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

许多蛋白质都经历了赖氨酸氨基的翻译后乙酰化。 组蛋白和转录因子的乙酰化，与其他修饰竞争 发生在相同的赖氨酸上，是公认的基因表达调节因子。 非核蛋白的乙酰化还没有得到广泛的分析。然而，它是 已经清楚的是，蛋白质乙酰化比最初认为的要普遍得多， 一些研究人员认为，它可能与后磷酸化一样重要- 细胞中非核活动的翻译调控。 缝隙连接是由连接蛋白亚基形成的通道，允许被动的 低分子亲水分子(约1 kDa或更小)之间的转移 相邻单元格。在脊椎动物中，由连接蛋白Cx43组成的缝隙连接执行 心脏和其他组织的重要功能。已知Cx43受多个 磷酸化事件，每个事件都被认为调节其生物发生的不同步骤，通道 门禁和/或营业额。我们最近发现，几种连接蛋白经历了 乙酰化。进一步的初步数据表明，连接蛋白乙酰化在其 营业额或交易，可能反映了乙酰化和其他 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，即缝隙连接人口贩运， 连接蛋白乙酰化调节其组装和/或周转。我们将测试我们的 假设有两个特定的目的：首先，我们将确定Cx43的赖氨酸是 乙酰化，在准备敲除的cdna克隆的过程中，以及其他可能 在我们确定的每个位置模拟乙酰化。在免疫学和生物化学方面 实验中，我们将利用放射性醋酸酯和泛乙酰赖氨酸标记 抗体可方便地监测Cx43的乙酰化状态。因此，具体目标1 将确定Cx43乙酰化的位点，并为其研究和扰动提供工具。 其次，我们将使用我们产生的乙酰化位点突变体开始建立 乙酰化的后果。我们将测试特定的乙酰化赖氨酸在Cx43中的作用 生物发生，组装成连接子，运输到质膜，组装成 斑块和营业额。我们提议的研究将增加我们对如何 乙酰化调节缝隙连接通道的结构和数量，可能还有其他 膜蛋白。在这两年期间完成的学习也将为 为进一步研究翻译后乙酰化对GJ功能的调节作用奠定了基础。 我们预计，我们在Cx43乙酰化方面的结果将直接影响我们的 理解和潜在地关于由基因突变引起的疾病的管理 Cx43和其他连接蛋白。从更广泛的角度来看，我们还将通过以下方式展示新角色 哪种蛋白质乙酰化调节与基因没有直接关系的细胞功能 表情。

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.