Dataset Information

Is transthyretin a regulator of Ubc9 SUMOylation?

ABSTRACT: Ageing and mutations of transthyretin (TTR), the thyroid hormones and retinol transporting protein lead to amyloidosis by destabilizing the structure of TTR. Because protein structure is regulated through posttranslational modifications, we investigated the Small Ubiquitin-like Modifier (SUMO)ylation of TTR. We chose the widely used Ubc9 fusion-directed SUMOylation system, which is based on a fusion of the SUMOylation substrate of interest with Ubc9, a sole SUMO conjugating enzyme. Surprisingly, despite our presumptions, we found that Ubc9 fused to TTR was SUMOylated at a unique set of lysine residues. Three unknown SUMOylation sites of Ubc9—K154, K18 and K65—were revealed by mass spectrometry (MS). The previously reported SUMOylation at K49 of Ubc9 was also observed. SUMOylation of the lysine residues of TTR fused to Ubc9 was hardly detectable. However, non-fused TTR was SUMOylated via trans-SUMOylation by Ubc9 fused to TTR. Interestingly, mutating the catalytic residue of Ubc9 fused to TTR did not result in complete loss of the SUMOylation signal, suggesting that Ubc9 linked to TTR is directly cross-SUMOylated by the SUMO-activating enzyme E1. Ubc9, TTR or fusion proteins composed of TTR and Ubc9 specifically affected the global SUMOylation of cellular proteins. TTR or Ubc9 alone increased global SUMOylation, whereas TTR and Ubc9 together decreased the amount of high-molecular weight (HMW) SUMO conjugates. Our data suggest that TTR may influence the SUMOylation of Ubc9, thereby altering signalling pathways in the cell.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Urszula Jankowska

LAB HEAD: Sylwia Kedracka-Krok

PROVIDER: PXD004515 | Pride | 2016-08-15

REPOSITORIES: Pride

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Is Transthyretin a Regulator of Ubc9 SUMOylation?

Wieczorek Elżbieta E Kędracka-Krok Sylwia S Sołtys Katarzyna K Jankowska Urszula U Hołubowicz Rafał R Seliga Justyna J Ożyhar Andrzej A

PloS one 20160808 8

Ageing and mutations of transthyretin (TTR), the thyroid hormones and retinol transporting protein lead to amyloidosis by destabilizing the structure of TTR. Because protein structure is regulated through posttranslational modifications, we investigated the Small Ubiquitin-like Modifier (SUMO)ylation of TTR. We chose the widely used Ubc9 fusion-directed SUMOylation system, which is based on a fusion of the SUMOylation substrate of interest with Ubc9, a sole SUMO conjugating enzyme. Surprisingly, ...[more]

PMID: 27501389

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Project description:The essential process of dosage compensation equalizes X-chromosome gene expression between C. elegans XO males and XX hermaphrodites through a dosage compensation complex (DCC) that resembles condensin. The DCC binds to both X chromosomes of hermaphrodites to repress transcription by half. Here we show that post-translational modification by the SUMO conjugation pathway is essential for sex-specific assembly of the DCC onto X. Depletion of the SUMO peptide in vivo severely disrupts binding of particular DCC subunits and causes changes in X-linked gene expression similar to those caused by disrupting genes encoding DCC subunits. Three DCC subunits are themselves SUMOylated, and depletion of SUMO preferentially reduces their binding to X, suggesting that SUMOylation of DCC subunits is essential for robust association with X. DCC SUMOylation is triggered by the signal that initiates DCC assembly onto X. The initial step of assembly--binding of X-targeting factors to recruitment sites on X (rex sites)--is independent of SUMOylation, but robust binding of the complete complex requires SUMOylation. SUMOylated DCC subunits are enriched at rex sites, and SUMOylation enhances interactions between X-targeting factors and condensin subunits that facilitate DCC binding beyond the low level achieved without SUMOylation. DCC subunits also participate in condensin complexes essential for chromosome segregation, but their SUMOylation occurs only in the context of the DCC. Our results reinforce a newly emerging theme in which multiple proteins of a complex are SUMOylated in response to a specific stimulus, leading to accelerated complex formation and enhanced function. ChIP-chip experiments using antibodies against DPY-27, SDC-3, DPY-30, DPY-26, DPY-28 and FLAG-tagged SDC-2 in wild-type and smo-1 RNAi treated mixed embryos, with IGG controls. Also, sequential ChIP-chip experiments: (1) ChIP using FLAG antibodies to determine the genome-wide binding sites for SUMOylated proteins, (2) ChIP using FLAG antibodies followed by re-ChIP of eluted protein-chromatin complexes with DPY-27 antibodies to determine genome-wide binding sites for SUMOylated DPY-27 (referred to as DPY-27 re-ChIP experiments), (3) ChIP using FLAG antibodies followed by re-ChIP of eluted protein-chromatin with IGG antibodies to determine background binding (referred to as IGG re-ChIP experiments, and (4) ChIP using DPY-27 antibodies as a control to assess the efficiency of DPY-27 binding and detection in control vs. FLAG-tagged strains.

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.

			Action	DRS
	15-07-22_Sumo_1_targeted_A.raw	Raw
	15-08-13_Sumo_4_Ubc9_TTR_targeted.raw	Raw
	Sumo_1.mgf	Mgf
	Sumo_1.mzid	Mzid
	Sumo_4.mgf	Mgf