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

新的蛋白质组学方法来了解人类疾病中泛素样修饰物的调节

• 批准号: RGPIN-2018-04193
• 负责人: Thibault, Pierre
• 金额: $ 4.66万
• 依托单位: Université de Montréal
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=711878
• 关键词: New; proteomic; methods; understand; regulation

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.

该申请提出了一项研究计划，旨在提供表征类泛素修饰物 (UBL) 的新工具。我们的分析策略涉及细胞系、试剂和方法的生成，这些细胞系、试剂和方法能够识别影响转录调控、细胞衰老、DNA 损伤和修复等重要细胞过程的蛋白质修饰。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发育下调蛋白 8 (NEDD8)。虽然结构相似，但这些蛋白质控制着不同的过程，并且 UBL 系统的改变与各种病理相关，例如病毒感染、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来富集细胞提取物中相应的修饰蛋白，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识别和分析。我们将应用这些工具来了解不同环境线索下人体细胞中这些 UBL 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，我们将利用新的 SUMO 残余物免疫亲和纯化方法来分析 SUMO 链的动态并识别人类细胞中旁系同源物特异性底物。这将通过使用稳定表达功能性 SUMO 旁系同源物的细胞系来实现，这些同源物含有促进 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识别 SUMO E3 连接酶和 SUMO 蛋白酶的目标底物，但这些底物的定义仍然不明确。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 将删除每个基因靶标后 SUMO 底物的丰度变化关联起来。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 可处理残留物的不同 UBL 的细胞的可用性将提供一种方便的工具，以位点特异性方式分析修饰蛋白的变化，并以时间方式定义 UBL 修饰的序列。我们预计这些进展不仅将解决蛋白质组学中未满足的需求，还将进一步加深我们对这些修饰对人类健康的功能和重要性的理解。