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

本应用程序提出了一项研究计划，旨在为表征泛素样修饰剂（UBLs）提供新的工具。我们的分析策略涉及细胞系、试剂和方法的生成，这些试剂和方法能够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。我们将重点关注UBL家族的三个成员，即泛素、小泛素样修饰因子（sumo）和神经前体细胞表达的发育下调蛋白8 （NEDD8）。尽管结构相似，但这些蛋白控制着不同的过程，UBL系统的改变与各种病理有关，如病毒感染、炎症性疾病和癌症。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，无法从细胞提取物中富集相应的修饰蛋白，因此它们的大规模鉴定仍然很困难。为了应对这些挑战，我们将开发新的分析方法，通过质谱（MS）对这些UBL底物进行位点特异性鉴定和分析。我们将运用这些工具来了解这些ubl在不同环境下在人类细胞中的调控和相互关系。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残体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相似特异性底物。这将通过稳定表达功能SUMO相似物的细胞系来实现，这些相似物含有有利于SUMO肽亲和富集和大规模质谱鉴定的突变。其次，我们将开发工具和方法来鉴定仍然不明确的SUMO E3连接酶和SUMO蛋白酶靶向的底物。我们将对表达SUMO突变体的细胞系进行CRISPR/Cas9基因组编辑，并通过MS关联每个基因靶缺失后SUMO底物的丰度变化。第三，我们将开发一种新的分析策略，结合功能性NEDD8突变体和免疫亲和富集来揭示蛋白质类化修饰及其与其他ubl的相互作用。具有亲和标签和ms可处理残体的表达不同UBL的细胞的可用性将提供一种方便的工具，以特定位点的方式分析修饰蛋白的变化，并及时定义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，而且将进一步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.