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

许多蛋白质会经历赖氨酸的<$-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，被公认为基因表达的调节剂。 非核蛋白的乙酰化尚未被广泛分析。但据 已经很清楚，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，它可能是重要的磷酸化后， 细胞中非核活性的翻译调节。 间隙连接是由连接蛋白亚基形成的通道，其允许在细胞内进行被动的细胞间连接。 低分子量亲水性分子（ca. 1 kDa或更小）之间 相邻的细胞在脊椎动物中，由连接蛋白Cx 43组成的差距连接， 心脏和其他组织的重要功能。已知Cx43受多个 磷酸化事件，每一个都被认为调节其生物发生的不同步骤， 门控和/或周转。我们最近发现几种连接蛋白 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，也许反映了乙酰化和其他 特定赖氨酸的共价修饰。 根据这些数据，我们提出了我们的工作假设，即间隙连接运输， 组装和/或转换通过连接蛋白乙酰化调节。我们将测试我们的 假设有两个具体的目的：首先，我们将确定Cx43的赖氨酸， 乙酰化，在制备敲除的cDNA克隆的过程中，以及其他可能 在我们发现的每个位点上进行模拟乙酰化。在免疫学和生物化学方面 在实验中，我们将利用放射性乙酸和全乙酰赖氨酸标记 抗体作为Cx43的乙酰化状态的方便监测器。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Cx43中特定乙酰化赖氨酸的作用， 生物发生，组装成连接子，运输到质膜，组装成 斑块和营业额。我们提出的研究将增加我们对如何 乙酰化调节间隙连接通道的结构和数量，并且可能调节其他的 膜蛋白在这两年期间完成的研究还将为 为今后通过翻译后乙酰化调控GJ功能的研究提供了方向。 我们预计，我们的Cx43乙酰化的结果将直接影响我们的研究。 了解，并可能对管理，由突变引起的疾病， 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颖的角色： 蛋白质乙酰化调节细胞功能，而不直接涉及基因 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.