Mechanistic Analyses of Protein Deacetylation

通过可逆的翻译后乙酰化，基因编码蛋白的功能能力得到了有力的扩展。我们的长期目标是了解蛋白质去乙酰化酶的催化机制和底物特异性，蛋白质去乙酰化酶水解组蛋白n端赖氨酸残基的epsilon-NH2侧链上的乙酰基，以及p53和HIV-Tat等蛋白质。蛋白质去乙酰化酶在转录抑制、染色质重塑和致癌蛋白、转录因子和有丝分裂蛋白的活性调节中起关键作用。这些酶的抑制剂是一种强大的抗癌药物，可以改变癌细胞的形态，通过刺激细胞分化和细胞周期机制，使转化的细胞恢复到正常的表型。了解这些重要蛋白质的机制、特异性和整体功能将对我们根除人类癌症的能力产生显著影响，并更好地理解转录的复杂生物学。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Aim 1中，我们将严格表征酿酒酵母Hos3的催化酰胺水解酶机制，Hos3是一种典型的I/II类锌依赖性组蛋白去乙酰化酶。在Aim 2中，对锌依赖性酶的分析将通过对酿酒酵母Hst2的全面表征来补充，Hst2是新发现的NAD+依赖性（类似Sir2） III类组蛋白去乙酰化酶的一个典型成员。最后，在Aim 3中，我们将使用明确定义的位点特异性修饰肽、全长组蛋白和合成核小体组装物来检查组蛋白去乙酰化酶的底物特异性。将确定微序列背景、翻译后修饰状态/位置、伴侣蛋白和DNA指示基团、同工异构体选择性以及组蛋白/核小体组成和构象对去乙酰化酶特异性的影响。总之，这些研究将为蛋白质去乙酰化酶的机制、抑制作用和底物特异性提供清晰的图像，并将指导基于机制的、同种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.