Deciphering the Code of N-terminal Post-translational Modification

破译N端翻译后修饰的密码

• 批准号: 9128022
• 负责人: Christine E Schaner-Tooley
• 金额: $ 1.42万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9128022
• 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; 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; Myosin Regulatory Light Chains; N-terminal; Nuclear; Nuclear Export; Nuclear Localization Signal; Pathway interactions; Peptides; Perception; Post-Translational Protein Processing; Process; Protein Array; Proteins; Reading; 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; human disease; innovation; knock-down; mutant; novel; overexpression; protein function; research study; tau Proteins; tumor; tumorigenesis

 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（由申请方提供）：数十年来，已记录了蛋白质α-氨基的翻译后修饰。然而，这些修饰很少被研究，并且被认为是组成性的，几乎没有调节作用。我们实验室最近的数据挑战了这些信念。我们已经鉴定了两种新的N-末端修饰酶，NRMT 1和NRMT 2，并表明它们的许多靶点是已知的疾病相关蛋白（包括视网膜母细胞瘤、SET、Tau、β-连环蛋白和共济失调蛋白-3）。我们已经构建了第一个可行的敲除小鼠的任何N-末端PTM（NRMT 1-/-），这有严重的发育缺陷。我们还鉴定了第一个蛋白质MYL 9，它可以是N-末端甲基化或N-末端乙酰化的。根据这些数据，我们的目标是证明N-末端翻译后修饰是动态的，具有不同的功能作用，并控制许多调节人类发育和疾病的途径。具体目标#1将使用我们新生成的NRMT 1-/-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将使用生物化学测定和质谱法的组合，首次表明N-末端甲基化和N-末端乙酰化是可互换的，并差异调节蛋白质功能。具体目标#3将使用等温量热法、蛋白质阵列和SILAC质谱法的组合来确定N端单甲基化、三甲基化和乙酰化是否促进不同的结合配偶体。这些实验将开始将N-末端翻译后修饰置于其相关的信号通路中，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创蛋白质调控的新领域，并为已知影响人类健康的许多细胞途径确定新的调控机制。