YY1 functions in vascular smooth muscle cells

YY1在血管平滑肌细胞中发挥作用

• 批准号: 6970316
• 负责人: ANNY A USHEVA-SIMIDJIYSKA
• 金额: $ 39万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-02-01 至 2009-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6970316
• 关键词: DNA binding protein; N acylation; cardiovascular injury; cell growth regulation; cell proliferation; chromatin immunoprecipitation; flow cytometry; laboratory rat; mass spectrometry; protein structure function; site directed mutagenesis; tissue /cell culture; vascular smooth muscle

DESCRIPTION (provided by applicant): Activation of smooth muscle cell (SMC) proliferation in response to vascular injury is a widely appreciated vascular problem in clinical medicine, but its basis remains obscure. Our hypothesis is that abnormal posttranslational modification of proteins makes a major contribution to the initiation of activated smooth muscle cellular proliferation associated with vascular neointimal formation following vascular injury. We are particularly interested in the possibility that O-GlcNAcylation of YY1, significantly elevated in the neontimal smooth muscle cells, regulates the YY1-Rb interaction and downstream transcription promoting gene expression that triggers smooth muscle cellular proliferation. We are interested in the possibility that the phosphorylation of Rb at specific amino acids also inhibits the YY1-Rb complexation contributing to the activation of smooth muscle cellular proliferation. The goal of this project is to identify how O-glycosylation of YY1 regulates the YY1-Rb interaction and the transcriptional changes that occur in SMC transition from quiescence to active proliferation. Aim 1: Determine the specific sites of YY1 O-GlcNAcylation in growth arrested and in growth stimulated SMC cultures. The position of O-GlcNAc attachments to YY1 in the fractions will be determined by applying a combination of automated triple quadruple and ion trapping tandem mass spectroscopy. Aim 2: Analysis of YY1 O-GlcNAcylation in response to vascular balloon injury in a rat carotid artery model. The rate and the position of YY1 O-glycosylation 2 days, 4 days, and 8 days after rat balloon injury will be determined. Aim 3: Structure-based site-directed mutagenesis for the identification of the YY1-Rb binding surface and its regulation by Rb phosphorylation. The phosphorylated Ser/Thr residues on pRb that inhibit YY1 binding will be identified. Aim 4: Determine how O-GlcNAcylation influences cellular YY1 functions applying flow cytometry, gene expression regulation, and chromatin immunoprecipitation (ChIP).

描述（由申请人提供）：血管损伤时平滑肌细胞（SMC）增殖的激活是临床医学中广泛认识到的血管问题，但其基础仍不清楚。我们的假设是蛋白质的异常翻译后修饰对血管损伤后与血管新生内膜形成相关的活化平滑肌细胞增殖的启动做出了重要贡献。我们特别感兴趣的可能性，O-GlcNAc酰基化的YY 1，显着升高的血管平滑肌细胞，调节YY 1-Rb相互作用和下游转录促进基因表达，触发平滑肌细胞增殖。我们感兴趣的可能性，Rb在特定的氨基酸磷酸化也抑制YY 1-Rb络合有助于激活平滑肌细胞增殖。本项目的目的是确定YY 1的O-糖基化如何调节YY 1-Rb相互作用以及SMC从静止到活跃增殖转变中发生的转录变化。目的1：确定生长停滞和生长刺激的SMC培养物中YY 1 O-GlcNAc酰化的特异性位点。将通过应用自动三重四重和离子捕获串联质谱法的组合来确定组分中O-GlcNAc与YY 1连接的位置。目的2：在大鼠颈动脉模型中响应于血管球囊损伤的YY 1 O-GlcNAc化的分析。将测定大鼠球囊损伤后2天、4天和8天YY 1 O-糖基化的速率和位置。目的3：基于结构的定点突变鉴定YY 1-Rb结合表面及其通过Rb磷酸化的调节。将鉴定pRb上抑制YY 1结合的磷酸化Ser/Thr残基。目标4：应用流式细胞术、基因表达调控和染色质免疫沉淀（ChIP）确定O-GlcNAc化如何影响细胞YY 1功能。