Dataset Information

Rat neutrophils phosphoproteome analysis

ABSTRACT: Intestinal ischemia reperfusion injury (iIRI) is a severe clinical condition presenting high morbidity and mortality worldwide. Some of the systemic consequences of iIRI can be prevented by applying ischemic preconditioning (iIPC), a series of short ischemia/reperfusion events preceding the major ischemia. Although neutrophils are key players in the physiopathology of ischemic injuries, neither the dysregulation presented by these cells in iIRI nor the protective effect of iIPC have their regulation mechanisms fully understood. Protein phosphorylation, as well as the regulation of the respective phosphatases and kinases are responsible for regulating a large number of cellular functions in the inflammatory response. Therefore, in the present study we analyzed the phosphoproteome of neutrophils from rats submitted to mesenteric ischemia and reperfusion, either submitted or not to IPC, compared to quiescent controls and sham laparotomy. Proteomic analysis was performed by multi-step enrichment of phosphopeptides, isobaric labeling and LC-MS/MS analysis. Bioinformatics was used to determine phosphosite and phosphopeptide abundance and clustering, as well as kinases and phosphatases sites and domains. We found that most of the phosphorylation-regulated proteins are involved in apoptosis and migration, and most of the regulatory kinases belong to CAMK and CMGC families. Some relevant regulatory proteins discussed are Vamp8, PKC delta, NDR1/Stk38 and Inpp5c/Shipp

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Rattus Norvegicus (ncbitaxon:10116)

SUBMITTER: Wagner Fontes

PROVIDER: MSV000084545 | MassIVE | Wed Nov 06 12:59:00 GMT 2019

SECONDARY ACCESSION(S): PXD016182

REPOSITORIES: MassIVE

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Publications

Phosphoproteomic Analysis of Rat Neutrophils Shows the Effect of Intestinal Ischemia/Reperfusion and Preconditioning on Kinases and Phosphatases.

Tahir Muhammad M Arshid Samina S Fontes Belchor B S Castro Mariana M Sidoli Simone S Schwämmle Veit V Luz Isabelle S IS Roepstorff Peter P Fontes Wagner W

International journal of molecular sciences 20200813 16

Intestinal ischemia reperfusion injury (iIRI) is a severe clinical condition presenting high morbidity and mortality worldwide. Some of the systemic consequences of IRI can be prevented by applying ischemic preconditioning (IPC), a series of short ischemia/reperfusion events preceding the major ischemia. Although neutrophils are key players in the pathophysiology of ischemic injuries, neither the dysregulation presented by these cells in iIRI nor the protective effect of iIPC have their regulati ...[more]

PMID: 32823483

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Project description:Ischemic preconditioning represents the most powerful mechanism of cardioprotection. The mechanisms mediating the second window of preconditioning (SWOP) differ from those mediating first window preconditioning. We hypothesized that chronic ischemia induced by repetitive ischemic stimuli would be mediated by yet different molecular mechanisms. Accordingly, conscious, chronically instrumented pigs (n=5/group) were submitted to a protocol of classical SWOP (two 10-min episodes of coronary artery occlusion followed by 24 hr reperfusion) and compared to pigs submitted to repetitive occlusion/reperfusion (RCO) by repeating 6 episodes of SWOP 12 hrs apart, and to a model of repetitive coronary stenosis (RCS), in which 6 episodes of 90 min coronary stenosis were performed 12 hrs apart. Microarray analysis was performed on the three models. There was an 85% homology in gene response between both models of RCO and RCS, whereas SWOP was qualitatively different. Both models of RCO and RCS but not SWOP showed a down-regulation of genes encoding proteins involved in oxidative metabolism, and an up-regulation of genes involved in protein synthesis and unfolded protein response, autophagy, heat shock response, protein secretion, and a strong activation of the NF-ÎºB signaling pathway. Two thirds of the genes regulated in the three models showed a gradual pattern of up- or down-regulation, in which RCO was quantitatively intermediary between RCS and SWOP. Therefore, the regulated genes in response to chronic, repetitive episodes of ischemia differ radically from classical first or second window preconditioning. Four groups of pigs were used for the study, i.e., control (n=5), SWOP (n=5), RCO (n=5), and RCS (n=5). SWOP was induced by two episodes of 10 min coronary artery occlusion (CAO), each followed by 10 min coronary artery reperfusion (CAR). RCO was induced by the same stimulus as SWOP (two cycles of 10 min CAO and 10 min CAR), but repeated six times every 12 hrs. RCS was induced by 90 min low-flow ischemia repeated six times every 12 hrs.