New proteomic methods to understand the regulation of ubiquitin-like modifiers in human diseases

本申请提出了一项研究计划，旨在为表征泛素样修饰物(UBL)提供新的工具。我们的分析策略包括生成细胞系、试剂和方法，以识别影响重要细胞过程的蛋白质修饰，如转录调节、细胞衰老、DNA损伤和修复。我们将重点介绍Ubl家族的三个成员，即泛素、小泛素样修饰物(SUMO)和神经前体细胞表达的发育下调蛋白8(NEDD8)。尽管这些蛋白质在结构上相似，但它们控制着不同的过程，UBL系统的变化与各种病理有关，如病毒感染、炎症性疾病和癌症。虽然这些示踪水平的修饰是高度动态的，但由于缺乏合适的亲和纯化方法来从细胞提取物中丰富相应的修饰蛋白，它们的大规模鉴定仍然困难。为了应对这些挑战，我们将开发新的分析方法，以实现通过质谱学(MS)对这些UBL底物进行现场特定识别和剖析。我们将应用这些工具来了解这些UBL在不同环境线索下在人类细胞中的调节和相互关系。为了实现这些目标，我们提出了三个相互关联的目标。首先，我们将利用我们新的相扑残留免疫亲和纯化方法来描述相扑链的动态，并在人类细胞中鉴定准对数特异性底物。这将通过稳定表达功能性相扑同源基因的细胞系来实现，这些细胞系含有突变，有助于相扑多肽的亲和力浓缩和大规模MS鉴定。其次，我们将开发工具和方法来确定相扑E3连接酶和相扑蛋白水解酶的靶标底物，这些底物仍然定义不清。我们将在表达相扑突变体的细胞系上使用CRISPR/Cas9基因组编辑，并通过MS关联相扑底物在每个基因靶标缺失时的丰度变化。第三，我们将开发一种新的分析策略，将功能性NEDD8突变体和免疫亲和力浓缩结合起来，以揭示蛋白质的相互作用及其与其他UBL的相互作用。具有亲和标签和MS易处理残基的表达不同UBL的细胞的可用性将提供一个方便的工具，以位置特定的方式描述修饰蛋白质的变化，并以时间方式定义UBL修饰的序列。我们预计，这些进展不仅将解决蛋白质组学中未得到满足的需求，而且还将加深我们对这些修饰在人类健康中的功能和重要性的理解。

This application presents a research program designed to provide new tools for characterizing ubiquitin-like modifiers (UBLs). Our analytical strategies involve the generation of cell lines, reagents, and methods that enable the identification of protein modifications affecting important cellular processes such as transcriptional regulation, cell senescence, DNA damage and repair. We will focus on three members of the UBL family namely ubiquitin, small ubiquitin-like modifiers (SUMOs), and neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8). Although similar in structures, these proteins control different processes, and alterations to the UBL system are associated with various pathologies, such as viral infection, inflammatory diseases and cancer. While these trace-level modifications are highly dynamic, their large-scale identification remain difficult due to the lack of suitable affinity purification methods that can enrich the corresponding modified proteins from cell extracts. To address these challenges, we will develop new analytical approaches that enable site-specific identification and profiling of these UBL substrates by mass spectrometry (MS). We will apply these tools to understand the regulation and interrelationship of these UBLs in human cells under different environmental cues. To fulfill these goals we propose three interrelated objectives. First, we will take advantage of our new SUMO remnant immunoaffinity purification approach to profile the dynamics of SUMO chains and identify paralog-specific substrates in human cells. This will be achieved using cell lines stably expressing functional SUMO paralogs that contain mutations facilitating the affinity enrichment and large-scale MS identification of SUMO peptides. Second, we will develop tools and methods to identify substrates targeted by SUMO E3 ligases and SUMO proteases that still remain ill-defined. We will use CRISPR/Cas9 genome editing on cell lines expressing SUMO mutants and correlate by MS the abundance changes of SUMO substrates upon deletion of each gene target. Third, we will develop a new analytical strategy that combines functional NEDD8 mutants and immunoaffinity enrichment to uncover protein neddylation and its interplay with other UBLs. The availability of cells expressing different UBLs with affinity tags and MS-tractable remnants will provide a convenient tool to profile the changes of modified proteins in a site-specific manner, and define the sequence of UBL modifications in a timewise fashion. We anticipate that these developments will not only address unmet needs in proteomics, but will also further our understanding of the function and importance of these modifications in human health.