PR002642 (Project)

Description:Skeletal muscle relies on mitochondrial proteostasis for energy production, which is maintained by two key quality-control systems: the ubiquitin-proteasome system (UPS) and mitochondria-derived vesicles. This study reveals that the E3 ligase complex CRL5Ozz and its substrate Alix both localize to mitochondria and cooperate to regulate the mitochondrial ATP transporter Slc25A4. CRL5Ozz targets Slc25A4 for proteasomal degradation, while Alix promotes Slc25A4 loading into exosomes for lysosomal degradation. Loss of Ozz or Alix in mice disrupts Slc25A4 levels, causing mitochondrial dysfunction, altered metabolism, and a shift in muscle fiber type from oxidative to glycolytic. This work uncovers a coordinated role of UPS and vesicle-mediated degradation in mitochondrial quality control. The goal of the study is to assess whether mitochondrial abnormalities observed in Alix−/− and Ozz−/− skeletal muscle are associated with impaired mitochondrial metabolism. Methods: Soleus muscles from 8-week-old mice were collected, rinsed briefly in ice-cold saline, patted dry, flash-frozen in liquid nitrogen, and stored at −80 °C. To extract both hydrophilic metabolites and lipids, an adapted three-phase solvent system (chloroform/methanol/water, 3:4:1, v/v/v) was used. Tissues were weighed and homogenized with zirconia beads using a Bead Ruptor Elite, followed by the addition of ice-cold saline and further mixing. After resting at 4 °C, samples were centrifuged at 21,000 ×g for 10 min. The aqueous phase was collected, frozen on dry ice, lyophilized, reconstituted in water/acetonitrile/formic acid (95:5:0.1, v/v/v), and analyzed by LC-MS. The organic phase was dried under nitrogen, reconstituted in chloroform/methanol (2:1, v/v), and analyzed by LC-MS. Results: High-throughput metabolomic analyses revealed approximately 87 significantly altered metabolites in Alix−/− and ccOzz−/− soleus muscles compared to WT controls. Heat maps generated using MetaboAnalyst 4.0 demonstrated distinct metabolic profiles among the three genotypes. Interestingly, several metabolites—including ADP, citrate, Citrulline, CoA, FAD, L-Acetylcarnitine, carnitine, oxoglutarate, and PEP, are regulated by the mitochondrial SLC25 solute carrier family. Pathway analysis further linked these metabolites to key metabolic pathways, including glycolysis, pyrimidine metabolism, the citric acid cycle, and others. Important, glycolysis was upregulated in Ozz−/− muscle relative to Alix−/− and WT, highlighting genotype-specific shifts in mitochondrial metabolism.