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

许多蛋白质经过翻译后赖氨酸氨基的乙酰化。

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.