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

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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.