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Quantitative proteomics identifies novel PIAS1 protein substrates involved in cell migration and motility

ABSTRACT: The Protein Inhibitor of Activated STAT 1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways, including STAT signaling, p53 pathway, and the steroid hormone signaling pathway. PIAS1 can SUMOylate PML (at Lys-65 and Lys-160) and PML-RARα promoting their ubiquitin-mediated degradation. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, such as prostate and lung cancers. To understand the mechanism of action of PIAS1, we developed a quantitative SUMO proteomic approach to identify potential substrates of PIAS1 in a system-wide manner. Our analyses enabled the profiling of 983 SUMO sites on 544 proteins, of which 204 SUMO sites on 123 proteins were identified as putative PIAS1 substrates. These substrates were found to be involved in different cellular processes, including transcriptional regulation, DNA binding and cytoskeleton dynamics. Further functional studies on Vimentin (VIM), a type III intermediate filament protein involved in cytoskeleton organization and cell motility, revealed that PIAS1 exerts its effects on cell migration and cell invasion through the SUMOylation of VIM at Lys-439 and Lys-445 residues. VIM SUMOylation was necessary for its dynamic disassembly, and cells expressing a non-SUMOylatable VIM mutant showed reduced levels of proliferation and migration. Our approach not only provides a novel strategy for the identification of E3 SUMO ligase substrates, but also yields valuable biological insights into the unsuspected role of PIAS1 and VIM SUMOylation on cell motility.

INSTRUMENT(S): Orbitrap Fusion, Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Kidney

SUBMITTER: Francis McManus

LAB HEAD: Pierre Thibault

PROVIDER: PXD011932 | Pride | 2020-02-06

REPOSITORIES: Pride

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Quantitative SUMO proteomics identifies PIAS1 substrates involved in cell migration and motility.

Li Chongyang C McManus Francis P FP Plutoni Cédric C Pascariu Cristina Mirela CM Nelson Trent T Alberici Delsin Lara Elis LE Emery Gregory G Thibault Pierre P

Nature communications 20200211 1

The protein inhibitor of activated STAT1 (PIAS1) is an E3 SUMO ligase that plays important roles in various cellular pathways. Increasing evidence shows that PIAS1 is overexpressed in various human malignancies, including prostate and lung cancers. Here we used quantitative SUMO proteomics to identify potential substrates of PIAS1 in a system-wide manner. We identified 983 SUMO sites on 544 proteins, of which 62 proteins were assigned as putative PIAS1 substrates. In particular, vimentin (VIM), ...[more]

PMID: 32047143

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Project description:SUMOylation is a key post-translational modification that regulates crucial cellular functions and pathological processes. It has emerged as a fundamental route that may drive different steps of human diseases. Indeed, alteration in expression or activity of one of the different SUMO pathway components may completely subvert cellular properties through fine-tuning modulation of protein(s) involved in cellular/tissue pathways, leading to alter cell proliferation, tissue regeneration, apoptosis resistance and metastatic potential. What is SUMOylation? SUMO, the Small Ubiquitin-like Modifier is a 97-amino acid protein that can be covalently attached to lysine residues on target proteins via an enzymatic cascade reaction named SUMOylation. SUMOylation requires an E1 (hetero-dimer SUMO-activating, SAE1/2), E2 (SUMO-specific conjugating, Ubc9), and, in most cases, E3 (SUMO ligase, PIASs) enzymes. There are four reported SUMO paralogues, SUMO-1 to SUMO-4, present in mammalian. All SUMO paralogues are transiently and reversibly conjugated to substrate proteins by the same enzymatic machinery but they can modify distinct targets although some proteins can be SUMOylated by either SUMO-1 or SUMO-2/3. The modifiers are expressed as immature peptides that must be cleaved prior to conjugation. This is carried out by isopeptidases, known as SENPs or SUMO specific proteases. The cleavage generates a C-terminal carboxyl group, SUMO-Gly-Gly, that will be attached via an isopeptide bond, formed between the epsilon-amino group of a Lys residue in the consensus sequence in target proteins designated ΨKxD/E, where Ψ is a large hydrophobic residue, K is the target lysine (Lys) and D/E are acidic residues. SUMO-2 and -3 have the ability to form poly-SUMO chains by covalent linkage of the C-terminal Gly-Gly residue to the internal Lys residue of their N-terminal consensus sequence motif. SUMO-1 lacks this consensus site and is unable to form chains but may act as a polySUMO chain terminator. SUMO-4 remains enigmatic since it has a restricted tissue distribution and may be unable to SUMOylate substrate proteins due to a proline residue that appears to prevent its maturation and conjugation. Heterodimeric activating enzyme E1 triggers SUMO proteins in an ATP-dependent way and transfers activated SUMO to a conserved catalytic cysteine in the conjugation enzyme E2. Ubc9, the only E2 identified delivers SUMO protein directly to the substrates. Ubc9 alone is sufficient for conjugation and ligation of the SUMO moiety to the substrates. However, the presence of SUMOylation E3 ligases, including the PIAS family, RanBP2, Polycomb2, TOPORS, TRAF7 and mitochondrial-anchored protein ligase (MPAL), stimulates the conjugation efficiency by promoting poly-SUMO chain formation and/or introducing additional SUMO acceptor sites. Despite being a covalent protein modification, SUMOylation is readily reversible due to the protease activity of SUMO specific deSUMOylation enzymes, SENPs. These enzymes exhibit specifically between the SUMO paralogues and have distinct subnuclear and subcellular localization patterns. Seven isoforms are known, including SENP1, SENP2, SENP3, SENP6 and SENP7. The SENPs contain distinct N-terminal domains that regulate their cellular localization, suggesting that each SENP may have a distinct set of substrates. In the last decade, the PTM with SUMO has emerged as a central regulatory mechanism for the control of cellular functions, including developmental and differentiation processes. However most of the studies have been performed on single eukaryotic cells from yeast to primary and model cells. The involvement of SUMOylation processes in regulating activity, function development and/or disorder in a more complex system like differentiated tissues (i.e: brain and cardiac muscle) is just emerging. At present, there is a completely lack of information regarding the functional significance of SUMOylation in skeletal muscle differentiation, regulation and diseases. Using a unique experimental Intensive Care Unit (ICU) rat model allowing long-term MV, diaphragm muscles were collected in rats control and exposed to controlled MV (CMV) for durations varying between 1 and 10 days. Endogenous SUMOylated diaphragm proteins were identified by mass-spectrometry and validated with in-vitro SUMOylation systems. Contractile, calcium regulator and mitochondrial proteins were of specific interest due to their putative involvement in VIDD. Differences were observed in the abundance of SUMOylated proteins between glycolytic and oxidative muscle fibers in control animals and high levels of SUMOylated proteins were present in all fibers during CMV. Finally, previously reported VIDD biomarkers and therapeutic targets were also identified in our datasets which may play an important role in response to muscle weakness seen in ICU patients.

Dataset Information

Quantitative proteomics identifies novel PIAS1 protein substrates involved in cell migration and motility

Publications

Quantitative SUMO proteomics identifies PIAS1 substrates involved in cell migration and motility.

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