Leigh syndrome is a fatal neurometabolic disorder caused by defects in mitochondrial function. mTOR inhibition with rapamycin attenuates disease progression in a mouse model of Leigh syndrome (Ndufs4 KO mouse); however, the mechanism of rescue is unknown. Here we identify PKC downregulation as a key event mediating the beneficial effects of rapamycin treatment of Ndufs4 KO mice. Assessing the impact of rapamycin on the brain proteome and phosphoproteome of Ndufs4 KO mice we find that rapamycin restores mitochondrial protein levels, inhibits signaling through both mTOR complexes, and reduces the abundance and activity of multiple protein kinase C (PKC) isoforms. Administration of PKC inhibitors increases survival, delays neurological deficits, prevents hair loss, and decreases inflammation in Ndufs4 KO mice. Thus, PKC may be a viable therapeutic target for treating severe mitochondrial disease. Reporting Summary Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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Proteomics has enabled the cataloguing of 100,000s of protein phosphorylation sites 1 , however we lack methods to systematically annotate their function. Phosphorylation has numerous biological functions, yet biochemically all involve changes in protein structure and interactions. These biochemical changes can be recapitulated by measuring the difference in stability between the protein and the phosphoprotein. Building on recent work, we present a method to infer phosphosite functionality by reliably measuring such differences at the proteomic scale. MAIN TEXTRecently, Huang et al. 2 developed the Hotspot Thermal Profiling (HTP) method to identify phosphosites that alter protein thermal stability, reporting 719 out of 2,883 (25%) phosphosites with significant effects. The reported melting temperatures (T m ) for phosphopeptides correlated poorly with the T m for their corresponding proteins (R 2 = 0.18) ( Fig. 1a), implying that many phosphosites function by structurally reshaping the proteome. However, the low T m reproducibility between replicates ( Supplementary Fig. 1a) suggests that this conclusion may be due to technical variation (Supplementary Discussion). The HTP workflow consists of phosphopeptide enrichment followed by separate isotopic labeling and mass spectrometric analysis to derive T m values for phosphopeptides and proteins, respectively. Because phosphopeptide samples also contained unmodified peptides, which are expected to have the same T m as the protein, we can use these peptides to assess technical variation between the two samples. Disconcertingly, our re-analysis revealed that 626 out of 3074 (20%) of the co-enriched unmodified peptides had significant stability effects, almost the same percentage as phosphopeptides (22%, 596 out of 2656 in our re-analysis) (Fig. 1b, Dataset S1). Additionally, the T m correlation of these peptides with their protein T m was similarly low ( R 2 = 0.18) to the correlation between phosphopeptides and protein ( Supplementary Fig. 1b). In the absence of a biological explanation, this suggests that the independent labeling and mass spectrometric analysis of peptide and phosphopeptide samples could have introduced substantial technical error precluding the comparison, and perhaps that the reported hits arise from a lack of stringency in the applied statistical analysis (Supplementary Discussion).

Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-min time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation because of chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.