Deciphering the Code of N-terminal Post-translational Modification

破译N端翻译后修饰的密码

基本信息

批准号：
9302821
负责人：
Christine E Schaner-Tooley
金额：
$ 25.62万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-01 至 2020-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9302821
关键词：
Acetylation Acetyltransferase Address Belief Binding Binding Proteins Biochemical Biological Assay Calorimetry Cell Culture Techniques Cell Proliferation Cell physiology Cells Chromatin Structure Code Consensus Sequence Cytotoxic agent DNA Binding DNA-Binding Proteins Data Deacetylase Defect Development Disease Disease Progression EMSA Embryo Enzymes Fibroblasts Frequencies Genes Genetic Transcription Goals Health Histone Code Histones Human Human Development ICAM1 gene Knock-out Knockout Mice Laboratories Lead MJD1 protein Mass Spectrum Analysis Mediating Methylation Modification Monitor Mono-S Mus Myosin Regulatory Light Chains N-terminal Nuclear Nuclear Export Nuclear Localization Signal Pathway interactions Peptides Perception Post-Translational Protein Processing Protein Array Proteins Recruitment Activity Regulation Retinoblastoma Role Signal Pathway Signal Transduction Specificity Tail Tertiary Protein Structure Testing Time Translating amino group base beta catenin cell growth regulation cell motility chromatin immunoprecipitation experimental study human disease innovation knock-down mutant novel overexpression protein function public health relevance tau Proteins tumor tumorigenesis

项目摘要

DESCRIPTION (provided by applicant): Post-translational modification of the α-amino group of proteins has been documented for many decades. However, these modifications are little studied and thought to be constitutive, with few regulatory roles. Recent data from our laboratory challenges these beliefs. We have identified two novel N-terminal modifying enzymes, NRMT1 and NRMT2, and shown that many of their targets are known disease-related proteins (including Retinoblastoma, SET, Tau, β-catenin, and ataxin- 3). We have constructed the first viable knockout mouse for any N-terminal PTM (NRMT1-/-), which has severe developmental defects. We have also identified the first protein, MYL9, which can be either N-terminally methylated or N-terminally acetylated. According to these data, we aim to demonstrate that N-terminal post- translational modifications are dynamic, serve distinct functional roles, and control many pathways regulating human development and disease. Specific Aim #1 will use our newly generated NRMT1-/- mouse embryonic fibroblasts to determine the functional differences between N-terminal mono- and trimethylation. Specific Aim #2 will use a combination of biochemical assays and mass spectrometry to show for the first time that N-terminal methylation and N-terminal acetylation are interchangeable and differentially regulate protein function. Specific Aim #3 will use a combination of isothermal calorimetry, protein arrays, and SILAC mass spectrometry to determine if N-terminal monomethylation, trimethylation, and acetylation promote different binding partners. These experiments will begin to place N-terminal post-translational modifications in their relevant signaling pathways and reveal how their misregulation leads to developmental defects and tumor formation. Successful completion of these aims will initiate a new field of protein regulation and identify novel regulatory mechanisms for many cellular pathways known to impact human health.

描述（由申请人提供）：蛋白质 α-氨基的翻译后修饰已被记录了数十年，然而，这些修饰很少被研究，并且被认为是组成性的，来自我们实验室挑战的最新数据也很少。我们已经鉴定出两种新型 N 末端修饰酶 NRMT1 和 NRMT2，并表明它们的许多靶标是已知的疾病相关蛋白（包括视网膜母细胞瘤、SET、Tau、 β-catenin 和 ataxin- 3) 我们构建了第一个可存活的 N 末端 PTM 敲除小鼠 (NRMT1-/-)，它具有严重的发育缺陷。 N-末端甲基化或N-末端乙酰化根据这些数据，我们的目的是证明N-末端翻译后修饰是动态的，具有不同的功能作用，并控制调节人类发育和发育的许多途径。具体目标 #1 将使用我们新生成的 NRMT1-/- 小鼠胚胎成纤维细胞来确定 N 末端单甲基化和三甲基化之间的功能差异。具体目标 #2 将结合生化测定和质谱来显示疾病。 N 末端甲基化和 N 末端乙酰化首次实现可互换且差异调节的蛋白质功能。具体目标 #3 将结合使用等温量热法、蛋白质阵列和技术。 SILAC 质谱法确定 N 端单甲基化、三甲基化和乙酰化是否促进不同的结合伴侣。这些实验将开始将 N 端翻译后修饰置于其相关信号通路中，并揭示它们的失调如何导致发育缺陷和肿瘤形成。这些目标的成功完成将开启蛋白质调控的新领域，并为许多已知影响人类健康的细胞途径确定新的调控机制。