Personalized phosphoproteomics identifies functional signaling

Needham, Elise J.; Hingst, Janne R.; Parker, Benjamin L.; Morrison, Kaitlin R.; Yang, Guang; Onslev, Johan; Kristensen, Jonas M.; Højlund, Kurt; Ling, Naomi X.Y.; Oakhill, Jonathan S.; Richter, Erik A.; Kiens, Bente; Petersen, Janni; Pehmøller, Christian; James, David E.; Wojtaszewski, Jørgen F. P.; Humphrey, Sean J.

doi:10.1038/s41587-021-01099-9

Cited by 62 publications

(59 citation statements)

References 46 publications

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“…Physical activity is well known to have vast whole-body metabolic benefits including, but not lim-ited to, the improvement of mitochondrial functional, the removal of reactive oxygen species, re-wiring of insulin signaling in skeletal muscle and the prevention of obesity (Holten et al, 2004, Needham et al, 2022, Richter and Hargreaves, 2013, Memme et al, 2021). However, how different types of physical exercise background may influence the basal metabolic rate of skeletal muscle has, until now, not been studied.…”

Section: Discussionmentioning

confidence: 99%

“…Mechanisms underlying the exercise–induced increase in SRX myosins remain unknown, however, previous studies have demonstrated the importance of post-translational modifications in the regulation of the myosin function (Alamo et al, 2016, Brito et al, 2011, McNamara et al, 2019a, McNamara et al, 2019b). It has been established that exercise can induce vast protein phosphoryla-tion, including that of sarcomeric proteins, and that different training methods can induce distinct training-specific phosphorylation profiles (Needham et al, 2022, Bódi et al, 2021). Further research into the PTMs that specifically occur in endurance-trained individuals could provide insight to why we see this result in EAs and into the mechanisms of control of myosin head conformation.…”

Section: Discussionmentioning

confidence: 99%

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Preponderant Myosin Super-Relaxed State In Skeletal Muscle From Endurance Athletes

Lewis

Tabrizian

Nielsen

et al. 2022

Preprint

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It has recently been established that myosin, the molecular motor protein, is able to exist in two conformations in relaxed skeletal muscle. These conformations are known as super-relaxed (SRX) and disordered-relaxed (DRX) states and are finely balanced to optimize skeletal muscle metabolism. Indeed, SRX myosins are thought to have a 10-fold reduction in ATP turnover compared to DRX myosins. Here, we investigated whether chronic physical activity in humans would be associated with changes in the proportions of SRX and DRX skeletal myosins. For that, we isolated muscle fibres from various athletic and sedentary populations and ran a loaded Mant-ATP chase proto-col. We observed that, in endurance-trained athletes, the amounts of myosin molecules in the SRX state was significantly greater than in age-matched sedentary individuals or than in strength athletes. To further assess whether this change would have an impact on the potency of a SRX-inducing pharmacological compound, Mavacamten, we performed similar analyses as above with and without the drug in muscle fibres from endurance athletes. Surprisingly, we found that 0.3 micromolar of Mavacamten had only marginal effects. Altogether, our results indicate that chronic endurance training-status influences resting skeletal myosin conformations, and Mavacamten potency. Our findings also emphasize that environmental stimuli such as exercise can re-wire the molecular metabolism of human skeletal muscle through myosin.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Preponderant Myosin Super-Relaxed State In Skeletal Muscle From Endurance Athletes

Lewis

Tabrizian

Nielsen

et al. 2022

Preprint

View full text Add to dashboard Cite

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“…Canonical insulin proteins were compiled as previously described 87 by compiling annotations from Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes, Reactome, and our previous publication 18 .…”

Section: Methodsmentioning

confidence: 99%

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance

Fazakerley

Gerwen

Cooke

et al. 2022

Preprint

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The failure of metabolic tissues to appropriately respond to insulin ("insulin resistance") is an early marker in the pathogenesis of type 2 diabetes. Protein phosphorylation is central to the adipocyte insulin response, but how adipocyte signaling networks are dysregulated upon insulin resistance is unknown. Here we employed phosphoproteomics to delineate insulin signal transduction in adipocyte cells and adipose tissue. Across a range of insults triggering insulin resistance, we observed marked rewiring of the insulin signaling network. This included both attenuated insulin-responsive phosphorylation, and the emergence of phosphorylation uniquely insulin-regulated in insulin resistance. Identifying signaling changes common to multiple insults revealed subnetworks likely containing causal drivers of insulin resistance. Focusing on defective GSK3 signaling initially observed in a relatively small subset of well-characterized substrates, we employed a pipeline for identifying context-specific kinase substrates. This facilitated robust identification of widespread dysregulated GSK3 signaling. Pharmacological inhibition of GSK3 partially reversed insulin resistance in cells and tissue explants. These data highlight that insulin resistance is a multi-nodal signaling defect that encompasses dysregulated GSK3 activity.

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“…To disentangle the functional role of specific phosphosites, there is a trend towards experimental designs that investigate the dynamic regulation of the phosphoproteome in many different cellular states, compartments and conditions [11][12][13] . This spurs ongoing efforts to increase the throughput of phosphoproteomics experiments, for example by means of shortening liquid chromatography gradients 6 .…”

Section: Introductionmentioning

confidence: 99%

Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients

Oliinyk

Meier

2022

Preprint

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Mass spectrometry-based phosphoproteomics has identified >150,000 post-translational phosphorylation sites in the human proteome. To disentangle their functional relevance, complex experimental designs that require increased throughput are now coming into focus. Here, we apply dia-PASEF on a trapped ion mobility (TIMS) mass spectrometer to analyze the phosphoproteome of a human cancer cell line in short liquid chromatography gradients. At low sample amounts equivalent to ~20 μg protein digest per analysis, we quantified over 12,000 phosphopeptides including ~8,000 class I phosphosites in one hour without a spectral library. Decreasing the gradient time to 15 min yielded virtually identical coverage of the phosphoproteome, and with 7 min gradients we still quantified about 80% of the class I sites with a median coefficient of variation <10% in quadruplicates. We attribute this in part to the increased peak capacity, which effectively compensates for the higher peptide density per time unit in shorter gradients. Our data shows a five-fold reduction in the number of co-isolated peptides with TIMS. In the most extreme case, these were positional isomers of nearby phosphosites that remained unresolved with fast chromatography. In summary, we demonstrate how key features of dia-PASEF translate to phosphoproteomics, resulting in high throughput and sensitivity.

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Personalized phosphoproteomics identifies functional signaling

Cited by 62 publications

References 46 publications

Preponderant Myosin Super-Relaxed State In Skeletal Muscle From Endurance Athletes

Preponderant Myosin Super-Relaxed State In Skeletal Muscle From Endurance Athletes

Phosphoproteomics reveals rewiring of the insulin signaling network and multi-nodal defects in insulin resistance

Ion mobility-resolved phosphoproteomics with dia-PASEF and short gradients

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