New technologies to understand the interplay of protein modifications in human diseases

这一应用主要集中在开发新的质谱学(MS)策略和数据挖掘方法，以描述影响重要细胞过程的蛋白质修饰的动态变化，如转录调节、蛋白质转位和降解，这些过程在癌症发展过程中经常被错误调控。在哺乳动物细胞中，大量的酶通过200多种不同类型的翻译后修饰(PTM)的共价连接来影响蛋白质的生物活性，其中许多PTM针对相同的氨基酸(例如，赖氨酸残基可以通过甲基化、乙酰化、泛素化或总甲基化来修饰)。尽管质谱法在提供关于PTMS性质和部位的准确信息方面一直是一项关键技术，但鉴于这些修饰的丰度低和动态性质，这些任务仍然具有挑战性。这些困难与缺乏方便的亲和层析方法来从复杂的细胞提取物中丰富修饰的蛋白质并确定不同类型的修饰之间的相互依赖而变得更加复杂。为了应对这些挑战，我们将开发特定的浓缩和检测方法来鉴定和分析人类细胞中的蛋白质磷酸化和相扑，并应用这些技术来确定Paralog特异的相扑底物，以及它们如何在As2O3和细胞因子的响应下被调节，这是已知影响蛋白质相思和随后的蛋白酶体降解的因素。开发的工具将用于识别新的底物，确定位点占有率的变化，并将蛋白质修饰与特定的酶活性相关联(例如，以特定的E3相扑连接酶或相扑蛋白酶为靶标的修改的相扑底物)。我们预计，作为这项提案的一部分所取得的进展，不仅将扩大大规模蛋白质组学和药物发现计划的范围和应用，还将对这些修饰在人类疾病中的调节和意义提供进一步的见解。

This application is primarily centered on the development of novel mass spectrometry (MS) strategies and data mining approaches to profile the dynamic changes in protein modifications affecting important cellular processes such as transcriptional regulation, protein translocation and degradation that are often misregulated during cancer development. In mammalian cells, a large array of enzymes affects the biological activities of proteins through the covalent attachment of more than 200 different types of post-translational modifications (PTMs), many of them targeting the same amino acid (e.g. lysine residues can be modified by methylation, acetylation, ubiquitylation or sumoylation). Although MS has been a key technology in providing precise information on the nature and site of PTMs, these tasks remain challenging in view of the low abundance and the dynamic nature of these modifications. These difficulties are compounded with the lack of convenient affinity chromatography approaches to enrich modified proteins from complex cell extracts and determine the interdependence between different types of modifications. To address these challenges, we will develop specific enrichment and detection methods to identify and profile protein phosphorylation and sumoylation in human cells, and apply these technologies to determine paralog-specific SUMO substrates and how they are regulated in response to As2O3 and cytokines, which are known to affect protein sumoylation and subsequent proteasome degradation. The tools developed will be use to identify novel substrates, determine changes in site occupancy and correlate protein modifications with specific enzyme activities (e.g. modified sumoylated substrates targeted by specific E3 SUMO ligase or SUMO proteases). We anticipate that developments made as part of this proposal will not only expand the scope and application of large-scale proteomics and drug discovery programs but will also provide further insights on the regulation and significance of these modifications in human diseases.