Insulin resistance (IR) and hyperglycemia is a cornerstone of metabolic disorders like type 2 diabetes, obesity, and cardiovascular disease. The exact causal molecular mechanisms are unclear but involve a dysregulation of signal transduction and activation of ER stress in skeletal muscle. Underpinning these are dynamic changes in protein:protein interactions (PPIs) but a global understanding of how these are altered in a state of insulin resistance (IR) is unknown. To investigate this, we performed an integrative analysis of proteome-wide PPI’s using protein correlation profiling mass spectrometry (PCP-MS) and cross-linking mass spectrometry (XL-MS).
For PCP-MS, control and palmitic acid-induced IR myotubes were gently lysed and protein complexes separated via blue native PAGE into 30 fractions followed by LC-MS/MS analysis. Pairwise correlations of 4,681 proteins were performed and protein complexes assembled with the CCProfiler workflow. For XL-MS, control and IR myotubes were directly cross-linked with t-butyl-PhoX. The use of Tandem Mass Tag multiplexed stable isotopes followed by extensive fractionation enabled the quantification of >16,000 unique cross-linked peptides. To validate these data, we performed the first ex vivo quantitative XL-MS study of skeletal muscles from control chow and IR high fat diet fed mice. This quantified >10,000 unique cross-linked peptides.
Integration of PCP-MS and XL-MS identified discrete changes in complex formation and PPI’s across various metabolic pathways. We also identified prominent changes in the interactions of PDIA6, an ER stress associated protein regulating oxidative modifications, including down-regulation in IR human skeletal muscle biopsies. In vivo overexpression of PDIA6 in mouse muscle using adeno-associated viral delivery was used to validate associations and is currently be investigated as a strategy to prevent oxidative modifications and ER stress following IR. Taken together, we provide the largest interactome map of skeletal muscle which will be a rich resource for identifying PPIs that regulate skeletal muscle insulin resistance.