Protein N-terminal Methylation Mechanisms and Inhibition

蛋白质 N 末端甲基化机制和抑制

基本信息

批准号：
9754194
负责人：
Rong Huang
金额：
$ 30.42万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-09-20 至 2021-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9754194
关键词：
Aging Amines Amino Acid Motifs Binding Biochemical Biochemical Pathway Bioinformatics Biological Biological Assay Biological Process Biology Catalysis Cell Extracts Cell physiology Cells Cellular Stress Cellular Stress Response Chemical Structure Chemicals Chromatin Complex Coupled Crystallization DNA Damage DNA Repair Data Defect Development Developmental Process Disease Enzymes Evaluation Exhibits Foundations Functional disorder Goals Immunoprecipitation Kinetics Knock-out Knockout Mice Lead Link Malignant Neoplasms Mediating Methylation Methyltransferase Mitosis Modification Molecular Molecular Structure Mutation Mutation Analysis N-terminal Pathogenesis Pathway interactions Peptides Pharmacology Phenotype Photoaffinity Labels Physiological Play Premature aging syndrome Property Protein Methyltransferases Proteins Reaction Reader Regulation Research Roentgen Rays Role Route Series Specificity Structure Structure-Activity Relationship Time Western Blotting Work analog cofactor database structure demethylation experimental study human disease inhibitor/antagonist interdisciplinary approach knock-down knowledge base mouse model mutant novel novel therapeutic intervention protein function public health relevance small molecule inhibitor success tool

项目摘要

Protein N-terminal methyltransferases (NTMTs) are a relatively new type of protein methyltransferase that catalyze the methylation of alpha-N-terminal amines of proteins starting with an X-P-K/R motif. The alpha-N- terminal methylation plays an essential role in regulating cell mitosis, chromatin interactions, and DNA repair. Its level is increased as response of cellular stress, aging, and developmental processes. Aberrant expression of NTMTs has been observed in various human diseases. However, the biological impact of protein alpha-N- terminal methylation and the molecular basis of NTMT catalysis are poorly defined. Our long-term research goal is to elucidate the biochemical pathways mediated by NTMTs that contribute to the pathogenesis of cancer and developmental defects, and to develop potent and specific NTMT inhibitors. The objective of this research to study mechanism, regulation, and recognition of protein alpha-N-terminal methylation through exploring and applying new chemical biology approaches. Three specific aims will be pursued: Aim 1. Elucidate the molecular and structural basis for substrate and product specificity of NTMTs. We will apply mutational and crystallographic analyses to understand how NTMT1 and 2 recognize their substrates and produce specific methylated products. In addition, we propose to identify a bioorthogonal pair of mutant NTMT1 and SAM analog and use it to obtain a comprehensive profile of physiological substrates for NTMT1. We will carry out biochemical assays and cellular methylation assays to confirm the relevance of identified substrates in cells. Aim 2. Develop potent and specific inhibitors for NTMTs. We will build on our preliminary data to complete synthesis and evaluation of a series of bisubstrate analogs for NTMTs, which will be used as probes to characterize the structural and functional differences of NTMT1 and 2, to investigate NTMT inhibition in cells in comparison with the effects of NTMT1 knockdown. We will extend our crystal structure database of NTMT ternary complexes and to screen for small molecule inhibitors for NTMTs. Aim 3. Identify readers and erasers for protein alpha-N-terminal methylation. There is no information on how this N-terminal methylation is `read' by interacting molecular partners and whether it is static or dynamic (`erasable'). We propose to prepare photoaffinity labeling probes as baits to identify interacting partners of protein alpha-N-terminal methylation in cell extracts. Identified interacting partners will be validated through binding studies and immunoprecipitation experiments. Bioinformatics analysis will be done to develop hypotheses for the biological functions of these interacting partners. Taken together, we believe that this research effort has the great potential to provide a clearer understanding of mechanisms and inhibition of NTMTs, and shed lights on the biological impact of protein N-terminal methylation. Accomplishment of the proposed work will also provide new chemical tools for both basic NTMT biology research and facilitate the development of novel therapeutic approaches to target NTMT1 and NTMT1-involved pathways.

蛋白质 N 末端甲基转移酶 (NTMT) 是一种相对新型的蛋白质甲基转移酶，催化以 X-P-K/R 基序开始的蛋白质 α-N 末端胺的甲基化。 α-N- 末端甲基化在调节细胞有丝分裂、染色质相互作用和 DNA 修复中发挥着重要作用。它的水平随着细胞应激、衰老和发育过程的反应而增加。异常表达已在多种人类疾病中观察到 NTMT 的存在。然而，蛋白质 α-N- 的生物学影响末端甲基化和 NTMT 催化的分子基础尚不清楚。我们的长期研究目标是阐明 NTMT 介导的生化途径，这些途径有助于疾病的发病机制癌症和发育缺陷，并开发有效且特异性的 NTMT 抑制剂。此举的目的研究蛋白质α-N-末端甲基化的机制、调控和识别探索和应用新的化学生物学方法。将追求三个具体目标：目标 1。阐明 NTMT 底物和产物特异性的分子和结构基础。我们将申请突变和晶体学分析，以了解 NTMT1 和 2 如何识别其底物并产生特定的甲基化产物。此外，我们建议鉴定一对生物正交突变体 NTMT1 和 SAM 类似物，并使用它来获得 NTMT1 生理底物的全面概况。我们将进行生化测定和细胞甲基化测定，以确认已识别的相关性细胞内的底物。目标 2. 开发有效且特异性的 NTMT 抑制剂。我们将在我们的初步基础上数据来完成一系列 NTMT 双底物类似物的合成和评估，这些类似物将用作探针来表征 NTMT1 和 2 的结构和功能差异，以研究 NTMT 抑制与 NTMT1 敲低的效果进行比较。我们将扩展我们的晶体结构数据库 NTMT 三元复合物并筛选 NTMT 的小分子抑制剂。目标 3. 确定读者并蛋白质α-N-末端甲基化的擦除器。没有关于 N 端甲基化是如何发生的信息通过相互作用的分子伙伴来“读取”，无论它是静态的还是动态的（“可擦除”）。我们建议准备光亲和标记探针作为诱饵来识别蛋白质α-N-末端甲基化的相互作用伙伴细胞提取物。确定的相互作用伙伴将通过结合研究和免疫沉淀进行验证实验。将进行生物信息学分析以提出这些生物功能的假设互动的伙伴。总而言之，我们相信这项研究工作具有巨大的潜力，可以提供更清楚地了解 NTMT 的机制和抑制，并阐明 NTMT 的生物学影响蛋白质 N 末端甲基化。拟议工作的完成还将为以下领域提供新的化学工具：既是基础 NTMT 生物学研究，又促进针对目标的新型治疗方法的开发 NTMT1 和 NTMT1 相关途径。