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

Abstract

the mitochondrial mutator mouse is a well-established model of premature aging. in addition to accelerated aging, these mice develop hypertrophic cardiomyopathy at ~13 months of age, presumably due to overt mitochondrial dysfunction. Despite evidence of bioenergetic disruption within heart mitochondria, there is little information about the underlying changes to the mitochondrial proteome that either directly underly or predict respiratory insufficiency in mutator mice. Herein, nLC-MS/MS was used to interrogate the mitochondria-enriched proteome of heart and skeletal muscle of aged mutator mice. the mitochondrial proteome from heart tissue was then correlated with respiratory conductance data to identify protein biomarkers of respiratory insufficiency. The majority of downregulated proteins in mutator mitochondria were subunits of respiratory complexes I and IV, including both nuclear and mitochondrial-encoded proteins. Interestingly, the mitochondrial-encoded complex V subunits, were unchanged or upregulated in mutator mitochondria, suggesting a robustness to mtDNA mutation. Finally, the proteins most strongly correlated with respiratory conductance were PPM1K, NDUFB11, and C15orf61. These results suggest that mitochondrial mutator mice undergo a specific loss of mitochondrial complexes i and iV that limit their respiratory function independent of an upregulation of complex V. Additionally, the role of PPM1K in responding to mitochondrial stress warrants further exploration. The mitochondrial polymerase gamma (Polg) is responsible for both replicating and maintaining the fidelity of the mitochondrial DNA (mtDNA) 1. Disruption of the proofreading capacity of this enzyme complex has been shown to lead to an accumulation of single point mutations and deletions throughout the mitochondrial genome 2-5. The mtDNA mutator mouse is a genetic knock-in model in which mice express a proofreading-deficient Polg harboring a D257A mutation, thus increasing mutational burden of the mtDNA 2,4. Mice homozygous (D257A +/+) for this transgene display a progeroid phenotype characterized by kyphosis, hair loss, hearing loss, pronounced cardiomyopathy, and reduced lifespan compared to wild-type littermates 2,4,6. At the cellular level, increased mtDNA mutational load and/or overt respiratory dysfunction has been associated with increased levels of reactive oxygen species, possibly due to a high amount of electron leak from the respiratory system 7-9. Our lab and others have demonstrated profound bioenergetic limitations in mitochondria isolated from adult D257A +/+ tissues 2,4,10. Recently we found that respiratory conductance, a measure of

Intrinsic oxidative phosphorylation limitations underlie cellular bioenergetics in leukemia.

“…To identify potential protein mediators responsible for reduced bioenergetic e ciency in leukemia, we conducted a proteomics screen using TMT-labeled peptides prepared from the same isolated mitochondria samples used for functional characterization. To control for group differences in percent mitochondrial enrichment, nLC-MS/MS raw data were searched using the MitoCarta 2.0 database, as previously described 36 . Using this approach, total mitochondrial protein abundance was similar between groups (Supp.…”

“…Mitochondrial pellets from leukemia cells and PBMC (approximately 250 µg of protein) were lysed in icecold 8 M Urea Lysis Buffer (8 M urea in 50 mM Tris, pH 8.0, 40 mM NaCl, 2 mM CaCl 2 , 1x cOmplete ULTRA mini EDTA-free protease inhibitor tablet), as described previously 36 . 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 5s bursts set at an amplitude of 30 (Q Sonica, Newtown, CT).…”

“…TMT labeling was performed as previously described 36 . The samples from isolated mitochondria were resuspended in 100 μL of 200 mM triethylammonium bicarbonate (TEAB), mixed with a unique 10-plex Tandem Mass Tag (TMT) reagent (0.8 mg re-suspended in 50 μL100% acetonitrile), and shaken for 4 hr at room temperature (Thermo Fisher).…”

“…nLC-MS/MS for TMT proteomics nLC-MS/MS was performed as described previously 36 Peptides were grouped to proteins using the rules of strict parsimony and proteins were ltered to 1% FDR using the Protein FDR Validator node of PD2.2. MS2 reporter ion intensities for all PSMs having coisolation interference below 0.5 (50% of the ion current in the isolation window) and an average S/N > 10 for reporter ions were summed together at the peptide and protein level.…”

“…The protein group tab in the PDv2.2 results was exported as tab delimited.txt. les, and analyzed based on a previously described work ow 36 . First, M2 reporter (TMT) intensities were summed together for each TMT channel, each channel's sum was divided by the average of all channels' sums, resulting in channelspeci c loading control normalization factors to correct for any deviation from equal protein input in the 10-plex experiments.…”