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

本申请提出了一个研究计划，旨在提供新的工具，用于表征泛素样修饰剂（UBL）。我们的分析策略涉及细胞系、试剂和方法的产生，这些方法能够鉴定影响重要细胞过程的蛋白质修饰，如转录调控、细胞衰老、DNA损伤和修复。我们将重点关注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（NEDD 8）。虽然结构相似，但这些蛋白质控制不同的过程，并且UBL系统的改变与各种病理学相关，例如病毒感染，炎性疾病和癌症。虽然这些痕量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，它们的大规模鉴定仍然很困难，该方法可以从细胞提取物中富集相应的修饰蛋白。为了应对这些挑战，我们将开发新的分析方法，使这些UBL底物的质谱（MS）的位点特异性识别和分析。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SUMO残基免疫亲和纯化方法来分析SUMO链的动力学，并鉴定人类细胞中的旁系同源物特异性底物。这将使用稳定表达功能性SUMO旁系同源物的细胞系来实现，所述功能性SUMO旁系同源物含有促进SUMO肽的亲和富集和大规模MS鉴定的突变。其次，我们将开发工具和方法来鉴定SUMO E3连接酶和SUMO蛋白酶靶向的底物，这些底物仍然不清楚。我们将在表达SUMO突变体的细胞系上使用CRISPR/Cas9基因组编辑，并通过MS关联每个基因靶点缺失后SUMO底物的丰度变化。第三，我们将开发一种新的分析策略，将功能性NEDD 8突变体和免疫亲和富集相结合，以揭示蛋白质neddylation及其与其他UBL的相互作用。表达具有亲和标签和MS易处理残留物的不同UBL的细胞的可用性将提供方便的工具来以位点特异性方式描绘修饰蛋白的变化，并以时间方式定义UBL修饰的序列。我们预计，这些发展不仅将解决蛋白质组学中未满足的需求，而且还将进一步了解这些修饰在人类健康中的功能和重要性。

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.