Protein N-terminal Methylation Mechanisms and Inhibition

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基序开始的蛋白质的α-N-末端胺的甲基化。阿尔法-N- 末端甲基化在调节细胞有丝分裂、染色质相互作用和DNA修复中起重要作用。 其水平随着细胞应激、衰老和发育过程的反应而增加。异常表达 NTMTs在各种人类疾病中被观察到。然而，蛋白质α-N- 末端甲基化和NTMT催化的分子基础还不清楚。我们的长期研究 目的是阐明NTMT介导的生化途径，这些途径有助于 癌症和发育缺陷，并开发有效和特异性NTMT抑制剂。的目的 研究蛋白质α-N-末端甲基化的机制、调节和识别， 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的底物和产物特异性的分子和结构基础。我们将应用 突变和晶体学分析，以了解NTMT 1和2如何识别其底物， 产生特定的甲基化产物。此外，我们建议确定一个生物正交对突变体 NTMT 1和SAM类似物，并使用它来获得NTMT 1的生理底物的全面概况。 我们将进行生物化学测定和细胞甲基化测定，以确认鉴定的 细胞中的底物。目标2.开发NTMT的有效和特异性抑制剂。我们将在初步调查的基础上 数据，以完成一系列NTMT的双底物类似物的合成和评价，其将用作 用于表征NTMT 1和2的结构和功能差异的探针，以研究NTMT抑制 与NTMT 1敲低的作用相比，我们将扩展我们的晶体结构数据库， NTMT三元复合物和筛选NTMT的小分子抑制剂。目标3：识别读者和 消除蛋白质α-N-末端甲基化。没有关于N-末端甲基化是如何 通过相互作用的分子伴侣“读取”，以及它是静态的还是动态的（“可擦除”）。我们建议准备 作为诱饵的光亲和标记探针，以鉴定蛋白质α-N-末端甲基化的相互作用伴侣， 细胞提取物将通过结合研究和免疫沉淀来验证所鉴定的相互作用伴侣 实验将进行生物信息学分析，以建立这些生物功能的假设。 互动伙伴总之，我们相信这项研究工作有很大的潜力， 更清楚地了解NTMT的机制和抑制作用，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方面提供新的化学工具： 基础NTMT生物学研究和促进新的治疗方法的发展， NTMT 1和NTMT 1参与的通路。

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.