Quantitative proteomics identifies substrates of SUMO E3 ligase PIAS proteins involved in cell growth and motility

Protein SUMOylation is a highly dynamic and reversible post-translational modification that targets lysine residues on a wide range of proteins involved in several essential cellular events, including protein translocation and degradation, mitotic chromosome segregation, DNA damage response, cell cycle progression, cell proliferation, and migration. Protein SUMOylation is an ATP-dependent enzymatic process that involves an E1 activating enzyme SAE1/2, a E2 conjugase UBC9, and usually facilitated by SUMO E3 ligases. The SP-RING family is the largest family of SUMO E3 ligases, encompassing seven mammalian protein inhibitor of activated STAT (PIAS) proteins. PIAS family was originally identified as specific inhibitors for signal transducer and activator of transcription (STAT), which involves gene transcriptional regulation. Recent studies showed that PIAS proteins also play important roles in the regulation of protein stability and signal transduction through the SUMOylation of target substrates. In addition, PIAS-mediated protein SUMOylation is also involved in several cellular processes, including DNA damage repair, immune response, cellular proliferation, and motility. Most notably, PIAS proteins are highly expressed in different cancer types and have been implicated in tumorigenesis. Several reports suggest that PIAS proteins could promote cancer cell growth and progression by regulating the SUMOylation of different substrates. To date, a number of substrates of PIAS ligases have been identified from several individual studies, and hundreds of specific SUMO E3 ligase substrates were identified from a human proteome microarray-based activity screen. However, how these substrates are selected, and which SUMOylation sites are targeted by these PIAS are still unknown.  To answer these questions, I started my investigation with PIAS1, one of the most well studied SUMO E3 ligases. By changing the expression level of PIAS1 in HeLa cells using gene overexpression or CRISPR/Cas9 gene knockout, I found PIAS1 had a physiological impact on cell proliferation and migration. I took advantage of the previously developed SUMO proteomics workflow to quantitatively profile global SUMOylome changes upon PIAS1 overexpression in a site-specific manner. I identified 983 SUMO sites on 544 proteins, of which 62 proteins were assigned as putative PIAS1 substrates. In particular, Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, was identified as PIAS1 substrates. Two SUMOylation sites mediated by PIAS1 at Lys-439 and Lys-445 residues were further evaluated and found to be necessary for dynamic disassembly and assembly of vimentin intermediate filaments, which further regulates cell migration and motility.  In the second study, I extended my investigation to all PIAS ligases and further found that all PIAS proteins impact cell proliferation and migration of breast cancer cell MDA-MB-231 after CRISPR/Cas9 gene knockout. I further optimized my SILAC-based quantitative SUMO proteomics approach and combined it with transcriptomics to gain a system-level understanding of the functional components involved in PIAS regulatory networks. A large subset of proteins/ genes involved in cell proliferation and migration were commonly regulated by all PIAS proteins, suggesting a redundancy of regulation within the PIAS family. In addition, each PIAS regulated a unique pool of substrates/genes involved in different cellular processes, such as DNA damage repair, chromatin remodeling, and SUMO chain formation, suggesting that each PIAS specifically regulates cellular functions. The trans-scale analyses between proteomics and transcriptomics shed light on the comprehensive pictures of the regulation networks by PIAS proteins beyond their direct enzymatic activity.  Overall, the quantitative SUMO proteomics approach provided a robust method for identifying substrates of PIAS SUMO E3 ligases. The combination of proteomic and transcriptomic analyzes made it possible to draw up a global portrait of the regulatory mechanisms governed by the PIAS proteins.

Keyphrases: SUMOylation, SUMO E3 ligase, PIAS proteins, Proteomics, Mass spectrometry, Cell proliferation, Cell migration