Mitochondrial PTRH2 controls the deubiquitinase TRABID to regulate mt-ND5 stability and metabolism

The integrin effector, PTRH2, associates with mitochondria in adherent cells where its function has not been elucidated (Jan Y, <i>et al</i>. 2004. A mitochondrial protein, Bit1, mediates apoptosis regulated by integrins and Groucho/TLE corepressors. <i>Cell</i>. 116:751-762; Griffiths GS, <i>et al</i>. 2011. Bit-1 mediates integrin-dependent cell survival through activation of the NF{kappa}B pathway. <i>J Biol Chem</i>. 286:14713-14723). <i>PTRH2</i> loss-of-function mutations cause multisystem disease in children through an unknown mechanism. We sought to determine the role of mitochondrial PTRH2. We used immunoprecipitation/mass spectrometric proteomics to identify PTRH2 interacting partners: TRABID (a deubiquitinase [DUB]) and respiratory complex I NADH: ubiquinone oxidoreductase core subunit 5 (mt-ND5). We show for the first time that mitochondrial PTRH2 regulates TRABID's ability to deubiquitylate mt-ND5. In cells lacking PTRH2 expression, mt-ND5 stability is significantly increased due to aberrant TRABID-mediated deubiquitylation of mt-ND5. This increase in mt-ND5 stability promotes complex I activity and ATP production, which under stress conditions leads to mitochondrial Ca²⁺ overload. Reexpression of mitochondrial PTRH2 blocks TRABID-mediated mt-ND5 deubiquitylation, resulting in mt-ND5 polyubiquitylation and proteasomal degradation. Inhibiting complex I or TRABID activity rescued <i>PTRH2</i> loss-of-function mutant patient cells from mitochondrial Ca²⁺ overload under stress. Immunostaining analysis of <i>ptrh2^+/+</i> and <i>ptrh2^-/-</i> mouse skeletal muscle revealed a negative relationship between PTRH2 and mt-ND5, confirming a regulatory role for PTRH2 in controlling mt-ND5 stability. Taken together, mitochondrial PTRH2 is a regulator of metabolic homeostasis that, when lost, promotes mitochondrial Ca²⁺ overload when cells are exposed to stress signals. Targeting mt-ND5 stability through PTRH2-mediated regulation of TRABID's DUB function provides a novel mechanistic approach to inhibit mitochondrial Ca²⁺ overload in diseases that occur due to dysregulated mitochondria.