Project PXD006085

PRIDE Assigned Tags:
Biological Dataset

Summary

Title

A proteomic approach to identify alterations in the SUMO network during controlled mechanical ventilation in rat diaphragm muscle

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.

Sample Processing Protocol

For each sample group a total of approximately 200 mg of diaphragm muscles tissue were collected. Solutions, muscle lysates, preparation of protein G-agarose beads coupled with monoclonal anti-SUMO1 and anti-SUMO2 produced by hybridoma cells, SUMO immunoprecipitation, peptide elution and recycling of the affinity matrixes were performed according with previous protocols (Becker J. et al. Nat Struct Mol Biol. 2013 and Barysch S.V. et al. Nat Protoc. 2014). In our procedure, before the incubation with protein G-agarose beads coupled with anti-SUMO antibodies, muscle lysates were also precleared with protein G-agarose beads coupled with anti-normal mouse IgG for 3 h, gently rotated at 4°C. Precleared muscle lysates were divided in two aliquots, one was incubated with protein G-agarose beads coupled with anti-SUMO1 antibodies (50% 21C7 and 50% 76-86) for SUMO1 complexes enrichment or with protein G-agarose beads coupled with anti-SUMO2/3 antibodies (8A2) for SUMO2/3 complexes enrichment. To analyze potential SUMO targets with mass spectrometry (MS), TCA-precipitated eluted samples were fractionated and separated with a SDS-PAGE using a 4-12% gradient gel and subsequent Coomassie blue staining. Next, proteins were in-gel digested. Briefly, gel sections were cut into small (1 mm3) pieces, washed several times with 25 mM ammonium bicarbonate and rehydrated with acetonitrile (ACN). Proteins were reduced with 10 mM dithiothreitol (DTT) and alkylated with 50mM iodoacetamide (IAA). Thereafter the proteins were digested by sequencing grade modified trypsin (Promega Corporation, Madison, WI, USA) at a concentration of 12.5 ng/µL in 25 mM ammonium bicarbonate pH 8 overnight at 37oC. The peptides were extracted by sonication in 60% ACN and 5% formic acid (FA). Finally, the extracted peptides were completely dried to completion and resolved in 0.1% FA before analysis by LC-MS/MS. The peptides were analyzed using a Q Exactive HF Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with an EASY-Spray Ion Source (Thermo Fisher Scientific). The peptides were separated by reversed phase LC using a Dionex UltiMate 3000 RSLC nano system (Thermo Fisher Scientific). The separation was performed on an Acclaim PepMap C18 (2 µm particles, 75 µm i.d. x 50 cm) column. Peptides were eluted with a 70 min long gradient: 2% B for 5 min, 2-20% B in 50 min, 20-32% B in 10 min, 32-95% B in 1 min, 95% B for 4 min. Solvent A was 0.1% FA in MilliQ water and solvent B was 0.1% FA in 20% in MilliQ water 80% ACN. The peptides were ionized by positive electrospray and then introduced to the MS. The MS was operated in positive ion mode (m/z 375-1500) using an automated gain control target of 3 × 10^6 at a resolution of 60 000 and an injection time of 60 ms. Data-dependent acquisition was applied. Consecutive higher-energy collisional dissociation (HCD) fragmentation spectra of the 20 most abundant ions were collected at a resolution of 15 000. These MS/MS spectra were generated with an automated gain control target of 1 × 10^5. The injection time was 200 ms, the normalized collision energy was set to 27 and the dynamic exclusion time was 20 s.

Data Processing Protocol

The acquired raw data files were analyzed with the Proteome Discoverer 1.4.0.288 (Thermo Fisher Scientific) software using the SEQUEST HT® (University of Washington, USA) search engine against proteins from Rattus norvegicus in the UniProtKB/SwissProt database downloaded 2015-08, containing 8046 sequences and 4074405 residues. The search parameters included: maximum 10 ppm and 0.6 Da error tolerance for the survey scan and MS/MS analysis, respectively; enzyme specificity was trypsin; maximum two missed cleavage sites allowed; cysteine carbamidomethylation was set as static modification; oxidation (M) and deamidation (N,Q) were set as variable modifications. The Percolator node and a decoy database were used to estimate false discovery rate (FDR) and an FDR of 5% for peptide identification was accepted. The protein identifications were based on at least two matching peptides per protein. Two approaches were used for the database analyses: first, each sample (gel section) was searched individually and thereafter the raw data for all 8 samples from one gel lane were combined into one search respectively using Multidimensional Protein Identification Technology (MudPIT) processing.

Contact

Sara Lind, Uppsala University
Sara Lind, Uppsala University Dept. Chemistry-Biomedical center, Analytical chemistry Box 599 SE-751 24 Uppsala ( lab head )

Submission Date

14/03/2017

Publication Date

05/04/2017

Instrument

Q Exactive HF

Software

Not available

Quantification

Not available

Experiment Type

Shotgun proteomics

Publication

    Namuduri AV, Heras G, Mi J, Cacciani N, Hörnaeus K, Konzer A, Bergström Lind S, Larsson L, Gastaldello S. A proteomic approach to identify alterations in the SUMO network during controlled mechanical ventilation in rat diaphragm muscle. Mol Cell Proteomics. 2017 Apr 3. pii: mcp.M116.066159 PubMed: 28373296