Determining histone deacetylase 8 substrates using non-natural amino acids

 描述（由申请人提供）：赖氨酸乙酰化是一种可逆的翻译后修饰（PTM），可影响蛋白质功能，包括稳定性、定位以及与其他蛋白质和DNA的相互作用。乙酰化水平受组蛋白N-乙酰转移酶（HAT）和组蛋白脱乙酰酶（HDAC）的相对活性调节。乙酰化赖氨酸侧链首先在组蛋白中被鉴定，在组蛋白中它们被提出调节基因表达，改变核小体中DNA的可及性。然而，在哺乳动物蛋白质组中已经鉴定出超过9000个乙酰化位点，这些蛋白质参与了从细胞内分泌到细胞稳态的各种细胞过程。异常乙酰化与T细胞淋巴瘤和几种癌症的发展有关，包括胃癌、前列腺癌、结肠癌和乳腺癌。确定每种HDAC同工酶的底物库对于靶向药物开发和了解哪种途径受到每种酶抑制的影响至关重要。HDAC 8，这个建议的重点，已被广泛的体外表征和研究，使HDAC 8一个理想的模型系统开发工具，以确定HDAC相互作用的合作伙伴。已经测量了HDAC 8对模拟潜在底物的乙酰化肽的活性;然而，很少有经验证的体内底物。最近的质谱分析已经产生了一个推定的体内HDAC-蛋白质相互作用图，但还不清楚这些相互作用的蛋白质是底物还是结合伴侣。本研究将填补体外HDAC 8表征和体内HDAC 8功能之间的差距，并深入了解体内HDAC 8底物和结合伴侣的相互作用。我建议（1）在HDAC 8的不同位置引入光活性非天然氨基酸;（2）通过质谱法捕获并分析与HDAC 8相互作用的短寿命和长寿命伴侣和底物（与B教授合作）。Martin）和（3）使用体外酶偶联测定法验证底物。这些实验将促进HDAC 8底物的鉴定。这些方法的发展和应用，以其他HDAC将导致更好地了解HDAC相互作用组和HDAC蛋白特异性抑制剂的发展。

 DESCRIPTION (provided by applicant): Lysine acetylation is a reversible post-translational modification (PTM) that affects protein function including stability, localization, and interaction with other proteins and DNA. Acetylation levels are regulated by the relative activity of histone N-acetyltransferases (HATs) and histone deacetylases (HDACs). Acetylated lysine side chains were first identified in histones, where they are proposed to regulate gene expression altering the accessibility to the DNA in nucleosomes. However, over 9000 acetylation sites have been identified in the mammalian proteome and these proteins have been implicated in a variety of cellular processes ranging from catabolism to cell homeostasis. Aberrant acetylation has been implicated in the development of T-cell lymphoma and several cancers, including gastric, prostate, colon, and breast cancers. Defining the substrate pool of each HDAC isozyme is critical for targeted drug development and understanding which pathways are affected by inhibition of each enzyme. HDAC8, the focus of this proposal, has been characterized and studied extensively in vitro, making HDAC8 an ideal model system for developing tools to identify HDAC interaction partners. HDAC8 activity toward acetylated peptides mimicking potential substrates has been measured; however, there are very few validated in vivo substrates. Recent mass spectrometry analyses have yielded a putative in vivo HDAC-protein interaction map, but it is not clear whether these interacting proteins are substrates or binding partners. The research in this proposal will bridge the gap between in vitro HDAC8 characterization and in vivo HDAC8 function and provide insight into in vivo HDAC8 substrate and binding partner interactions. I propose to (1) incorporate photo-active non-natural amino acids at different positions of HDAC8; (2) trap and analyze short and long-lived interacting partners and substrates with HDAC8 through mass spectrometry (in collaboration with Prof. B. Martin) and (3) validate substrates using an in vitro enzyme coupled assay. These experiments will facilitate identification of HDAC8 substrates. Development and application of these methods to other HDACs will lead to a better understanding of the HDAC interactome and development of HDAC-protein specific inhibitors.