Protein Acylation and Methylation Mechanisms

描述（由申请人提供）：该提案是R 01 GM 62437的竞争性更新申请，其结合了化学方法、酶学分析和细胞研究的使用，以增强我们对调节蛋白质酰化和甲基化的酶的理解。现在普遍认为，涉及赖氨酸的翻译后修饰（PTM） 组蛋白和其它蛋白质上的乙酰化和可逆甲基化是表观遗传学的核心。这种表观遗传修饰酶被视为癌症和其他疾病的有吸引力的药物靶标。生长素释放肽O-酰基转移酶（GOAT）催化辛酰基连接到肽激素生长素释放肽的Ser侧链的不寻常PTM，并且该酶的抑制剂可用于治疗肥胖症和糖尿病。尽管人们越来越多地努力了解这些PTM的机制和功能以及催化它们的酶，但我们在这些领域的理解存在重大差距。填补这些知识空白有可能使人们更清楚地了解基本的生物医学过程，并有机会促进新的治疗方法和疾病诊断策略的发展。这项建议有四个具体目标。1.使用新的化学和生物化学方法阐明组蛋白乙酰转移酶（HAT）蛋白底物选择性的分子基础。我们将开发和应用新技术来产生组蛋白-CoA结合物，并利用蛋白质微阵列来阐明HAT-底物相互作用的结构和功能特征。2.开发和应用改进的化学工具来分析LSD 1组蛋白去甲基化。将制备合成抑制剂和炔丙基-组蛋白的组合以询问LSD 1细胞功能和分子相互作用。3.阐明赖氨酸乙酰化对S-腺苷高半胱氨酸水解酶和肌醇单磷酸脱氢酶-2的影响。表达的蛋白质连接将用于将乙酰基-赖氨酸位点特异性地安装到这些代谢酶中，以剖析蛋白质乙酰化和细胞代谢之间的潜在重要节点。4.定义胃饥饿素O-酰基转移酶的主要结构特征。膜拓扑映射和双底物类似物交联的组合将用于创建这种关键代谢调节酶的蓝图。我们相信，这项研究工作有可能大大扩展我们对蛋白质翻译后修饰机制和功能的理解，并确定治疗代谢和肿瘤疾病的新治疗机会。

DESCRIPTION (provided by applicant): This proposal is a competing renewal application of R01 GM62437 that combines the use of chemical approaches, enzymologic analysis, and cellular studies to enhance our understanding of enzymes regulating protein acylation and methylation. It is now well- accepted that post-translational modifications (PTMs) involving lysine acetylation and reversible methylation on histones and other proteins are central to epigenetics. Such epigenetic modifying enzymes are viewed as attractive drug targets for cancer and other diseases. The ghrelin O-acyltransferase (GOAT) enzyme catalyzes the unusual PTM of octanoyl attachment to a Ser side chain of the peptide hormone ghrelin and inhibitors of this enzyme may be useful in the treatment of obesity and diabetes. Although there has been increasing efforts to understand the mechanisms and functions of these PTMs and the enzymes that catalyze them, there are major gaps in our understanding in these areas. Filling these knowledge gaps has the potential to provide a clearer understanding of basic biomedical processes and has the opportunity to enhance the development of novel therapeutic approaches and disease diagnostic strategies. There are four Specific Aims in this proposal. 1. Elucidate the molecular basis for protein substrate selectivity for histone acetyltransferases (HAT) using new chemical and biochemical approaches. We will develop and apply new techniques to generate histone-CoA conjugates and exploit protein microarrays to clarify structural and functional features of HAT-substrate interactions. 2. Develop and apply improved chemical tools for analyzing LSD1 histone demethylation. A combination of synthetic inhibitors and propargyl-histones will be prepared to interrogate LSD1 cellular functions and molecular interactions. 3. Clarify the effects of Lys acetylation on S-adenosyl homocysteine hydrolase and inositol monophosphate dehydrogenase-2. Expressed protein ligation will be used to install acetyl-Lys site-specifically into these metabolic enzymes to dissect potentially important nodes between protein acetylation and cellular metabolism. 4. Define major structural features of ghrelin O-acyltransferase. A combination of membrane topology mapping and bisubstrate analog crosslinking will be used to create a blueprint of this key metabolic regulatory enzyme. We believe that this research effort has the potential to greatly expand our understanding of protein post-translational modification mechanisms and functions and identify new therapeutic opportunities for treating metabolic and neoplastic diseases.