Mitochondria are critical for regulating metabolism and energy expenditure. These organelles are comprised of roughly 2,000 proteins. Dysfunction of mitochondria and its components has been associated with many pathologies including cardiovascular, neurodegenerative, and metabolic diseases. While previous reports have aimed to characterize the mitochondrial proteome, the relationship between the mitochondrial proteome and function has not been experimentally established on a systematic level. This lack of understanding impedes the contextualization and translation of proteomic data to the molecular derivations of mitochondrial diseases.
To traverse this knowledge gap, we analyzed the mitochondrial proteomic profile from four different tissue types – two mitochondrial proteomes from identical genetic codons (mouse heart and mouse liver), two cardiac mitochondrial proteomes from unique genomes (mouse heart and human heart), and one well-studied metazoan model system (drosophila). By linking mitochondrial protein abundance with biochemical pathways, we were able to identify the core functionalities of these mitochondria. Using bioinformatics analyses, we identified significant enrichment of disease-associated genes and their products. Correlational analyses suggested that the mitochondrial proteome design is primarily driven by cellular environment. Taken together, these results link the mitochondrial composition with function, providing a prospective resource for mitochondrial pathophysiology and developing novel therapeutic targets in medicine.
As mitochondrial dysfunction is exacerbated by dysfunctional protein quality control and often further contributes to pathology, we addressed the mechanisms underlying the maintenance of healthy mitochondrial architecture. This requires a steady balance between protein synthesis and degradation – or turnover. It is well-documented that mitochondrial autophagy (mitophagy) is the primary mechanism to degrade dysfunctional mitochondria via lysosomes. However, as failure to contain or replenish mitochondrial proteins damaged by reactive oxygen species directly underlies many pathological phenotypes, developing therapies to target mitochondria on an individual protein level is of interest. Therefore, we designed a metabolic heavy water (2H2O) labeling strategy to study individual protein turnover rates in vivo. We calculated the turnover rates for 458 proteins in mouse cardiac and hepatic mitochondria and revealed distinct tissue-specific turnover kinetics with protein half-lives spanning from hours to months. These results indicate that mitochondria are not turned over only as individual units; excess mitochondrial proteins are synthesized in the cytosol to be imported into the mitochondria when needed. Therefore, mitochondria possess a mixture of previously- and newly-synthesized proteins. Our study demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.
To further study the structure of mitochondria, we sought to investigate how alterations in mitochondrial morphology and dynamics (fission and fusion) affect metabolism and physiology. Proper mitochondrial function is required to maintain metabolic homeostasis and cellular energetic capacity and mitochondrial dysfunction has been associated with the development of insulin resistance in glucoregulatory tissues. Our laboratory has recently shown that heat shock protein 72 (HSP72) is an important molecular link between mitochondrial function, cellular metabolism, and insulin action. Indeed, HSP72 protein levels are reduced in muscle from obese and diabetic patients, and HSP72 levels are inversely associated with the degree of insulin resistance and adiposity. Findings from our laboratory show that HSP72 regulates Parkin action including mitochondrial quality control and reveal that the deletion of either HSP72 or Parkin induces mitochondrial dysfunction and skeletal muscle insulin resistance. However, the molecular phenotypes encompassing the HSP72-mitochondria-glucose homeostasis paradigm have been to date, exclusively established in male model systems. In contrast to the obesity-insulin resistance phenotype of male HSP72 knockout (KO) mice, our findings show that female mice lacking HSP72 possess a lean phenotype with enhanced insulin sensitivity. Interestingly, loss of HSP72 promotes increased muscle ERα expression in female mice; this is a likely mediator of improved mitochondrial function and insulin action in female HSP72 KO mice fed a normal chow diet. Our studies lay the important foundation for the rational design of novel therapeutic strategies that can be used to combat metabolic-related diseases in women.
Together, our findings suggest that mitochondria and its proteomic profile are highly dynamic in composition and kinetics. These properties are dependent on not only the host organism, but also the organ milieu. In addition, our findings highlight the sex-specific role of mitochondrial dysfunction in the onset of metabolic diseases. Therefore, developing therapeutic strategies to target mitochondrial function, health, and dynamics are crucial to ameliorate complications associated with metabolic diseases, including type 2 diabetes and insulin resistance in men and women.