Respiratory Phenomics across Multiple Models of Protein Hyperacylation in Cardiac Mitochondria Reveals a Marginal Impact on Bioenergetics

Abstract

SUMMARY Acyl CoA metabolites derived from the catabolism of carbon fuels can react with lysine residues of mitochondrial proteins, giving rise to a large family of post-translational modifications (PTMs). Mass spectrometry-based detection of thousands of acyl-PTMs scattered throughout the proteome has established a strong link between mitochondrial hyperacylation and cardiometabolic diseases; however, the functional consequences of these modifications remain uncertain. Here, we use a comprehensive respiratory diagnostics platform to evaluate three disparate models of mitochondrial hyperacylation in the mouse heart caused by genetic deletion of malonyl CoA decarboxylase (MCD), SIRT5 demalonylase and desuccinylase, or SIRT3 deacetylase. In each case, elevated acylation is accompanied by marginal respiratory phenotypes. Of the >60 mitochondrial energy fluxes evaluated, the only outcome consistently observed across models is a ~15% decrease in ATP synthase activity. In sum, the findings suggest that the vast majority of mitochondrial acyl PTMs occur as stochastic events that minimally affect mitochondrial bioenergetics.

Subcellular proteomics combined with bioenergetic phenotyping reveals protein biomarkers of respiratory insufficiency in the setting of proofreading-deficient mitochondrial polymerase

“…Mitochondrial lysis, protein digestion, and peptide labeling for label-free (heart) or TMT (skeletal muscle) quantitative proteomics. Heart mitochondrial pellets, as well as skeletal muscle mitochondrial pellets (approximately 250 µg of protein) from D257A +/+ and WT mice (n = 4/group -heart; n = 3/group -skeletal muscle) were lysed in ice-cold 8 M Urea Lysis Buffer (8 M urea in 40 mM Tris, pH 8.0, 30 mM NaCl, 1 mM CaCl 2 , 1x cOmplete ULTRA mini EDTA-free protease inhibitor tablet), as described previsouly 23 . The Samples were frozen on dry ice and thawed for three freeze-thaw cycles and further disrupted by sonication with a probe sonicator in three 5 second bursts (Q Sonica #CL-188; amplitude of 30).…”

“…tMt labeling. TMT labeling was performed as previously described 23 . The six samples from skeletal muscle isolated mitochondria were re-suspended in 100 μL of 200 mM triethylammonium bicarbonate (TEAB), mixed with a unique 6-plex Tandem Mass Tag (TMT) reagent (0.8 mg re-suspended in 50 μL100% acetonitrile), and shaken for 4 hours at room temperature (ThermoFisher Scientific; Cat# 90064).…”

“…nLC-MS/MS for label-free proteomics. As described previously 23 , with some modification, all samples were suspended in 0.1% formic acid at a concentration of 0.25 µg/µL, following peptide quantification (ThermoFisher Cat# 23275). Samples were subjected to nanoLC-MS/MS analysis using an UltiMate 3000…”

“…Data analysis for tMt proteomics. As described previously 23 , with some modification, Proteome Discoverer 2.2 (PDv2.2) was used for raw data analysis, with default search parameters including oxidation (15.995 Da on M) as a variable modification and carbamidomethyl (57.021 Da on C) and TMT6plex (229.163 Da on peptide N-term and K) as fixed modifications, and 2 missed cleavages (full trypsin specificity). Data were searched against the Uniprot mouse proteome database, as well as the mouse Mito Carta 2.0 database 14 .…”

“…Protein and peptide groups tabs in the PDv2.2 results were exported as tab delimited.txt. files, and analyzed based on a previously described workflow 23,24 , with some modification. First, peptide group M1 precursor (label-free) or M2 reporter (TMT) intensities for each peptide group were summed together for each sample or TMT channel, each sample/channel's sum was divided by the average of all samples/channels' sums, resulting in sample/channel-specific loading control normalization factors to correct for any deviation from equal protein input in the label-free or six-plex experiments.…”