Mechanistic Analyses of Protein Deacetylation

基因编码的蛋白质的功能能力通过可逆的翻译后乙酰化而得到有力的扩展。 我们的长期目标是了解蛋白质脱乙酰酶的催化机制和底物特异性，这些酶水解组蛋白N-末端内赖氨酰残基的ε-NH 2侧链的乙酰基以及蛋白质如p53和HIV-Tat。 蛋白质脱乙酰酶在转录抑制、染色质重塑以及致癌蛋白、转录因子和有丝分裂蛋白活性的调节中发挥关键作用。 这些酶的抑制剂是强大的抗癌剂，其引起癌细胞的形态变化，通过刺激细胞分化和细胞周期机制将转化的细胞恢复为正常样表型。 了解这些重要蛋白质的机制、特异性和全局功能将对我们根除人类癌症的能力产生显著影响，并使我们更好地理解复杂的转录生物学。 在这个建议中，我们试图获得一个完整的视图组蛋白去乙酰化酶的机制，特异性和抑制。 在目标1中，我们将严格表征来自S的Hos 3的催化酰胺水解酶机制。cerevisiae，一种典型的I/II类锌依赖性组蛋白脱乙酰酶。 在目标2中，锌依赖酶的分析将通过S.酿酒酵母Hst 2，新发现的NAD+依赖性（Sir 2样）III类组蛋白脱乙酰酶的原型成员。 最后，在目标3中，我们将使用定义明确的位点特异性修饰肽，全长组蛋白，和组成定义的合成核小体组件检查组蛋白脱乙酰酶的底物特异性。 将确定微序列背景、翻译后修饰状态/位置、伴侣蛋白和DNA定向基团、异构体选择性以及组蛋白/核小体组成和构象对脱乙酰酶特异性的影响。 总之，这些研究将提供蛋白脱乙酰酶机制，抑制和底物特异性的清晰图像，并将指导基于机制的异构体选择性抑制剂的开发。

The functional capacity of genetically-encoded proteins is powerfully expanded by reversible posttranslational acetylation. Our long term goal is to understand the catalytic mechanism and substrate specificity of protein deacetylases, the enzymes which hydrolyze acetyl groups from the epsilon-NH2 side chain of lysyl residues within histone N-termini as well as proteins such as p53 and HIV-Tat. Protein deacetylases play a critical role in transcriptional repression, chromatin remodeling, and modulation of activities of oncogenic proteins, transcription factors, and mitotic proteins. Inhibitors of these enzymes are powerful anticancer agents that bring about morphological changes in cancer cells, reverting transformed cells to a normal-like phenotype by stimulating cellular differentiation and cell cycle machinery. Understanding the mechanisms, specificity, and global function of these important proteins will have pronounced impact on our ability to eradicate human cancer and better our understanding of the complex biology of transcription. In this proposal we seek to gain a complete view of histone deacetylase mechanism, specificity, and inhibition. In Aim 1 we will rigorously characterize the catalytic amidohydrolase mechanism of Hos3 from S. cerevisiae, a prototypical Class I/II zinc-dependent histone deacetylase. In Aim 2, analysis of the zinc-dependent enzymes will be complemented by a full characterization of S. cerevisiae Hst2, a prototypical member of the newly discovered NAD+-dependent (Sir2- like) Class III histone deacetylases. Finally, in Aim 3 we will examine the substrate specificity of histone deacetylases using well defined site-specifically modified peptides, full-length histones, and compositionally defined synthetic nucleosome assemblies. The effects of microsequence context, posttranslational modification state/position, partner protein and DNA directive groups, isoform selectivity, and histone/nucleosome composition and conformation on deacetylase specificity will be determined. Together, these studies will provide a clear picture of protein deacetylase mechanism, inhibition, and substrate specificity, and will direct the development of mechanism-based, isoform-selective inhibitors.