Deciphering the Code of N-terminal Post-translational Modification

 描述(由申请人提供)：α-氨基蛋白质的翻译后修饰已经被记录了几十年。然而，这些修改很少被研究，也被认为是结构性的，几乎没有监管作用。来自我们实验室的最新数据挑战了这些信念。我们已经鉴定了两个新的N末端修饰酶，NRMT1和NRMT2，并表明它们的许多靶点都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)。我们已经构建了第一个具有严重发育缺陷的N端PTM(NRMT1-/-)基因敲除小鼠。我们还鉴定了第一个蛋白质MYL9，它可以是N-末端甲基化的，也可以是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，起着不同的功能作用，并控制着许多调节人类发育和疾病的途径。具体目标#1将使用我们新产生的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。特殊目标#2将使用生化分析和质谱学的组合，首次证明N-末端甲基化和N-末端乙酰化是可以互换的，并以不同的方式调节蛋白质的功能。特定目标#3将使用等温量热法、蛋白质阵列和SILAC质谱仪的组合来确定N-末端单甲基化、三甲基化和乙酰化是否促进不同的结合伙伴。这些实验将开始在它们的相关信号通路中放置N末端的翻译后修饰，并揭示它们的错误调控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。

 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.