Dataset Information

SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage

ABSTRACT: Small Ubiquitin-like Modifiers play critical roles in the DNA Damage Response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment with the DNA damaging agent MMS. We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in response to MMS. In contrast to yeast, where a response is centered on homologous recombination, we identified dynamically SUMOylated interaction networks of chromatin modifiers, transcription factors, DNA repair factors and nuclear body components. SUMOylated chromatin modifiers include JARID1B/KDM5B, JARID1C/KDM5C, p300, CBP, PARP1, SetDB1 and MBD1. Whereas SUMOylated JARID1B was ubiquitylated by the SUMO-targeted ubiquitin ligase RNF4 and degraded by the proteasome in response to DNA damage, JARID1C was SUMOylated and recruited to the chromatin to demethylate histone H3K4.

INSTRUMENT(S): LTQ Orbitrap Velos, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Permanent Cell Line Cell, Hela Cell

DISEASE(S): Cervix Carcinoma

SUBMITTER: Ivo Hendriks

LAB HEAD: Alfred C.O. Vertegaal

PROVIDER: PXD001810 | Pride | 2015-03-16

REPOSITORIES: pride

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Dataset's files

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			Action	DRS
	20091127_Velos1_JVO_SUMO-IP-GeLCMS-MMS1_S01.raw	Raw
	20091127_Velos1_JVO_SUMO-IP-GeLCMS-MMS1_S02.raw	Raw
	20091127_Velos1_JVO_SUMO-IP-GeLCMS-MMS1_S03.raw	Raw
	20091127_Velos1_JVO_SUMO-IP-GeLCMS-MMS1_S04.raw	Raw
	20091127_Velos1_JVO_SUMO-IP-GeLCMS-MMS1_S05.raw	Raw

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SUMO-2 Orchestrates Chromatin Modifiers in Response to DNA Damage.

Hendriks Ivo A IA Treffers Louise W LW Verlaan-de Vries Matty M Olsen Jesper V JV Vertegaal Alfred C O ACO

Cell reports 20150312 10

Small ubiquitin-like modifiers play critical roles in the DNA damage response (DDR). To increase our understanding of SUMOylation in the mammalian DDR, we employed a quantitative proteomics approach in order to identify dynamically regulated SUMO-2 conjugates and modification sites upon treatment with the DNA damaging agent methyl methanesulfonate (MMS). We have uncovered a dynamic set of 20 upregulated and 33 downregulated SUMO-2 conjugates, and 755 SUMO-2 sites, of which 362 were dynamic in re ...[more]

PMID: 25772364

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Project description:Genotoxic agents can cause replication fork stalling in dividing cells due to DNA lesions, eventually leading to replication fork collapse when the damage is not repaired. Small Ubiquitin-like Modifiers (SUMOs) are known to counteract replication stress, nevertheless, only a small number of relevant SUMO target proteins are known. To address this, we have purified and identified SUMO-2 target proteins regulated by replication stress in human cells. The developed methodology enabled single step purification of His10-SUMO-2 conjugates under denaturing conditions with high yield and high purity. The methodology is generic and is widely applicable in the ubiquitin field. Following statistical analysis on five biological replicates, a total of 566 SUMO-2 targets were identified. After 2 hours of Hydroxyurea treatment, 10 proteins were up-regulated for SUMOylation and 2 proteins were down-regulated for SUMOylation, whereas after 24 hours, 35 proteins were up-regulated for SUMOylation and 13 proteins were down-regulated for SUMOylation. A site-specific approach was used to map over 1,000 SUMO-2 acceptor lysines in target proteins. A large subset of these identified proteins function in one network that consists of interacting replication factors, transcriptional regulators, DNA damage response factors including MDC1, ATR-interacting protein ATRIP, the Bloom syndrome protein and the BLM-binding partner RMI1, the crossover junction endonuclease EME1, BRCA1 and CHAF1A. Furthermore, centromeric proteins and signal transducers were dynamically regulated by SUMOylation upon replication stress. Our results uncover a comprehensive network of SUMO target proteins dealing with replication damage and provide a framework for detailed understanding of the role of SUMOylation to counteract replication stress. Ultimately, our study reveals how a post-translational modification is able to orchestrate a large variety of different proteins to integrate different nuclear processes with the aim of dealing with the induced DNA damage

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.