with normal control cells;
图5L示出与WT动物相比,来自SIRT2敲除动物的心脏组织样品中BAF55的BAF155水平显著增强;Figure 5L shows that BAF155 levels of BAF55 were significantly enhanced in heart tissue samples from SIRT2 knockout animals compared with WT animals;
图5M-图5P示出WT-SIRT2的过表达降低了BAF155的外源乙酰化水平(图5M),而转染SIRT2的无活性突变体(H187YQ167A)则没有效果(图5N);此外,与WT动物相比,来自SIRT2敲除动物的心脏组织样本中BAF155的乙酰化水平显著增强,无论是否由AngII诱导(图5O);外源性BAF155的乙酰化水平在AngII处理下降低,而外源性BAF155的乙酰化水平随着AngII诱导的外源性SIRT2过表达而进一步下降(图5P);Figure 5M-Figure 5P shows that overexpression of WT-SIRT2 reduced the exogenous acetylation level of BAF155 (Figure 5M), while transfection of an inactive mutant of SIRT2 (H187YQ167A) had no effect (Figure 5N); in addition, with Compared with WT animals, the acetylation level of BAF155 in heart tissue samples from SIRT2 knockout animals was significantly enhanced, whether induced by AngII or not (Fig. 5O); the acetylation level of exogenous BAF155 was reduced under AngII treatment, while exogenous The acetylation level of sexual BAF155 further decreased with AngII-induced exogenous SIRT2 overexpression (Figure 5P);
图5Q-图5U分别示出K948在整个进化过程中发现,从莫哈文果蝇到哺乳动物物种(图5Q);特异性识别BAF155的乙酰化K948的抗体(5R);施用TSA和NAM后,BAF155-K948的乙酰化水平增加(图5S);此外,外源转染四种乙酰转移酶后,只有CBP增加了BAF155-K948的乙酰化水平(图5T);同时,外源转染WT-SIRT2而不是失活的SIRT2(H187YQ167A)降低了BAF155-K948的乙酰化水平(图5U);总之,以上数据表明K948受到CBP和SIRT2的特异性调控;Figures 5Q-5U respectively show that K948 is found throughout evolution, from Mohave Drosophila to mammalian species (Figure 5Q); an antibody specifically recognizing acetylated K948 of BAF155 (5R); after administration of TSA and NAM , the acetylation level of BAF155-K948 increased (Figure 5S); in addition, after exogenous transfection of four acetyltransferases, only CBP increased the acetylation level of BAF155-K948 (Figure 5T); at the same time, exogenous transfection of WT -SIRT2 but not inactivated SIRT2 (H187YQ167A) reduced the acetylation level of BAF155-K948 (Figure 5U); in summary, the above data indicate that K948 is specifically regulated by CBP and SIRT2;
图6示出BAF155-K948在体内的SIRT2去乙酰化,其中:Figure 6 shows SIRT2 deacetylation by BAF155-K948 in vivo, where:
图6A在SIRT2敲除小鼠(SIRT2-KO)、SIRT2过表达小鼠(SIRT2-TG)和野生型动物(SIRT2-WT)示出SIRT2调节的小鼠心脏赖氨酸乙酰化变化;Figure 6A shows SIRT2-regulated mouse cardiac lysine acetylation changes in SIRT2 knockout mice (SIRT2-KO), SIRT2 overexpression mice (SIRT2-TG) and wild-type animals (SIRT2-WT);
图6B和图6C分别示出与WT相比,在SIRT2敲除后BAF155-K948的乙酰化水平在给予或不给予AngII的情况下增加,而BAF155-K948的乙酰化水平在SIRT2-TG小鼠心脏组织中降低;Figure 6B and Figure 6C respectively show that compared with WT, the acetylation level of BAF155-K948 increased with or without AngII administration after SIRT2 knockout, while the acetylation level of BAF155-K948 increased in SIRT2-TG mice. Decreased in cardiac tissue;
图7示出SIRT2通过WWP2促进BAF155-K948和PARP1-K294泛素化,其中:Figure 7 shows that SIRT2 promotes BAF155-K948 and PARP1-K294 ubiquitination via WWP2, where:
图7A示出,BAF155丰度在SIRT2下调后增加;申请人使用shSIRT2的三个片段(61965、61966和61967)观察到类似的趋势,并使用61966-shSIRT2片段进行后续实验; Figure 7A shows that BAF155 abundance increases after SIRT2 downregulation; Applicants observed similar trends using three fragments of shSIRT2 (61965, 61966, and 61967), and used the 61966-shSIRT2 fragment for subsequent experiments;
图7B示出与给予MG132的SIRT2-KO动物相比,SIRT2-WT小鼠心脏组织中BAF155上调的速率和程度升高;Figure 7B shows that the rate and extent of BAF155 upregulation in the heart tissue of SIRT2-WT mice was increased compared with SIRT2-KO animals administered MG132;
图7C示出SIRT2-WT小鼠心脏组织在CHX给药后BAF155表达显著降低,而SIRT2-KO小鼠组随着时间的推移保持非常高的BAF155表达;Figure 7C shows that BAF155 expression in SIRT2-WT mouse heart tissue significantly decreased after CHX administration, while the SIRT2-KO mouse group maintained very high BAF155 expression over time;
图7D示出与通过泛素-蛋白酶体途径降解一致,与Flag-对照质粒组相比,在Myc-SIRT2过表达和MG132处理后检测到BAF155的泛素化水平增强;Figure 7D shows that consistent with degradation through the ubiquitin-proteasome pathway, enhanced ubiquitination levels of BAF155 were detected after Myc-SIRT2 overexpression and MG132 treatment compared with the Flag-control plasmid group;
图7E示出BAF155的泛素化水平在WT-SIRT2-Flag过表达后增加,但H187YQ167A-SIRT2-Flag(无脱乙酰活性的突变SIRT2);Figure 7E shows that the ubiquitination level of BAF155 increased after overexpression of WT-SIRT2-Flag, but not H187YQ167A-SIRT2-Flag (mutated SIRT2 without deacetylation activity);
图7F和图7G分别示出示出随着四种乙酰转移酶的过表达,仅在过表达CBP的细胞中观察到BAF155丰度上调(图7F)和泛素化水平降低(7G);Figures 7F and 7G respectively show that with the overexpression of four acetyltransferases, upregulation of BAF155 abundance (Figure 7F) and reduction in ubiquitination levels (7G) were only observed in cells overexpressing CBP;
图7H示出与BAF155-K948R对应物相比,SIRT2过表达增强了BAF155-WT细胞中的泛素化水平,表明BAF155在K948处被SIRT2去乙酰化,导致在同一位点泛素化并促进BAF155的降解;Figure 7H shows that SIRT2 overexpression enhanced ubiquitination levels in BAF155-WT cells compared with the BAF155-K948R counterpart, indicating that BAF155 is deacetylated by SIRT2 at K948, leading to ubiquitination at the same site and promoting Degradation of BAF155;
图7I-图7O证明WWP2参与SIRT2介导的去乙酰化诱导的BAF155降解,HA-WWP2在NC和shSIRT2H9c2细胞系中表达;其中,BAF155的丰度在NC细胞系中逐渐减少,而在shSIRT2细胞中保持在高水平(图7I);与NC细胞相比,由WWP2介导的BAF155泛素化水平在shSIRT2处理中降低(图7J);此外,Myc-SIRT2过表达导致BAF155和WWP2之间的结合升高,但与K948R-BAF155过表达无关(图7K);上述发现进一步表明SIRT2通过WWP2促进了BAF155的泛素化;此外,申请人旨在验证BAF155-PARP1损伤复合物受SIRT2-WWP2调节;申请人发现PARP1的泛素化水平在Flag-SIRT2和HA-WWP2过表达后显著增加,并随着Flag-BAF155的进一步过表达而降低(图7L);然后,过度表达K249R-PARP1(突变PARP1没有由BAF155和WWP2介导的泛素化位点),在PARP1上观察到的泛素化水平可以忽略不计(图7L);上述结果表明,SIRT2通过WWP2介导的K948泛素化促进BAF155的降解,WWP2主要通过K249泛素化调节PARP1的降解;申请人进一步检查了SIRT2对PARP1-BAF155复合物的影响;随着Myc-SIRT2的过表达,PARP1与BAF155的结合减少(图7M);与先前报道的结果一致,在有或没有AngII诱导的情况下,在shBAF155-H9C2细胞系中PARP1的表达降低(图7N),但在shSIRT2-H9C2细胞中BAF155的表达增加而PARP1的表达降低(图7O);与shBAF155H9C2细胞相比,PARP1在shBAF155和shSIRT2H9C2细胞中的表达更高(图7N),但低于shSIRT2H9C2细胞(图7O);这些发现表明SIRT2通过促进BAF155的降解使BAF155-PARP1复合物不稳定;Figure 7I-Figure 7O demonstrates that WWP2 is involved in SIRT2-mediated deacetylation-induced BAF155 degradation. HA-WWP2 is expressed in NC and shSIRT2H9c2 cell lines; among them, the abundance of BAF155 gradually decreased in NC cell lines, while in shSIRT2 cells maintained at high levels in NC cells (Fig. 7I); compared with NC cells, the level of BAF155 ubiquitination mediated by WWP2 was reduced in shSIRT2 treatment (Fig. 7J); in addition, Myc-SIRT2 overexpression resulted in a negative relationship between BAF155 and WWP2 Binding was elevated, but not associated with K948R-BAF155 overexpression (Figure 7K); the above findings further suggest that SIRT2 promotes ubiquitination of BAF155 via WWP2; furthermore, the applicants aimed to verify that the BAF155-PARP1 damage complex is regulated by SIRT2-WWP2 ; Applicants found that the ubiquitination level of PARP1 significantly increased after overexpression of Flag-SIRT2 and HA-WWP2 and decreased with further overexpression of Flag-BAF155 (Figure 7L); then, overexpression of K249R-PARP1 (mutated PARP1 has no ubiquitination site mediated by BAF155 and WWP2), and the level of ubiquitination observed on PARP1 was negligible (Figure 7L); the above results indicate that SIRT2 promotes BAF155 through WWP2-mediated ubiquitination of K948 Degradation, WWP2 mainly regulates the degradation of PARP1 through K249 ubiquitination; Applicants further examined the effect of SIRT2 on the PARP1-BAF155 complex; with the overexpression of Myc-SIRT2, the binding of PARP1 to BAF155 was reduced (Figure 7M); Consistent with previously reported results, PARP1 expression was decreased in shBAF155-H9C2 cell lines with or without AngII induction (Fig. 7N), but BAF155 expression was increased and PARP1 expression was decreased in shSIRT2-H9C2 cells ( Figure 7O); PARP1 expression was higher in shBAF155 and shSIRT2H9C2 cells compared with shBAF155H9C2 cells (Figure 7N), but lower than that in shSIRT2H9C2 cells (Figure 7O); these findings indicate that SIRT2 complexes BAF155-PARP1 by promoting the degradation of BAF155 Things are unstable;
图8示出SIRT2敲除和转基因小鼠中差异蛋白的蛋白质组学分析表明,SIRT2在体内通过WWP2促进BAF155和PARP1的泛素化,其中:Figure 8 shows proteomic analysis of differential proteins in SIRT2 knockout and transgenic mice indicating that SIRT2 promotes the ubiquitination of BAF155 and PARP1 through WWP2 in vivo, where:
图8A示出与给予或不给予AngII的SIRT2-WT小鼠样品相比,用AngII治疗后BAF155与PARP1的结合增强,并且在SIRT2-KO小鼠心脏组织中显著增强;Figure 8A shows that the binding of BAF155 to PARP1 was enhanced after treatment with AngII compared with SIRT2-WT mouse samples with or without AngII administration, and was significantly enhanced in the heart tissue of SIRT2-KO mice;
图8B和8C分别示出在SIRT2-WT小鼠心脏组织中,与WT小鼠样品相比,BAF155和PARP1的泛素化水平在AngII治疗后降低,并且在SIRT2-KO小鼠心脏组织中显著降低; Figures 8B and 8C show that in SIRT2-WT mouse heart tissue, respectively, the ubiquitination levels of BAF155 and PARP1 were reduced after AngII treatment compared with WT mouse samples, and significantly in SIRT2-KO mouse heart tissue. reduce;
图8D-图8H示出,与SIRT2-WT动物相比,在给予或不给予AngII的SIRT2-KO小鼠的心脏组织中,WWP2与BAF155和PARP1的结合降低(图8D);相反,与给予或不给予AngII的SIRT2-WT组相比,BAF155与PARP1的结合在SIRT2-TG小鼠心脏组织中降低(图8E);与SIRT2-WT小鼠相比,SIRT2-TG小鼠心脏组织中BAF155和PARP1的泛素化水平显著升高(图8F和8G);此外,与SIRT2-WT动物相比,在给予或不给予AngII的SIRT2-TG小鼠的心脏组织中,WWP2与BAF155和PARP1的结合增加(图8H)。Figures 8D-8H show that compared with SIRT2-WT animals, the binding of WWP2 to BAF155 and PARP1 was reduced in the heart tissue of SIRT2-KO mice administered or not administered AngII (Fig. 8D); conversely, compared with SIRT2-WT animals, Or compared with the SIRT2-WT group without AngII administration, the binding of BAF155 to PARP1 was reduced in the heart tissue of SIRT2-TG mice (Figure 8E); compared with SIRT2-WT mice, BAF155 in the heart tissue of SIRT2-TG mice and PARP1 ubiquitination levels were significantly increased (Figures 8F and 8G); furthermore, compared with SIRT2-WT animals, WWP2 interacted with BAF155 and PARP1 in the heart tissue of SIRT2-TG mice administered or not administered AngII. Binding increased (Fig. 8H).
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.
实施例1Example 1
一、材料和方法1. Materials and methods
1.1 BAF155、WWP2和SIRT2敲除和转基因小鼠1.1 BAF155, WWP2 and SIRT2 knockout and transgenic mice
条件性心肌细胞特异性敲除(KO)小鼠,包括Myh6Cre+、BAF155Fl/Fl(BAF155-cKO)和Myh6Cre-、BAF155Fl/Fl(BAF155-cWT)、BAF155-WT和BAF155-TG小鼠(CAG启动子)获自上海南方模式生物科技股份有限公司;Conditional cardiomyocyte-specific knockout (KO) mice, including Myh6Cre+, BAF155Fl/Fl (BAF155-cKO) and Myh6Cre-, BAF155Fl/Fl (BAF155-cWT), BAF155-WT and BAF155-TG mice (CAG-initiated Sub) obtained from Shanghai Southern Model Biotechnology Co., Ltd.;
条件性心肌细胞特异性敲除小鼠,包括Myh6Cre+、WWP2Fl/Fl(WWP2-cKO)和Myh6Cre-、WWP2Fl/Fl(WWP2-cWT)动物、SIRT2-KO小鼠获得自DengCX,澳门大学健康科学学院。Conditional cardiomyocyte-specific knockout mice, including Myh6Cre+, WWP2Fl/Fl (WWP2-cKO) and Myh6Cre-, WWP2Fl/Fl (WWP2-cWT) animals, and SIRT2-KO mice were obtained from DengCX, Faculty of Health Sciences, University of Macau .
SIRT2转基因(SIRT2-TG)小鼠(CAG启动子)获得自上海南方模式生物科技股份有限公司。SIRT2 transgenic (SIRT2-TG) mice (CAG promoter) were obtained from Shanghai Southern Model Biotechnology Co., Ltd.
在这项研究中,纳入了8至10周大的无特定病原体(SPF)雄性小鼠。在AngII和NaCl输注小鼠模型中,BAF155-cKO、BAF155-cWT、BAF155-WT、BAF155-TG、WWP2-cKO、WWP2-cWT、SIRT2-WT、SIRT2-KO和SIRT2-TG小鼠(每组6总共n=120)被随机分配到各组并进行麻醉(异氟醚在氧气中的浓度为2%;1,500毫升/分钟)。随后按照制造商的指示在肩胛中部切开并皮下植入渗透性微型泵(Alzet),诱导了心脏重塑。在接下来的14天中,通过颈椎脱位对小鼠实施安乐死。所有动物研究均经中国医科大学动物学科委员会批准(方案号2019026)。In this study, 8- to 10-week-old specific pathogen-free (SPF) male mice were included. In the AngII and NaCl infusion mouse model, BAF155-cKO, BAF155-cWT, BAF155-WT, BAF155-TG, WWP2-cKO, WWP2-cWT, SIRT2-WT, SIRT2-KO and SIRT2-TG mice (per Group 6 (total n = 120) were randomly assigned to groups and anesthetized (2% isoflurane in oxygen; 1,500 ml/min). Cardiac remodeling was then induced by incision and subcutaneous implantation of an osmotic minipump (Alzet) in the midscapula according to the manufacturer's instructions. Over the next 14 days, the mice were euthanized by cervical dislocation. All animal studies were approved by the Animal Science Committee of China Medical University (Protocol No. 2019026).
1.2.蛋白质组学和乙酰化,泛素化蛋白质组学1.2. Proteomics and acetylation, ubiquitination proteomics
1.2.1蛋白质提取1.2.1 Protein extraction
将标本在液氮中粉碎并置于5-mL管中,补充四倍体积的裂解缓冲液(8M尿素、1%蛋白酶抑制剂混合物、3μMTSA,50mMNAM)并在冰上用Scientz超声均质器超声处理3次.随后在12,000g和4℃下离心清除10分钟。使用BCA试剂盒测量所得上清液的蛋白质浓度。Specimens were pulverized in liquid nitrogen and placed in 5-mL tubes, supplemented with four volumes of lysis buffer (8 M urea, 1% protease inhibitor cocktail, 3 μM TSA, 50 mM NAM) and sonicated on ice with a Scientz ultrasonic homogenizer. Process 3 times. Subsequently clear by centrifugation at 12,000g and 4°C for 10 minutes. The protein concentration of the resulting supernatant was measured using a BCA kit.
1.2.2胰蛋白酶消化1.2.2 Trypsin digestion
每个样品中等量的总蛋白质被酶解,并且调整体积以在整个样品组中保持一致。滴加TCA至终浓度为20%,然后涡旋混合,4℃沉淀2h。以4500g离心5分钟,所得沉淀用预冷的丙酮洗涤两次。随后干燥沉淀,通过超声处理将沉淀重新 悬浮在200mMTEAB中,将胰蛋白酶以1:50(蛋白酶:蛋白质,w/w)添加到每个样品中进行过夜消化。以5mM添加二硫苏糖醇(DTT),然后在56℃下孵育30分钟。然后,加入11mM的碘乙酰胺(IAA),然后在室温下在黑暗中孵育15分钟。An equal amount of total protein per sample was enzymatically digested, and the volume was adjusted to be consistent across the entire sample set. TCA was added dropwise to a final concentration of 20%, then vortexed and allowed to settle at 4°C for 2 h. Centrifuge at 4500g for 5 minutes, and the resulting precipitate is washed twice with pre-cooled acetone. The pellet was then dried and reconstituted by sonication. Resuspend in 200mM TEAB and add trypsin at 1:50 (protease:protein, w/w) to each sample for overnight digestion. Dithiothreitol (DTT) was added at 5mM and incubated at 56°C for 30 minutes. Then, 11 mM iodoacetamide (IAA) was added and incubated in the dark at room temperature for 15 minutes.
1.2.3翻译后修饰肽的富集1.2.3 Enrichment of post-translationally modified peptides
将肽溶解在IP缓冲液(100mM NaCl、1mM EDTA、50mM Tris-HCl、0.5%NP-40、pH 8.0)中,与预洗的抗赖氨酸乙酰化和抗泛素残留抗体树脂(PTM-104和PTM-1104;Hangzhou Jingjie PTM-Bio),并在4℃下轻轻摇晃过夜。分别用IP缓冲液和去离子水洗涤抗体树脂。最后,富集肽用0.1%三氟乙酸洗脱3次,并用C18ZipTips清洗。The peptides were dissolved in IP buffer (100mM NaCl, 1mM EDTA, 50mM Tris-HCl, 0.5% NP-40, pH 8.0) and mixed with prewashed anti-lysine acetylation and anti-ubiquitin residue antibody resin (PTM- 104 and PTM-1104; Hangzhou Jingjie PTM-Bio) and shake gently at 4°C overnight. Wash the antibody resin with IP buffer and deionized water respectively. Finally, the enriched peptides were eluted three times with 0.1% trifluoroacetic acid and washed with C18ZipTips.
1.2.4 LC-MS/MS分析1.2.4 LC-MS/MS analysis
将胰蛋白酶肽溶解在液相色谱流动相A中,并在NanoElute超高性能液体系统上进行分离。流动相A和B分别为0.1%甲酸和2%乙腈的水溶液和0.1%甲酸的乙腈溶液。使用设置为XXX的梯度以450nL/min的恒定流速洗脱肽。洗脱梯度设置为:0-72min,7%-24%B;72-84分钟,24%-32%B;84-87分钟,32%-80%B;87-90分钟,80%B.在注入毛细管离子源进行电离和TIMS-TOFPro质谱仪(离子源电压,1.6kV;扫描范围,100-1700大)。为数据采集启用了并行累积串行碎片(PASEF)模式。选择电荷状态为0到5的前体进行碎裂,每个循环进行10次PASEFMS/MS扫描。MS/MS扫描的动态排除时间为30秒,以防止对同一母离子进行多次扫描。Tryptic peptides were dissolved in liquid chromatography mobile phase A and separated on the NanoElute Ultra High Performance Liquid System. Mobile phases A and B were 0.1% formic acid and 2% acetonitrile in water and 0.1% formic acid in acetonitrile, respectively. Peptides were eluted at a constant flow rate of 450 nL/min using a gradient set to XXX. The elution gradient settings were: 0-72 min, 7%-24% B; 72-84 min, 24%-32% B; 84-87 min, 32%-80% B; 87-90 min, 80% B. Ionization was performed with an injected capillary ion source and a TIMS-TOFPro mass spectrometer (ion source voltage, 1.6 kV; scan range, 100-1700 mA). Parallel accumulated serial fragmentation (PASEF) mode is enabled for data acquisition. Precursors with charge states 0 to 5 were selected for fragmentation, and 10 PASEFMS/MS scans were performed per cycle. MS/MS scans have a dynamic exclusion time of 30 seconds to prevent multiple scans of the same precursor ion.
1.2.5数据库搜索1.2.5 Database search
Maxquant(v1.6.15.0)针对Swissprot蛋白质序列数据库(Mus_musculus_10090_SP_20201214.fasta)搜索原始质谱数据,其中包含反向诱饵条目和常见污染蛋白质。胰蛋白酶/P消化最多允许2个缺失的切割,每个肽至少需要7个氨基酸。母离子的质量误差容限分别为10ppm和子离子为20ppm。半胱氨酸烷基化(氨基甲酰甲基[C])被认为是一种固定修饰。可变修饰是蛋氨酸氧化和n-末端乙酰化。赖氨酸乙酰化和赖氨酸上的二甘氨酸也被设置为可变修饰,用于相应的修饰富集分析。蛋白质和PSM鉴定的FDR均为1%。Maxquant (v1.6.15.0) searches raw mass spectrometry data against the Swissprot protein sequence database (Mus_musculus_10090_SP_20201214.fasta), which contains reverse decoy entries and common contaminating proteins. Trypsin/P digestion allows up to 2 missing cleavages, requiring at least 7 amino acids per peptide. The mass error tolerance is 10 ppm for precursor ions and 20 ppm for product ions. Cysteine alkylation (carbamoylmethyl [C]) is considered a fixed modification. Variable modifications are methionine oxidation and N-terminal acetylation. Lysine acetylation and diglycine on lysine were also set as variable modifications for corresponding modification enrichment analysis. The FDR for both protein and PSM identification was 1%.
1.2.6基因本体(GO)分析1.2.6 Gene Ontology (GO) analysis
UniProt-GOA数据库(www.http://www.ebi.ac.uk/GOA/)用于GO注释。首先,获得的蛋白质识别根据其单端口ID映射到GOID。对于UniProt-GOA中没有注释的蛋白质,InterProScan用于基于蛋白质序列比对注释GO功能。蛋白质被分配到作为GO术语的生物过程、细胞成分和分子功能。在各个类别中,使用双尾Fisher检验来评估差异表达蛋白质与所有检测到的蛋白质的富集度;校正后的p<0.05表明有统计学意义。UniProt-GOA database (www.http://www.ebi.ac.uk/GOA/) was used for GO annotation. First, the obtained protein identifications are mapped to GOIDs based on their single-port IDs. For proteins that are not annotated in UniProt-GOA, InterProScan is used to annotate GO functions based on protein sequence alignment. Proteins were assigned to biological processes, cellular components, and molecular functions as GO terms. Within each category, a two-tailed Fisher test was used to evaluate the enrichment of differentially expressed proteins against all detected proteins; a corrected p<0.05 indicates statistical significance.
1.2.7亚细胞定位1.2.7 Subcellular localization
Wolfpsort,最新版本的PSORT/PSORTII,用于预测真核蛋白的亚细胞定位。Wolfpsort, the latest version of PSORT/PSORTII, is used to predict the subcellular localization of eukaryotic proteins.
1.3组织病理学评估1.3 Histopathological evaluation
心肌组织样本经过福尔马林(4%)固定4小时,石蜡包埋和5μm切片。二甲苯脱蜡后,用分级乙醇进行再水化,然后进行H&E和Masson三色(G1340;Solarbio,中国)染色。 Myocardial tissue samples were fixed in formalin (4%) for 4 hours, embedded in paraffin, and sectioned at 5 μm. After dewaxing in xylene, they were rehydrated with graded ethanol, and then stained with H&E and Masson's trichrome (G1340; Solarbio, China).
通过免疫荧光检查冷冻的心肌组织切片。在用5μMWGA(Thermo,USA)染色后获得的图像中评估心肌细胞的横截面积。H9c2细胞被给予10-5μMAngII或接受Nacl处理48小时。然后,将细胞用4%福尔马林固定并在室温下用WGA(5μM)处理10分钟,然后进行荧光显微镜分析。Frozen myocardial tissue sections were examined by immunofluorescence. The cross-sectional area of cardiomyocytes was evaluated in images obtained after staining with 5 μM MWGA (Thermo, USA). H9c2 cells were given 10-5 μM AngII or treated with Nacl for 48 hours. Then, cells were fixed with 4% formalin and treated with WGA (5 μM) for 10 min at room temperature before fluorescence microscopy analysis.
1.4.超声心动图和左心室功能(LVEF)评估1.4. Echocardiography and left ventricular function (LVEF) assessment
Myh6Cre+、BAF155Fl/Fl、Myh6Cre-、BAF155Fl/Fl、BAF155-WT和BAF155-TG组的心脏功能在VisualSonicsVevo2100实时高分辨率活体显微成像系统(Visualsonic,加拿大)。用1.5%异氟醚麻醉24只小鼠,然后通过40MHz传感器连续供氧进行心脏功能分析。通过二维M模式记录检查LVEF。心脏功能测定基于室间隔尺寸(IVSd)、左心室后壁尺寸(PWTd)、收缩期LV内部尺寸(LVD)、舒张期LV内部尺寸(LVDd)和LV质量测量值。此外,测定了左心室射血分数(EF%)和缩短分数(FS%)。The cardiac functions of Myh6Cre+, BAF155Fl/Fl, Myh6Cre-, BAF155Fl/Fl, BAF155-WT and BAF155-TG groups were measured on the VisualSonics Vevo2100 real-time high-resolution intravital microscopy system (Visualsonic, Canada). Twenty-four mice were anesthetized with 1.5% isoflurane and then continuously supplied with oxygen through a 40MHz sensor for cardiac function analysis. Check LVEF by 2D M-mode recording. Cardiac function measurements were based on interventricular septal dimension (IVSd), left ventricular posterior wall dimension (PWTd), systolic LV internal dimension (LVD), diastolic LV internal dimension (LVDd), and LV mass measurements. In addition, left ventricular ejection fraction (EF%) and fractional shortening (FS%) were determined.
1.5.细胞和治疗1.5. Cells and Treatment
HEK293T和H9c2细胞(ATCC)在含10%FBS(HyClone)的高葡萄糖Dulbecco改良Eagle培养基中培养,温度为37℃,加湿5%CO2培养箱。HEK293T and H9c2 cells (ATCC) were cultured in high glucose Dulbecco's modified Eagle medium containing 10% FBS (HyClone) at 37°C in a humidified 5% CO2 incubator.
1.6.质粒构建、抗体和试剂1.6. Plasmid construction, antibodies and reagents
表1列出了各种质粒Various plasmids are listed in Table 1
表1
Table 1
Lipofectamine3000(Invitrogen,USA)用于转染。慢病毒用于shRNA转导。表2列出了研究中使用的所有抗体。Lipofectamine 3000 (Invitrogen, USA) was used for transfection. Lentivirus is used for shRNA transduction. Table 2 lists all antibodies used in the study.
表2
Table 2
MG132(A2585),蛋白酶体抑制剂,和放线菌酮(CHX,A8244)购自Apexbio(USA),并用DMSO溶解。使用DMSO中的AngII(A9525;Sigma,USA),浓度为10μM。AGK2由Selleck(美国)提供。MG132 (A2585), a proteasome inhibitor, and cycloheximide (CHX, A8244) were purchased from Apexbio (USA) and dissolved with DMSO. AngII (A9525; Sigma, USA) in DMSO was used at a concentration of 10 μM. AGK2 is provided by Selleck (USA).
1.7.PARP1和BAF155泛素化定量1.7. Quantification of PARP1 and BAF155 ubiquitination
使用1%SDS缓冲液(TrispH7.5、0.5mMEDTA和1mMDTT)裂解小鼠心肌组织样本,煮沸10分钟,然后将Tris-HCL(pH8.0)饱和。用HA标记(HA)-泛素、全长人Myc-PARP1和突变体Myc-PARP1质粒转染的细胞;或全长人Flag-BAF155和突变Flag-BAF155质粒也如上所述被裂解。细胞裂解物与抗PARP1或抗BAF155抗体(1μg/mg细胞裂解物;4℃)和蛋白A/G(B23202)或抗Myc(B26302)或抗标记免疫沉淀磁珠连续孵育(Biotool,美国)12小时。Mouse myocardial tissue samples were lysed using 1% SDS buffer (TrispH7.5, 0.5mMEDTA and 1mMDTT), boiled for 10 minutes, and then saturated with Tris-HCL (pH8.0). Cells transfected with HA-tagged (HA)-ubiquitin, full-length human Myc-PARP1 and mutant Myc-PARP1 plasmids; or full-length human Flag-BAF155 and mutant Flag-BAF155 plasmids were also cleaved as described above. Cell lysates were sequentially incubated with anti-PARP1 or anti-BAF155 antibodies (1 μg/mg cell lysate; 4°C) and protein A/G (B23202) or anti-Myc (B26302) or anti-Flag immunoprecipitation magnetic beads (Biotool, USA) 12 Hour.
1.8.免疫共沉淀1.8. Co-immunoprecipitation
小鼠心肌组织样本和细胞用Flag裂解缓冲液[50mMTris、137mMNaCl、1mMEDTA、10mMNaF、0.1mMNa3VO4、1%NP-40、1mM二硫苏糖醇[DTT]和10%甘油裂解,pH7.8]含有蛋白酶抑制剂(Bimake)。将得到的裂解物与30μl抗Flag/Myc亲和凝胶(B23102/B26302,Biotool;4℃下12小时或足够的抗体(1μg/mg细胞裂解物;2-3小时)和蛋白A/G用于免疫沉淀(B23202;Biotool)在30μl在4℃下12小时。免疫沉淀的复合物通过SDS-PAGE分离,然后电转移到PVDF膜上。膜被封闭(5%牛血清白蛋白)在环境中1小时,然后连续孵育初级(4℃,过夜)和二级(环境,1小时)抗体。泛素化的BAF155和PARP1,用抗Myc、抗标志、抗PARP1或抗-BAF155抗体,分别用抗HA抗体进行检测。ImageJv1.46(美国国立卫生研究院)用于信号量化,然后标准化为GAPDH和微管蛋白表达。Mouse myocardial tissue samples and cells were lysed with Flag lysis buffer [50mM Tris, 137mM NaCl, 1mM EDTA, 10mM NaF, 0.1mM Na3VO4, 1% NP-40, 1mM dithiothreitol [DTT], and 10% glycerol, pH 7.8]. Protease inhibitor (Bimake). The resulting lysate was incubated with 30 μl of anti-Flag/Myc affinity gel (B23102/B26302, Biotool; 12 hours at 4°C or sufficient antibody (1 μg/mg cell lysate; 2-3 hours) and protein A/G). Immunoprecipitate (B23202; Biotool) in 30 μl for 12 hr at 4°C. Immunoprecipitated complexes were separated by SDS-PAGE and then electrotransferred to PVDF membrane. Membrane was blocked (5% bovine serum albumin) in ambient 1 hr, followed by sequential incubations with primary (4°C, overnight) and secondary (ambient, 1 hr) antibodies. Ubiquitinated BAF155 and PARP1 were incubated with anti-Myc, anti-Flag, anti-PARP1 or anti-BAF155 antibodies, respectively. HA antibodies were used for detection. ImageJv1.46 (National Institutes of Health) was used for signal quantification and then normalized to GAPDH and tubulin expression.
1.9.统计分析1.9. Statistical analysis
数据显示为平均值±标准差(SD)。通过F-和Brown-Forsythe检验分别评估两组和多组的方差同质性。进行夏皮罗-威尔克检验以评估正态性。两组的正态分布和偏态分布数据分别通过学生t检验和韦尔奇t检验进行比较。对分别涉及一个和两个参数的多组比较进行单向方差分析和双向方差分析,然后进行事后Bonferroni检验。P值针对多重比较进行了适当调整。采用SPSS22.0(SPSS,USA)进行统计分析,P<0.05表示有统计学意义。Data are shown as mean ± standard deviation (SD). Homogeneity of variances across two and multiple groups was assessed by F- and Brown-Forsythe tests, respectively. The Shapiro-Wilk test was performed to assess normality. Normally distributed and skewed distributed data of the two groups were compared by Student's t test and Welch's t test, respectively. One-way ANOVA and two-way ANOVA followed by post hoc Bonferroni test were performed for multiple group comparisons involving one and two parameters respectively. P values were appropriately adjusted for multiple comparisons. SPSS22.0 (SPSS, USA) was used for statistical analysis, and P<0.05 indicated statistical significance.
二 结果与分析2. Results and Analysis
2.1.心肌特异性BAF155敲除可显著缓解小鼠模型中的心肌肥大和心力衰竭2.1. Myocardial-specific BAF155 knockout can significantly alleviate cardiac hypertrophy and heart failure in mouse models
为了确定BAF155在小鼠心脏重塑中的功能,将BAF155-cWT和心肌特异性BAF155敲除(BAF155-cKO)小鼠暴露于持续的0.9%NaCl和AngII(2mg/kg/天)两周(图1A)。免疫荧光和蛋白质印迹显示BAF155在心脏组织中被特异性敲除(图1B-C)。与BAF155-cWT小鼠相比,BAF155-cKO显著减轻了AngII诱导的心功能不全(图1D),这反映在小鼠中EF%(图1E)和FS%(图1F)的增加。H&E和 WGA染色数据显示,在BAF155-cWT小鼠中,AngII提高了心肌细胞的大小以及心肌细胞的横截面积,而BAF155-cKO小鼠中给予AngII的这种变化并不显著(图1G)。与BAF155-cWT小鼠相比,BAF155-cKO显著抑制AngII诱导的HW/BW和HW/TL比值升高(图1H-I),这表明BAF155的心脏特异性敲除减轻了小鼠的心脏肥大。此外,与BAF155-cWT小鼠相比,BAF155-cKO显著降低了AngII诱导的小鼠ANP、BNP、cleavedcaspase-3和PARP1的表达(图1J)。同样,与BAF155-cWT小鼠相比,BAF155-cKO小鼠的AngII诱导的心肌纤维化水平显著降低(图1K),心肌纤维化相关蛋白α-SMA和胶原蛋白I的表达降低(图1L)。总体而言,这些结果表明BAF155的心脏特异性敲除减轻了小鼠的心脏重塑。To determine the function of BAF155 in mouse cardiac remodeling, BAF155-cWT and myocardial-specific BAF155 knockout (BAF155-cKO) mice were exposed to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks ( Figure 1A). Immunofluorescence and Western blotting showed that BAF155 was specifically knocked out in cardiac tissue (Fig. 1B-C). BAF155-cKO significantly attenuated AngII-induced cardiac dysfunction (Fig. 1D) compared with BAF155-cWT mice, which was reflected by increases in EF% (Fig. 1E) and FS% (Fig. 1F) in mice. H&E and WGA staining data showed that AngII increased cardiomyocyte size and cardiomyocyte cross-sectional area in BAF155-cWT mice, whereas this change was not significant in BAF155-cKO mice given AngII (Fig. 1G). Compared with BAF155-cWT mice, BAF155-cKO significantly inhibited the AngII-induced increase in HW/BW and HW/TL ratios (Figure 1H-I), indicating that cardiac-specific knockout of BAF155 alleviated cardiac hypertrophy in mice. . Furthermore, BAF155-cKO significantly reduced AngII-induced expression of ANP, BNP, cleavedcaspase-3, and PARP1 in mice compared with BAF155-cWT mice (Fig. 1J). Similarly, compared with BAF155-cWT mice, the level of AngII-induced myocardial fibrosis in BAF155-cKO mice was significantly reduced (Figure 1K), and the expression of myocardial fibrosis-related proteins α-SMA and collagen I was reduced (Figure 1L) . Overall, these results indicate that cardiac-specific knockout of BAF155 alleviates cardiac remodeling in mice.
2.2.BAF155过表达显著加重小鼠模型中的心肌肥大和心力衰竭2.2. BAF155 overexpression significantly aggravates cardiac hypertrophy and heart failure in mouse models
接下来,将BAF155-WT和BAF155转基因小鼠(BAF155-TG)暴露于持续的0.9%NaCl和AngII(2mg/kg/天)两周(图2A)。蛋白质印迹显示BAF155在心脏组织中特异性过表达(图2B)。与BAF155-WT小鼠相比,BAF155-TG显著加重了AngII诱导的心功能不全(图2C),这反映在小鼠中EF%(图2D)和FS%(图2E)的增加。H&E和WGA染色数据显示,与BAF155-WT小鼠相比,AngII给药导致BAF155-TG小鼠心肌细胞大小和心肌细胞横截面积升高(图2F)。与BAF155-WT小鼠相比,BAF155-TG显著加重了AngII诱导的HW/BW和HW/TL比值升高(图2G-H),这表明BAF155的过表达导致小鼠心脏肥大。此外,与BAF155-WT小鼠相比,AngII诱导的ANP、BNP、cleavedcaspase-3和PARP1的表达在BAF155-TG小鼠中均升高(图2I)。同样,与BAF155-WT小鼠相比,BAF155-TG显著加重了AngII诱导的小鼠心肌纤维化(图2J),并上调了α-SMA和胶原蛋白I(图2K)。总体而言,这些结果表明BAF155的过表达加重了小鼠的心脏重塑。Next, BAF155-WT and BAF155 transgenic mice (BAF155-TG) were exposed to continuous 0.9% NaCl and AngII (2 mg/kg/day) for two weeks (Fig. 2A). Western blotting showed that BAF155 was specifically overexpressed in cardiac tissue (Fig. 2B). BAF155-TG significantly exacerbated AngII-induced cardiac dysfunction (Fig. 2C), as reflected by increases in EF% (Fig. 2D) and FS% (Fig. 2E) in mice compared with BAF155-WT mice. H&E and WGA staining data showed that AngII administration resulted in an increase in cardiomyocyte size and cardiomyocyte cross-sectional area in BAF155-TG mice compared with BAF155-WT mice (Figure 2F). Compared with BAF155-WT mice, BAF155-TG significantly exacerbated AngII-induced increases in HW/BW and HW/TL ratios (Fig. 2G-H), indicating that overexpression of BAF155 causes cardiac hypertrophy in mice. In addition, AngII-induced expression of ANP, BNP, cleavedcaspase-3, and PARP1 were all increased in BAF155-TG mice compared with BAF155-WT mice (Fig. 2I). Similarly, BAF155-TG significantly aggravated AngII-induced myocardial fibrosis in mice (Fig. 2J) and upregulated α-SMA and collagen I (Fig. 2K) compared with BAF155-WT mice. Overall, these results indicate that overexpression of BAF155 exacerbates cardiac remodeling in mice.
2.3.PARP1在生理条件下充当关键的BAF155结合蛋白2.3.PARP1 acts as a key BAF155-binding protein under physiological conditions
验证了BAF155和PARP1的生物学关联。首先,通过免疫共沉淀评估内源性BAF155与PARP1的相互作用(图3A-B)。此外,BAF155与PARP1的相互作用是由AngII诱导的(图3C)。此外,PARP1与PARP1的476-779个氨基酸结构域相互作用,即PARP-A-螺旋结构域(图3D)。上述发现表明BAF155-PARP1轴可能调节心脏重塑。The biological association of BAF155 and PARP1 was verified. First, the interaction of endogenous BAF155 with PARP1 was assessed by co-immunoprecipitation (Fig. 3A-B). Furthermore, the interaction of BAF155 with PARP1 was induced by AngII (Fig. 3C). Furthermore, PARP1 interacted with the 476-779 amino acid domain of PARP1, the PARP-A-helical domain (Fig. 3D). The above findings suggest that the BAF155-PARP1 axis may regulate cardiac remodeling.
2.4.BAF155通过抑制E3泛素连接酶WWP2稳定PARP1并减少PARP1-K2492.4.BAF155 stabilizes PARP1 and reduces PARP1-K249 by inhibiting E3 ubiquitin ligase WWP2 泛素化ubiquitination
为了探索BAF155对PARP1的调节机制,申请人首先检查了BAF155是否调节PARP1蛋白的表达。如图4A所示,BAF155的表达升高导致PARP1的同步表达。一致地,用56438shBAF155RNA(SEQ ID NO:2TGGGAAGCGTCGAAATCAGAA)沉默的BAF155显著下调PARP1表达(图4B)。此外,在Flag对照组中PARP1的丰度逐渐降低,而BAF155过表达在增加CHX的情况下维持PARP1表达,CHX是用于抑制PARP1蛋白合成的转录抑制剂(图4C)。上述发现表明,BAF155通过阻断蛋白酶体途径抑制PARP1降解。此外,在MG132处理后,与Flag对照细胞相比,PARP1的表达在BAF155过表达细胞中(速率和程度)增加(图4D)。这些数据表明BAF155的过表达通过抑制蛋白酶体途径 的降解来维持PARP1的表达。与通过泛素-蛋白酶体途径降解一致,与Flag-对照质粒组相比,用Flag-BAF155转染后PARP1的泛素化水平降低(图4E)。同时,PARP1的泛素化水平因BAF155敲低而增加(图4F)。In order to explore the regulatory mechanism of BAF155 on PARP1, Applicants first examined whether BAF155 regulates the expression of PARP1 protein. As shown in Figure 4A , elevated expression of BAF155 resulted in synchronized expression of PARP1. Consistently, BAF155 silenced with 56438shBAF155 RNA (SEQ ID NO: 2TGGGAAGCGTCGAAATCAGAA) significantly downregulated PARP1 expression (Fig. 4B). Furthermore, the abundance of PARP1 gradually decreased in the Flag control group, while BAF155 overexpression maintained PARP1 expression in the presence of increased CHX, a transcriptional inhibitor used to inhibit PARP1 protein synthesis (Fig. 4C). The above findings indicate that BAF155 inhibits PARP1 degradation by blocking the proteasome pathway. Furthermore, after MG132 treatment, PARP1 expression was increased (rate and extent) in BAF155-overexpressing cells compared with Flag control cells (Fig. 4D). These data suggest that overexpression of BAF155 inhibits the proteasome pathway degradation to maintain PARP1 expression. Consistent with degradation through the ubiquitin-proteasome pathway, the ubiquitination level of PARP1 was reduced after transfection with Flag-BAF155 compared with the Flag-control plasmid group (Fig. 4E). At the same time, the ubiquitination level of PARP1 was increased by BAF155 knockdown (Fig. 4F).
申请人之前的研究发现,K249上的泛素化是PARP1的关键修饰之一。因此,为了进一步证明PARP1的泛素化被BAF155抑制,申请人过表达PARP1-WT、PARP1-K249R以及Flag-control和Flag-BAF155。如图4G所示,BAF155降低了PARP1-WT组的泛素化水平,但在PARP1-K249R细胞中没有,表明BAF155抑制了K249上的PARP1泛素化。Applicants' previous studies found that ubiquitination on K249 is one of the key modifications of PARP1. Therefore, to further demonstrate that ubiquitination of PARP1 is inhibited by BAF155, Applicants overexpressed PARP1-WT, PARP1-K249R as well as Flag-control and Flag-BAF155. As shown in Figure 4G, BAF155 reduced the ubiquitination level in the PARP1-WT group but not in PARP1-K249R cells, indicating that BAF155 inhibited PARP1 ubiquitination on K249.
接下来,申请人旨在确定哪种E3泛素化连接酶介导BAF155抑制的PARP1泛素化。在申请人之前的工作中,申请人发现WWP2是PARP1的特异性E3泛素化连接酶,并泛素化PARP1-K249位点。同时,WWP2也是BAF155的E3泛素化连接酶。在MG132的处理下,通过外源性免疫分析,阻断了PARP1和BAF155的降解,导致BAF155和PARP1与WWP2的相互作用增强(图4H)。此外,在BAF155敲低后,PARP1与WWP2的结合增加(图4I),而BAF155的过表达导致PARP1与WWP2的结合减少(图4J)。当WWP2过表达时,与Flag对照组相比,过表达Flag-BAF155后PARP1的泛素化水平下降(图4K)。Next, Applicants aimed to determine which E3 ubiquitination ligase mediates BAF155-inhibited PARP1 ubiquitination. In the applicant's previous work, the applicant discovered that WWP2 is a specific E3 ubiquitination ligase of PARP1 and ubiquitinates the PARP1-K249 site. At the same time, WWP2 is also the E3 ubiquitination ligase of BAF155. Under treatment of MG132, the degradation of PARP1 and BAF155 was blocked by exogenous immunoassay, resulting in enhanced interaction of BAF155 and PARP1 with WWP2 (Fig. 4H). Furthermore, the binding of PARP1 to WWP2 was increased after BAF155 knockdown (Fig. 4I), while overexpression of BAF155 led to a decrease in the binding of PARP1 to WWP2 (Fig. 4J). When WWP2 was overexpressed, the ubiquitination level of PARP1 decreased after overexpressing Flag-BAF155 compared with the Flag control group (Figure 4K).
申请人之前的研究发现PARP1-K249的泛素化依赖于E3泛素化WWP2。有趣的是,PARP1-K249的修饰依赖于SIRT2在同一位点的上游脱乙酰化。因此,为了验证BAF155对PARP1的分子调控机制,在小鼠心脏组织中进行了免疫沉淀。PARP1与WWP2和SIRT2的结合在用AngII治疗后得到增强,并且在BAF155-cKO小鼠心脏组织中显著增加,与有或没有AngII治疗的WT相比(图4L)。就PARP1的泛素化水平而言,申请人的数据显示AngII在WT小鼠心脏组织中诱导泛素化(图4M)。此外,与WT小鼠相比,PARP1的泛素化在BAF155-cKO小鼠心脏组织中显著增加(图4M)。相反,与WT小鼠相比,在BAF155-TG小鼠心脏组织中,PARP1与WWP2和SIRT2的结合比WT降低(图4N)。与WT小鼠相比,BAF155-TG小鼠心脏组织中PARP1的泛素化显著降低(图4O)。Applicants' previous studies found that ubiquitination of PARP1-K249 is dependent on E3 ubiquitination of WWP2. Interestingly, modification of PARP1-K249 relies on upstream deacetylation of SIRT2 at the same site. Therefore, in order to verify the molecular regulatory mechanism of BAF155 on PARP1, immunoprecipitation was performed in mouse heart tissue. The binding of PARP1 to WWP2 and SIRT2 was enhanced upon treatment with AngII and significantly increased in heart tissue of BAF155-cKO mice compared with WT with or without AngII treatment (Fig. 4L). In terms of ubiquitination levels of PARP1, Applicants' data show that AngII induces ubiquitination in WT mouse heart tissue (Figure 4M). Furthermore, ubiquitination of PARP1 was significantly increased in heart tissue of BAF155-cKO mice compared with WT mice (Fig. 4M). In contrast, PARP1 binding to WWP2 and SIRT2 was reduced in heart tissue of BAF155-TG mice compared with WT mice (Fig. 4N). Ubiquitination of PARP1 was significantly reduced in heart tissue of BAF155-TG mice compared with WT mice (Fig. 4O).
2.5.蛋白质组学分析鉴定出BAF155上的K948受SIRT2特异性调控2.5. Proteomic analysis identified K948 on BAF155 as specifically regulated by SIRT2
内源性和外源性免疫共沉淀证实SIRT2与BAF155相互作用(图5A-C)。此外,在AngII的诱导下,SIRT2和BAF155之间的相互作用得到增强(图5D和E)。根据UniProt数据库,BAF155包含五个功能域,包括Chromo、SWIRM、SANT、Glu-rich和Pro-rich域。使用内源性SIRT2和全长Flag-tagged-BAF155或各种截短的Flag-BAF155质粒,申请人证明BAF155的SWIRM结构域是SIRT2的结合位点(图5F)。Endogenous and exogenous co-immunoprecipitation confirmed that SIRT2 interacts with BAF155 (Fig. 5A-C). Furthermore, the interaction between SIRT2 and BAF155 was enhanced upon induction by AngII (Fig. 5D and E). According to the UniProt database, BAF155 contains five functional domains, including Chromo, SWIRM, SANT, Glu-rich and Pro-rich domains. Using endogenous SIRT2 and full-length Flag-tagged-BAF155 or various truncated Flag-BAF155 plasmids, Applicants demonstrated that the SWIRM domain of BAF155 is the binding site for SIRT2 (Figure 5F).
接下来,申请人重点研究了BAF155乙酰化的调控机制。在用曲古抑菌素A(TSA)和烟酰胺(NAM)治疗后,BAF155的乙酰化水平增加,它们是组蛋白去乙酰化酶HDACI和III以及去乙酰化酶的去乙酰化酶家族的抑制剂(图5G)。为了鉴定BAF155的特异性乙酰转移酶,分别转染了四种乙酰转移酶,包括p300(300-kDaE1A结合蛋白)、CBP、PCAF(p300/CBP相关因子)和GCN5(KAT2A)。如图5H所示,CBP的过表达,而不是其他乙酰转移酶的过表达,显著增加了 BAF155的乙酰化水平。此外,CBP在内源性和外源性条件下都与BAF155相互作用(图5I和5J)。因此,BAF155被证明是CBP赖氨酸乙酰化的底物。接下来,申请人分析了用或不用20μmol/LAGK2(一种常用的SIRT2特异性抑制剂)处理的正常对照和shSIRT2细胞的整体乙酰化变化。与之前的结果一致,与正常对照细胞相比,BAF155的乙酰化水平在shSIRT2细胞和用AGK2处理的细胞中上调(图5K)。上述调节在WT和SIRT2敲除小鼠的心脏组织中得到验证,与WT动物相比,来自SIRT2敲除动物的心脏组织样品中BAF55的BAF155水平显著增强(图5L)。此外,WT-SIRT2的过表达降低了BAF155的外源乙酰化水平(图5M),而转染SIRT2的无活性突变体(H187YQ167A)则没有效果(图5N)。此外,与WT动物相比,来自SIRT2敲除动物的心脏组织样本中BAF155的乙酰化水平显著增强,无论是否由AngII诱导(图5O)。外源性BAF155的乙酰化水平在AngII处理下降低,而外源性BAF155的乙酰化水平随着AngII诱导的外源性SIRT2过表达而进一步下降(图5P)。Next, the applicant focused on studying the regulatory mechanism of BAF155 acetylation. Acetylation levels of BAF155 are increased after treatment with trichostatin A (TSA) and nicotinamide (NAM), members of the histone deacetylase HDACI and III and deacetylase families of inhibitor (Figure 5G). To identify the specific acetyltransferase of BAF155, four acetyltransferases were transfected, including p300 (300-kDaE1A binding protein), CBP, PCAF (p300/CBP-related factor), and GCN5 (KAT2A). As shown in Figure 5H, overexpression of CBP, but not other acetyltransferases, significantly increased Acetylation levels of BAF155. Furthermore, CBP interacted with BAF155 under both endogenous and exogenous conditions (Figures 5I and 5J). Therefore, BAF155 was shown to be a substrate for CBP lysine acetylation. Next, Applicants analyzed global acetylation changes in normal control and shSIRT2 cells treated with or without 20 μmol/LAGK2, a commonly used SIRT2-specific inhibitor. Consistent with previous results, the acetylation level of BAF155 was upregulated in shSIRT2 cells and cells treated with AGK2 compared with normal control cells (Fig. 5K). The above regulation was verified in heart tissue of WT and SIRT2 knockout mice, with BAF155 levels significantly enhanced in heart tissue samples from SIRT2 knockout animals compared with WT animals (Fig. 5L). Furthermore, overexpression of WT-SIRT2 reduced the exogenous acetylation level of BAF155 (Fig. 5M), whereas transfection of an inactive mutant of SIRT2 (H187YQ167A) had no effect (Fig. 5N). Furthermore, acetylation levels of BAF155 were significantly enhanced in heart tissue samples from SIRT2 knockout animals compared with WT animals, regardless of whether induced by AngII (Fig. 5O). The acetylation level of exogenous BAF155 decreased under AngII treatment, and the acetylation level of exogenous BAF155 further decreased with AngII-induced overexpression of exogenous SIRT2 (Fig. 5P).
K948可能代表受SIRT2调节的BAF155的重要乙酰化位点。K948在整个进化过程中发现,从莫哈文果蝇到哺乳动物物种(图5Q)。为了进一步研究乙酰化对K948的关键作用,产生了特异性识别BAF155的乙酰化K948的抗体(图5R)。施用TSA和NAM后,BAF155-K948的乙酰化水平增加(图5S)。此外,外源转染四种乙酰转移酶后,只有CBP增加了BAF155-K948的乙酰化水平(图5T)。同时,外源转染WT-SIRT2而不是失活的SIRT2(H187YQ167A)降低了BAF155-K948的乙酰化水平(图5U)。总之,以上数据表明K948受到CBP和SIRT2的特异性调控。K948 may represent an important acetylation site for BAF155 regulated by SIRT2. K948 is found throughout evolution, from Drosophila melanogaster to mammalian species (Figure 5Q). To further investigate the critical role of acetylation on K948, an antibody specifically recognizing acetylated K948 of BAF155 was generated (Fig. 5R). The acetylation level of BAF155-K948 increased after administration of TSA and NAM (Fig. 5S). In addition, only CBP increased the acetylation level of BAF155-K948 after exogenous transfection of four acetyltransferases (Fig. 5T). At the same time, exogenous transfection of WT-SIRT2 but not inactivated SIRT2 (H187YQ167A) reduced the acetylation level of BAF155-K948 (Fig. 5U). Taken together, the above data indicate that K948 is specifically regulated by CBP and SIRT2.
2.6.对SIRT2敲除和转基因小鼠中差异蛋白的乙酰化蛋白质组学分析证明了2.6. Acetylation proteomic analysis of differential proteins in SIRT2 knockout and transgenic mice demonstrated BAF155-K948在体内的SIRT2去乙酰化SIRT2 deacetylation by BAF155-K948 in vivo
为了全面了解SIRT2调节的小鼠心脏赖氨酸乙酰化变化,在SIRT2敲除小鼠(SIRT2-KO)、SIRT2过表达小鼠(SIRT2-TG)和野生型动物(SIRT2-WT)(图6A)。To comprehensively understand SIRT2-regulated changes in cardiac lysine acetylation in mice, SIRT2 knockout mice (SIRT2-KO), SIRT2 overexpression mice (SIRT2-TG), and wild-type animals (SIRT2-WT) (Figure 6A ).
在生物学实验中验证了BAF155-K948在SIRT2敲除和转基因小鼠中的乙酰化蛋白质组学分析。免疫沉淀测定证实,与WT相比,在SIRT2敲除后BAF155-K948的乙酰化水平在给予或不给予AngII的情况下增加(图6B),而BAF155-K948的乙酰化水平在SIRT2-TG小鼠心脏组织中降低(图6C)。Acetylation proteomic analysis of BAF155-K948 in SIRT2 knockout and transgenic mice was validated in biological experiments. Immunoprecipitation assay confirmed that the acetylation level of BAF155-K948 was increased after SIRT2 knockdown with or without AngII administration compared with WT (Fig. 6B), while the acetylation level of BAF155-K948 was smaller in SIRT2-TG. decreased in mouse heart tissue (Fig. 6C).
2.7.SIRT2通过WWP2促进BAF155-K948和PARP1-K294泛素化2.7.SIRT2 promotes BAF155-K948 and PARP1-K294 ubiquitination through WWP2
为了研究SIRT2对BAF155-PARP1凋亡复合物的调节作用,分析了SIRT2-WT和SIRT2-KO心脏组织以及NC和shSIRT2H9c2细胞中BAF155的丰度。如图7A所示,BAF155丰度在SIRT2下调后增加。申请人使用shSIRT2的三个片段(61965-shSIRT2 SEQ ID NO:3CTATGCAAACTTGGAGAAATA、61966-shSIRT2 SEQ ID NO:4GACCAAAGAGAAAGAGGAACA、61967-shSIRT2 SEQ ID NO:5GTGGAAAAGAGTACACGATGA)观察到类似的趋势,并使用61966-shSIRT2片段进行后续实验(图7A)。此外,与给予MG132的SIRT2-KO动物相比,SIRT2-WT小鼠心脏组织中BAF155上调的速率和程度升 高(图7B)。同时,SIRT2-WT小鼠心脏组织在CHX给药后BAF155表达显著降低,而SIRT2-KO小鼠组随着时间的推移保持非常高的BAF155表达(图7C)。与通过泛素-蛋白酶体途径降解一致,与Flag-对照质粒组相比,在Myc-SIRT2过表达和MG132处理后检测到BAF155的泛素化水平增强(图7D)。BAF155的泛素化水平在WT-SIRT2-Flag过表达后增加,但H187YQ167A-SIRT2-Flag(无脱乙酰活性的突变SIRT2)(图7E)。相反,随着四种乙酰转移酶的过表达,仅在过表达CBP的细胞中观察到BAF155丰度上调(图7F)和泛素化水平降低(图7G)。To investigate the regulatory effect of SIRT2 on the BAF155-PARP1 apoptotic complex, the abundance of BAF155 in SIRT2-WT and SIRT2-KO heart tissues as well as NC and shSIRT2H9c2 cells was analyzed. As shown in Figure 7A, BAF155 abundance increased after SIRT2 downregulation. Applicants observed similar trends using three fragments of shSIRT2 (61965-shSIRT2 SEQ ID NO: 3CTATGCAAACTTGGAGAAATA, 61966-shSIRT2 SEQ ID NO: 4GACCAAAGAGAAAGAGGAACA, 61967-shSIRT2 SEQ ID NO: 5GTGGAAAAGAGTACACGATGA) and followed up using the 61966-shSIRT2 fragment experiment (Figure 7A). Furthermore, the rate and extent of BAF155 upregulation in the heart tissue of SIRT2-WT mice increased compared with SIRT2-KO animals administered MG132. high (Fig. 7B). Meanwhile, BAF155 expression in SIRT2-WT mouse heart tissue significantly decreased after CHX administration, while the SIRT2-KO mouse group maintained very high BAF155 expression over time (Fig. 7C). Consistent with degradation through the ubiquitin-proteasome pathway, enhanced ubiquitination levels of BAF155 were detected after Myc-SIRT2 overexpression and MG132 treatment compared with the Flag-control plasmid group (Fig. 7D). The ubiquitination level of BAF155 increased after overexpression of WT-SIRT2-Flag but not H187YQ167A-SIRT2-Flag (mutated SIRT2 without deacetylation activity) (Fig. 7E). In contrast, with the overexpression of the four acetyltransferases, upregulation of BAF155 abundance (Figure 7F) and reduced ubiquitination levels (Figure 7G) were only observed in cells overexpressing CBP.
为了直接显示SIRT2促进BAF155的泛素化,申请人确定了由SIRT2调节的BAF155的去乙酰化位点。如图7H所示,与BAF155-K948R对应物相比,SIRT2过表达增强了BAF155-WT细胞中的泛素化水平,表明BAF155在K948处被SIRT2去乙酰化,导致在同一位点泛素化并促进BAF155的降解。简而言之,K948被SIRT2确认为BAF155的主要去乙酰化位点。To directly show that SIRT2 promotes ubiquitination of BAF155, Applicants identified deacetylation sites of BAF155 that are regulated by SIRT2. As shown in Figure 7H, SIRT2 overexpression enhanced ubiquitination levels in BAF155-WT cells compared with the BAF155-K948R counterpart, indicating that BAF155 is deacetylated by SIRT2 at K948, leading to ubiquitination at the same site And promote the degradation of BAF155. Briefly, K948 was identified by SIRT2 as the major deacetylation site of BAF155.
正如申请人上面提到的,BAF155与PARP1一样,共享相同的E3泛素连接酶WWP2。接下来,为了探索WWP2是否参与SIRT2介导的去乙酰化诱导的BAF155降解,HA-WWP2在NC和shSIRT2H9c2细胞系中表达。有趣的是,BAF155的丰度在NC细胞系中逐渐减少,而在shSIRT2细胞中保持在高水平(图7I)。与NC细胞相比,由WWP2介导的BAF155泛素化水平在shSIRT2处理中降低(图7J)。此外,Myc-SIRT2过表达导致BAF155和WWP2之间的结合升高,但与K948R-BAF155过表达无关(图7K)。上述发现进一步表明SIRT2通过WWP2促进了BAF155的泛素化。此外,申请人旨在验证BAF155-PARP1损伤复合物受SIRT2-WWP2调节。申请人发现PARP1的泛素化水平在Flag-SIRT2和HA-WWP2过表达后显著增加,并随着Flag-BAF155的进一步过表达而降低(图7L)。然后,过度表达K249R-PARP1(突变PARP1没有由BAF155和WWP2介导的泛素化位点),在PARP1上观察到的泛素化水平可以忽略不计(图7L)。上述结果表明,SIRT2通过WWP2介导的K948泛素化促进BAF155的降解,WWP2主要通过K249泛素化调节PARP1的降解。申请人进一步检查了SIRT2对PARP1-BAF155复合物的影响。随着Myc-SIRT2的过表达,PARP1与BAF155的结合减少(图7M)。与先前报道的结果一致,在有或没有AngII诱导的情况下,在shBAF155-H9C2细胞系中PARP1的表达降低(图7N),但在shSIRT2-H9C2细胞中BAF155的表达增加而PARP1的表达降低(图7O)。与shBAF155H9C2细胞相比,PARP1在shBAF155和shSIRT2H9C2细胞中的表达更高(图7N),但低于shSIRT2H9C2细胞(图7O)。这些发现表明SIRT2通过促进BAF155的降解使BAF155-PARP1复合物不稳定。As applicants mentioned above, BAF155 shares the same E3 ubiquitin ligase WWP2 as PARP1. Next, to explore whether WWP2 is involved in SIRT2-mediated deacetylation-induced BAF155 degradation, HA-WWP2 was expressed in NC and shSIRT2H9c2 cell lines. Interestingly, the abundance of BAF155 gradually decreased in NC cell lines while remaining at high levels in shSIRT2 cells (Figure 7I). The level of BAF155 ubiquitination mediated by WWP2 was reduced in shSIRT2 treatment compared with NC cells (Fig. 7J). Furthermore, Myc-SIRT2 overexpression led to increased binding between BAF155 and WWP2, but not K948R-BAF155 overexpression (Fig. 7K). The above findings further indicate that SIRT2 promotes the ubiquitination of BAF155 through WWP2. Additionally, applicants aimed to verify that the BAF155-PARP1 damage complex is regulated by SIRT2-WWP2. Applicants found that the ubiquitination level of PARP1 increased significantly after overexpression of Flag-SIRT2 and HA-WWP2 and decreased with further overexpression of Flag-BAF155 (Figure 7L). Then, by overexpressing K249R-PARP1 (mutated PARP1 without ubiquitination sites mediated by BAF155 and WWP2), negligible levels of ubiquitination were observed on PARP1 (Fig. 7L). The above results indicate that SIRT2 promotes the degradation of BAF155 through WWP2-mediated K948 ubiquitination, and WWP2 mainly regulates the degradation of PARP1 through K249 ubiquitination. Applicants further examined the effect of SIRT2 on the PARP1-BAF155 complex. With overexpression of Myc-SIRT2, PARP1 binding to BAF155 was reduced (Fig. 7M). Consistent with previously reported results, PARP1 expression was decreased in shBAF155-H9C2 cell lines with or without AngII induction (Fig. 7N), but BAF155 expression was increased and PARP1 expression was decreased in shSIRT2-H9C2 cells ( Figure 7O). Compared with shBAF155H9C2 cells, PARP1 expression was higher in shBAF155 and shSIRT2H9C2 cells (Fig. 7N), but lower than that in shSIRT2H9C2 cells (Fig. 7O). These findings suggest that SIRT2 destabilizes the BAF155-PARP1 complex by promoting the degradation of BAF155.
3.8.对SIRT2敲除和转基因小鼠中差异蛋白的蛋白质组学分析表明,SIRT2在体3.8. Proteomic analysis of differential proteins in SIRT2 knockout and transgenic mice showed that SIRT2 内通过WWP2促进BAF155和PARP1的泛素化Promotes ubiquitination of BAF155 and PARP1 via WWP2
在小鼠心脏组织中进行了免疫沉淀;与给予或不给予AngII的SIRT2-WT小鼠样品相比,用AngII治疗后BAF155与PARP1的结合增强,并且在SIRT2-KO小鼠心脏组织中显著增强(图8A)。此外,在SIRT2-WT小鼠心脏组织中,与WT小鼠样品相比,BAF155和PARP1的泛素化水平在AngII治疗后降低,并且在SIRT2-KO小鼠心脏组织中显著降低(图8B,8C)。此外,与SIRT2-WT动物相比, 在给予或不给予AngII的SIRT2-KO小鼠的心脏组织中,WWP2与BAF155和PARP1的结合降低(图8D)。相反,与给予或不给予AngII的SIRT2-WT组相比,BAF155与PARP1的结合在SIRT2-TG小鼠心脏组织中降低(图8E)。与SIRT2-WT小鼠相比,SIRT2-TG小鼠心脏组织中BAF155和PARP1的泛素化水平显著升高(图8F和8G)。此外,与SIRT2-WT动物相比,在给予或不给予AngII的SIRT2-TG小鼠的心脏组织中,WWP2与BAF155和PARP1的结合增加(图8H)。Immunoprecipitation was performed in mouse heart tissue; BAF155 binding to PARP1 was enhanced after treatment with AngII and significantly enhanced in SIRT2-KO mouse heart tissue compared with samples from SIRT2-WT mice administered with or without AngII (Figure 8A). Furthermore, in SIRT2-WT mouse heart tissue, the ubiquitination levels of BAF155 and PARP1 were reduced after AngII treatment compared with WT mouse samples, and were significantly reduced in SIRT2-KO mouse heart tissue (Figure 8B, 8C). Furthermore, compared with SIRT2-WT animals, The binding of WWP2 to BAF155 and PARP1 was reduced in the heart tissue of SIRT2-KO mice administered with or without AngII (Fig. 8D). In contrast, the binding of BAF155 to PARP1 was reduced in the heart tissue of SIRT2-TG mice compared with the SIRT2-WT group administered with or without AngII (Fig. 8E). Compared with SIRT2-WT mice, the ubiquitination levels of BAF155 and PARP1 were significantly increased in the heart tissue of SIRT2-TG mice (Figures 8F and 8G). Furthermore, WWP2 binding to BAF155 and PARP1 was increased in the heart tissue of SIRT2-TG mice administered or not administered AngII compared with SIRT2-WT animals (Fig. 8H).
讨论和结论Discussion and conclusion
申请人将SWI/SNF染色质重塑复合物的亚基BAF155(SMARCC1)鉴定为PARP1的重要体内相互作用蛋白。申请人的工作表明,BAF155通过干扰PARP1和WWP2、E3泛素连接酶以及PARP1的以下泛素化之间的相互作用来稳定PARP1。因此,上述证据指出了一种可能的新机制,即通过共定位来维持PARP1的高局部浓度和激活状态,以稳定其相互作用的蛋白质。Applicants identified BAF155 (SMARCC1), a subunit of the SWI/SNF chromatin remodeling complex, as an important in vivo interacting protein of PARP1. Applicants' work shows that BAF155 stabilizes PARP1 by interfering with the interaction between PARP1 and WWP2, E3 ubiquitin ligases, and the subsequent ubiquitination of PARP1. Therefore, the above evidence points to a possible new mechanism to maintain high local concentration and activation state of PARP1 through colocalization to stabilize its interacting proteins.
基于申请人的模型机制,低活性的SIRT2在BAF155的K948和PARP1的K249上保留了较高水平的乙酰化,从而使BAF155和PARP1保持相互作用状态并防止被WWP2泛素化。Based on the applicant's model mechanism, low-activity SIRT2 retains higher levels of acetylation on K948 of BAF155 and K249 of PARP1, thereby allowing BAF155 and PARP1 to maintain an interactive state and prevent ubiquitination by WWP2.
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.
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