Dataset Information

Phosphoproteomic screening of exercise-like treatments reveals drug interactions regulating protein secretion

ABSTRACT: Exercise triggers skeletal muscle signalling pathways that modulate the release of circulating factors to cause systemic health benefits. Understanding these mechanisms could lead to better strategies for treating cardiometabolic diseases. We previously showed that acute exercise induces >1000 changes in protein phosphorylation in human muscle. Here we employed a strategy to deconvolute this network by analysing phosphoproteomes of rat L6 myotubes treated with small molecules that mimic different aspects of exercise signalling. Bioinformatics suggested that combining β-adrenergic and calcium agonists would yield a phosphoproteome most closely resembling exercise. Experimental analysis supported this but also revealed a surprising divergence in signalling from that observed with either drug alone. Dual stimulation promoted multisite phosphorylation of SERBP1, a regulator of Serpine1 mRNA stability, a pro-thrombotic fibrotic secreted protein. Secretomic analysis of L6 myotubes treated with β-adrenergic and calcium agonists revealed a significant decrease in SERPINE1 secretion and other deleterious secretory factors. This provides a novel approach to dissect the beneficial effects of exercise and demonstrates an underappreciated effect of exercise to reduce the circulating levels of certain factors, providing new insights into exercise benefits and their therapeutic potential.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Rattus Norvegicus (rat)

TISSUE(S): Myotube

SUBMITTER: Benjamin Parker

LAB HEAD: Benjamin Parker

PROVIDER: PXD014322 | Pride | 2019-09-26

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	160824exerciseliketreatmentssamplenames.xlsx	Xlsx
	171010ITcombosamplenames.xlsx	Xlsx
	20160824_QEplus_BenP_Elise_A10.raw	Raw
	20160824_QEplus_BenP_Elise_A2.raw	Raw
	20160824_QEplus_BenP_Elise_A3.raw	Raw

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Publications

Phosphoproteomics of Acute Cell Stressors Targeting Exercise Signaling Networks Reveal Drug Interactions Regulating Protein Secretion.

Needham Elise J EJ Humphrey Sean J SJ Cooke Kristen C KC Fazakerley Daniel J DJ Duan Xiaowen X Parker Benjamin L BL James David E DE

Cell reports 20191101 6

Exercise engages signaling networks to control the release of circulating factors beneficial to health. However, the nature of these networks remains undefined. Using high-throughput phosphoproteomics, we quantify 20,249 phosphorylation sites in skeletal muscle-like myotube cells and monitor their responses to a panel of cell stressors targeting aspects of exercise signaling in vivo. Integrating these in-depth phosphoproteomes with the phosphoproteome of acute aerobic exercise in human skeletal ...[more]

PMID: 31693893

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Project description:Endurance exercise improves cardiac performance and affords protection against cardiovascular diseases but the signalling events that mediate these benefits are largely unexplored. Phosphorylation is an widely studied post-translational modification involved in intracellular signalling, and to discover novel phosphorylation events associated with exercise we have profiled the cardiac phosphoproteome response to a standardised exercise test to peak oxygen uptake (VO2peak). Male Wistar rats (346 ± 18 g) were assigned to 3 independent groups (n= 6, in each) that were familiarised with running on a motorised treadmill within a metabolic chamber. Animals performed a graded exercise test and were killed either immediately (0 h) after or 3 h after terminating the test at a standardised physiological end point (i.e. peak oxygen uptake; VO2peak). Control rats were killed at a similar time of day to the exercised animals, to minimise possible circadian effects. Cardiac proteins were digested with trypsin and phosphopeptides were enriched by selective binding to titanium dioxide (TiO2). Phosphopeptides were analysed by liquid chromatography and high-resolution tandem mass spectrometry, and phosphopeptides were quantified by MS1 intensities and identified against the UniProt knowledgebase using MaxQuant (v1.3.0.5). The VO2peak of rats in the 0 h and 3 h groups was 66 ± 5 ml•kg-1•min-1 and 69.8 ± 5 ml•kg-1•min-1, respectively. Proteome profiling detected 1169 phosphopeptides and one-way ANOVA found 141 significant (P<0.05 with a false discovery rate of 10 %) differences. Almost all (97 %) of the phosphosites that were responsive to exercise are annotated in the PhosphoSitePlus database but, importantly, the majority of these have not previously been associated with the cardiac response to exercise. More than two-thirds of the exercise-responsive phosphosites were different from those identified in previous phosphoproteome profiling of the cardiac response to β1-adrenergic receptor stimulation. Moreover, we report entirely new phosphorylation sites on 4 cardiac proteins, including S81 of muscle LIM protein, and identified 7 exercise-responsive kinases, including myofibrillar protein kinases such as obscurin, titin and the striated-muscle-specific serine/threonine kinase (SPEG) that may be worthwhile targets for future investigation.

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