Profile substrates and inhibitors of protein lysine methyltransferase

 描述(由申请人提供)：在定义细胞命运时，需要在表观遗传水平上编排许多生物事件。蛋白质甲基转移酶(PMTs)是重要的表观遗传调控因子，它以S-腺苷-L-蛋氨酸(SAM)为辅因子，使特定的精氨酸/赖氨酸残基甲基化。虽然许多重组PMT在体外具有活性，但它们的细胞活性在很大程度上取决于不同的PMT异构体或复合体的存在。这种依赖于上下文的复杂性使得仅仅依靠传统方法来阐明PMT的目标具有挑战性。PMT灵活的底物识别模式，以及它们高度保守的SAM结合基序，也使开发高效和选择性的PMT抑制剂变得具有挑战性。该项目的一个长期目标是阐明和操纵甲基转移酶参与的表观遗传学用于疾病诊断和治疗。本申请的目的是使用SAM模拟物作为探针来分析PMT的特定细胞类型的底物，并以特定的方式干扰PMT。在这里，将实施蛋白质甲基化的综合生物正交谱分析(BPPM)，以在相关的生物背景下剖析指定的PMTs的底物。与特定生物过程相关的甲基化事件的一个亚类将用成熟的方法进一步验证。它们的功能作用将在癌症、恶性肿瘤或干细胞重新编程的背景下进行研究。同时，将寻求稳定的SAM类似物作为针对PMTs的靶向特异性抑制剂。该提案的完成将明确揭示多种PMT的底物，并在相关细胞环境中表征高质量的PMT抑制剂。这些底物分析和抑制物识别策略的影响还在于它们对其他甲基转移酶的普遍适用性。

 DESCRIPTION (provided by applicant): Numerous biological events need to be orchestrated at an epigenetic level upon defining cellular fate. Among the key epigenetic regulators are protein methyltransferases (PMTs), which methylate specific Arg/Lys residues with S-adenosyl-L- methionine (SAM) as a cofactor. Whereas many recombinant PMTs are active in vitro, their cellular activities can largely depend on the presence of distinct PMT isoforms or complexes. Such context- dependent complexity makes it challenging to solely rely on conventional methods to elucidate the targets of PMTs. The flexible substrate-recognizing patterns of PMTs, together with their highly- conserved SAM-binding motifs, also made it challenging to develop PMT inhibitors with potency and selectivity. A long-term goal of the project is to elucidate and manipulate methyltransferase- involved epigenetic for disease diagnosis and treatments. The objective of this application is to use SAM mimics as probes to profile cell-type-specific substrates of PMTs and to perturb PMTs in a specific manner. Here an integrative Bioorthogonal Profiling of Protein Methylation (BPPM) will be implemented to dissect the substrates of designated PMTs in relevant biological contexts. A subtype of methylation events associated with specific biological processes will be further validated with well-established methods. Their functional roles will be examined in the context of cancer malignancy or stem cell reprogramming. In parallel, stable SAM analogues will be pursued as target-specific inhibitors against PMTs. The completion of this proposal will unambiguously reveal the substrates of multiple PMTs and characterize high-quality PMT inhibitors in relevant cellular contexts. The impact of these substrate-profiling and inhibitor-identifying strategies further lie n their general applicability to other methyltransferases.