Enzymology of Post-translational Modifications

翻译后修饰的酶学

基本信息

批准号：
8237003
负责人：
CAROL A FIERKE
金额：
$ 43.02万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
1988
资助国家：
美国
起止时间：
1988-07-01 至 2014-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8237003
关键词：
Acetylation Active Sites Address Affect Affinity Affinity Chromatography Amino Acids Antifungal Agents Benzophenones Binding Biological Biological Process C-terminal Candida albicans Cardiovascular Diseases Catalysis Cell Differentiation process Cell physiology Cells Chemicals Chimeric Proteins Clinical Clinical Trials Code Complement Cysteine Cysteine-Rich Domain Deacetylase Deacetylation Dependence Development Diabetes Mellitus Dimethylallyltranstransferase Disease Dissociation Drug Design Enzymatic Biochemistry Enzymes Gel Gene Expression Goals Histone Acetylation Histones Homologous Gene Hydrolysis In Vitro Individual Isoenzymes Kinetics Label Lysine Malignant Neoplasms Mass Spectrum Analysis Measurement Membrane Metals Modification Molecular Mutagenesis N-acetylglucosamine deacetylase Oxidation-Reduction Palmitoyl Coenzyme A Pathway interactions Peptides Phenylalanine Post-Translational Protein Processing Protein Acetylation Protein Isoprenylation Proteins Proteolysis RNA Interference Reaction Regulation Regulatory Pathway Relative (related person)Role Site Site-Directed Mutagenesis Specificity Stress Structure Substrate Specificity Transferase Yeasts base carboxymethylation cofactor crosslink drug development enzyme activity enzyme substrate fluorophore histone acetyltransferase in vivo inhibitor/antagonist isoprenoid nervous system disorder new therapeutic target novel palmitoylation pathogen prenylation protein farnesyltransferase protein function protein geranylgeranyltransferase protein transport public health relevance response small molecule thioester

项目摘要

DESCRIPTION (provided by applicant): Post-translational modifications are essential regulators of protein function in cells. In many cases, the mechanisms and substrate specificities of enzymes that catalyze these modifications are not well understood, despite their involvement in a multitude of diseases, including cancer, diabetes, cardiovascular disease and neurological disorders. Our studies will focus on two classes of post-translational modifications, lipidation (including prenylation and palmitoylation) and acetylation, and the enzymes that regulate these modifications. The majority of protein prenylation, whereby an isoprenoid group is appended near the C-terminus of a target protein, is catalyzed by protein farnesyltransferase (FTase) and protein geranylgeranyltransferase type I (GGTase-I). Palmitoylation is catalyzed by protein palmitoyl transferases (PATs), such as Akr1p, and is a thioester exchange reaction between palmitoyl-CoA and a cysteine on a substrate protein to form a palmitoyl thioester. Both prenylation and palmitoylation target proteins to membranes and are essential for protein trafficking. Protein acetylation is regulated by competition between histone acetyltransferases, that catalyze protein N-acetylation, and histone deacetylases (HDACs), that catalyze hydrolysis of 5-N-acetyl lysine. Acetylation affects multiple cellular processes, including gene expression and cell differentiation. All of these enzymes are current targets for drug development for a variety of diseases; HDAC inhibitors are approved for clinical use and FTase and GGTase inhibitors are currently in clinical trials. However, the full complement of in vivo substrates for these enzymes is unknown, and the functional basis for substrate selectivity by each enzyme is undefined. Moreover, the effects of these modifications on protein function and cellular pathways are frequently unclear. Therefore, significant questions remain for each enzyme, including their in vivo specificity and regulation. Here, we propose to: (1) investigate determinants of prenyltransferase substrate specificity for mammalian and Candida albicans enzymes and analyze the effects of individual prenylation pathway modifications on protein localization and function; (2) investigate the catalytic mechanism of the protein palmitoyltransferase Akr1p; (3) investigate the metal selectivity of deacetylases HDAC8 and LpxC in vivo and in vitro and the potential for regulation by metal switching in response to metal availability and redox stress; and (4) identify substrates and evaluate determinants of HDAC8 and HDAC11 substrate specificity, both in vitro and in vivo, by chemical crosslinking and activity-based probes. Our long-term goals are: (1) to determine the catalytic mechanism and molecular basis of specificity for each of these post-translational modifying enzymes; and (2) to examine the extent, biological function and regulation of prenylation, palmitoylation, and acetylation in the cell. Overall, our studies will assist in targeted drug design by describing the specific interactions that dictate substrate specificity and by identifying novel regulatory pathways as targets for disease therapies. PUBLIC HEALTH RELEVANCE: Post-translational modifications are essential regulators of protein function. Protein lipidation and acetylation are implicated in a multitude of diseases, including cancer, cardiovascular disease and neurological disorders. Identification of the protein substrates and downstream biological function will enhance the development of new therapeutics targeting these pathways.

描述（由申请人提供）：翻译后修饰是细胞中蛋白质功能的基本调节剂。在许多情况下，尽管它们参与了多种疾病，包括癌症，糖尿病，心血管疾病和神经系统疾病，但促成这些修饰的酶的机制和底物特异性尚未得到很好的了解。我们的研究将集中于两类翻译后修饰，脂质化（包括原苯基化和棕榈酰化）和乙酰化，以及调节这些修饰的酶。大多数蛋白介烯基化，从而将类吸收蛋白附在靶蛋白的C末端附近，是由蛋白Farnesylansylansferase（FTase）和蛋白质geranylgeranylylylansyltansferase催化的（GGTase-I）。棕榈酰化是由蛋白质棕榈酰转移酶（PATS）催化的，例如AKR1P，是棕榈酰-COA和底物蛋白上半胱氨酸之间的硫酯交换反应，形成了硫基硫酰基硫酰基硫酯。前酰化和棕榈酰化靶蛋白均靶向膜，对于蛋白质运输至关重要。蛋白质乙酰化受组蛋白乙酰转移酶之间的竞争调节，乙酰基转移酶，催化蛋白N-乙酰化和组蛋白脱乙酰基酶（HDACS），从而催化5-N-乙酰赖氨酸的水解。乙酰化会影响多种细胞过程，包括基因表达和细胞分化。所有这些酶都是目前用于各种疾病的药物开发靶标。 HDAC抑制剂被批准用于临床使用，FTase和GGTase抑制剂目前正在临床试验中。但是，这些酶的体内底物的完整补体尚不清楚，并且每个酶的底物选择性的功能基础都是不确定的。此外，这些修饰对蛋白质功能和细胞途径的影响通常不清楚。因此，每个酶（包括其体内特异性和调节）仍然存在重大问题。在这里，我们建议：（1）研究哺乳动物和白色念珠菌酶的前转移酶底物特异性的决定因素，并分析单个预苯途径修饰对蛋白质定位和功能的影响；（2）研究蛋白质棕榈转移酶AKR1P的催化机制；（3）研究脱乙酰基酶HDAC8和LPXC在体内和体外的金属选择性，并在金属转换中对金属可用性和氧化还原应激进行调节的潜力；（4）通过化学交联和基于活性的探针确定体外和体内HDAC8和HDAC11底物特异性的底物并评估底物的决定因素。我们的长期目标是：（1）确定这些翻译后修饰酶的特异性的催化机制和分子基础；（2）检验细胞中原酰化，棕榈酰化和乙酰化的程度，生物学功能和调节。总体而言，我们的研究将通过描述决定底物特异性的特定相互作用并确定新型调节途径作为疾病疗法的靶标，来帮助有针对性的药物设计。公共卫生相关性：翻译后修饰是蛋白质功能的重要调节剂。蛋白质脂化和乙酰化与多种疾病有关，包括癌症，心血管疾病和神经系统疾病。鉴定蛋白质底物和下游生物学功能将增强针对这些途径的新疗法的发展。