Connexin acetylation: Impact on gap junction function and HDAC inhibitor respons

连接蛋白乙酰化：对间隙连接功能和 HDAC 抑制剂反应的影响

• 批准号: 7934520
• 负责人: JEANNETTE CHLOE BULINSKI
• 金额: $ 31.61万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7934520
• 关键词: Acetylation; Antibodies; Biochemical; Biogenesis; Biology; Cardiac; Cell membrane; Cell physiology; Cells; Complementary DNA; Connexin 43; Connexins; Connexon; Data; Deacetylation; Degradation Pathway; Disease; Event; Gap Junctions; Gene Expression; Health; Histone Acetylation; Histone Deacetylase Inhibitor; Human; Hypertrophy; Infarction; Knock-out; Knowledge; Laboratories; Light; Lysine; Malignant Neoplasms; Mediator of activation protein; Membrane; Membrane Proteins; Modification; Monitor; Mutation; Nuclear; Nuclear Protein; Nuclear Proteins; Phosphorylation; Post-Translational Protein Processing; Post-Translational Regulation; Process; Property; Protein Acetylation; Proteins; Regulator Genes; Research Personnel; Role; Site; Staging; Structure; Testing; Therapeutic; Tissues; Ursidae Family; Vertebrates; Work; amino group; base; designer antibody; disease-causing mutation; gap junction channel; human disease; intercellular communication; membrane assembly; molecular mass; mutant; novel; research study; tool; trafficking; transcription factor

Many proteins undergo post-translational acetylation of the ¿-amino group of lysine. Acetylation of histones and transcription factors, in competition with other modifications occurring on the same lysines, is well-established as a regulator of gene expression. Acetylation of non-nuclear proteins has not been extensively analyzed. However, it is already clear that protein acetylation is far more common than originally thought, with some researchers suggesting that it could be as important as phosphorylation in the post- translational regulation of non-nuclear activities in cells. Gap junctions are channels formed from connexin subunits that permit the passive transfer of low molecular mass hydrophilic molecules (ca. 1 kDa or less) between adjacent cells. In vertebrates the gap junctions comprised of the connexin, Cx43, perform vital functions in cardiac and other tissues. Cx43 is known to be regulated by multiple phosphorylation events, each thought to regulate different steps in its biogenesis, channel gating, and/or turnover. We recently discovered that several connexins undergo acetylation. Further preliminary data suggest that connexin acetylation functions in its turnover or trafficking, perhaps reflecting a competition between acetylation and other covalent modifications of specific lysines. From these data, we propose our Working Hypothesis, that gap junction trafficking, assembly, and/or turnover are modulated by connexin acetylation. We will test our hypothesis in two Specific Aims: First, we will determine the lysines of Cx43 that are acetylated, in the process preparing cDNA clones that knock out, and others that possibly mimic, acetylation at each of the sites we identify. In immunological and biochemical experiments, we will utilize labeling with radioactive acetate and pan-acetyl lysine antibodies as convenient monitors of the acetylation state of Cx43. Thus, Specific Aim 1 will define the sites of Cx43 acetylation and provide tools for its study and perturbation. Second, we will use the acetylation-site mutants we generate to begin to establish acetylation's consequences. We will test the role of particular acetylated lysines in Cx43 biogenesis, assembly into connexons, trafficking to the plasma membrane, assembly into plaques, and turnover. Our proposed studies will increase our understanding of how acetylation modulates the structure and amount of gap junction channels and, likely, other membrane proteins. The studies accomplished during this two-year period will also pave the way for future studies on modulation of GJ function via post-translational acetylation. We anticipate that our results on Cx43 acetylation will bear directly on our understanding, and potentially on the management, of diseases caused by mutations in Cx43 and other connexins. In a broader view, we will also demonstrate novel roles by which protein acetylation regulates cellular functions not directly involved with gene expression.

许多蛋白质经历了赖氨酸 - 氨基的翻译后乙酰化。 在与其他修饰的竞争中，组蛋白和转录因子的乙酰化 出现在同一歌词上，作为基因表达的调节剂良好。 非核蛋白质的乙酰化尚未广泛分析。但是，是 已经很明显，蛋白质乙酰化比最初想象的要普遍得多， 一些研究人员认为，在后的后，它可能与磷酸化一样重要 细胞中非核活性的翻译调节。 差距连接是由连接蛋白亚基形成的通道，允许被动 低分子质量亲水分子（约1 kDa或更少）的转移 相邻细胞。在脊椎动物中，连接蛋白CX43完成的间隙连接 心脏和其他组织中的重要功能。已知CX43由多个调节 磷酸化事件，每个人都认为调节其生物发生不同步骤，通道 门控和/或营业额。我们最近发现几种连接蛋白发生 乙酰化。进一步的初步数据表明，连接蛋白乙酰化在其 营业额或贩运，也许反映了乙酰化与其他之间的竞争 特定赖氨酸的共价修饰。 从这些数据中，我们提出了我们的工作假设，即差距交通运输， 组装和/或营业额通过连接蛋白乙酰化调节。我们将测试我们的 两个具体目的的假设：首先，我们将确定CX43的赖氨酸 乙酰化，在制备cDNA克隆的过程中，其他可能 在我们识别的每个位点模拟，乙酰化。在免疫和生化 实验，我们将使用放射性乙酸盐和泛乙酰赖氨酸使用标签 抗体作为CX43的乙酰化状态的方便监测器。那是特定目标1 将定义CX43乙酰化的位点，并为其研究和扰动提供工具。 其次，我们将使用我们生成的乙酰化位点突变体开始建立 乙酰化的后果。我们将测试CX43中特定乙酰化歌词的作用 生物发生，组装成连接子，贩运到质膜，组装到 斑块和营业额。我们提出的研究将提高我们对 乙酰化调节间隙连接通道的结构和量，可能是其他 膜蛋白。在这两年期间完成的研究也将铺路 通过翻译后乙酰化对GJ功能调节的未来研究方式。 我们预计我们对CX43乙酰化的结果将直接置于我们的 理解，并有可能在管理中，是由突变引起的疾病 CX43和其他连接素。从更广泛的角度来看，我们还将通过 哪种蛋白乙酰化调节了不直接参与基因的细胞功能 表达。