Mitochondria are important organelles of eukaryotic cells, and they play a key role in the regulation of cellular energy metabolism, biosynthesis, and cell death (including apoptosis and programmed cell necrosis). In addition, mitochondria are also involved in important physiological processes such as the tricarboxylic acid cycle, fatty acid and amino acid oxidation, and steady state regulation of calcium ions. Because mitochondria play an important role in maintaining homeostasis, their dysfunction is related to many human diseases, such as neurodegenerative diseases, cancer, heart disease, and diabetes. Because the abnormal expression of mitochondrial proteins is the main reason for its dysfunction, mitochondrial proteomics analyzes the dynamic changes of mitochondrial protein composition, expression level and modification state from the overall perspective, in order to clarify the expression pattern of all proteins in mitochondria And functional patterns, including protein expression and existence, structure and function, and protein interactions, so as to explore mitochondrial physiological functions and related diseases at the protein level Ill connection. Mitochondrial proteomics can systematically study the differences in the distribution and expression of mitochondrial proteins in normal and diseased tissues, thereby laying a theoretical foundation for studying the molecular mechanisms of mitochondrial-related diseases and the development of drugs targeting mitochondria.
Figure 1. Schematic diagram of mitochondria.
Mitochondrial protein extraction method
In order to achieve specific extraction of mitochondrial sub-interval proteins, researchers have genetically engineered anti-ascorbate peroxidase (APEX) to specifically "fish" membrane gaps or matrix proteins. As shown in Figure 2, genetically engineered APEX can target the mitochondrial matrix and oxidize a variety of phenol derivatives to generate hydroxyl radicals. These radicals have a short life (<1 ms) and a small labeling radius (<20 nm). Electron-rich amino acids such as Tyr, Trp, His, etc. are covalently bound to label neighboring proteins. By this method, 495 proteins of the mitochondrial matrix of HEK cells can be identified, of which 31 are newly discovered proteins. The coverage and specificity of the matrix proteome captured by this method reached 85% and 94%, respectively. The reason for the lower coverage may be that some protein side chains are too densely embedded to interact with hydroxyl radicals. Researchers further extracted and identified the mitochondrial membrane gap proteome by APEX combined with cell culture stable isotope labeling method, and identified a total of 127 membrane gap proteins (nine of which are newly discovered mitochondrial proteins) with a specificity as high as 94 %, Which achieves an efficient measurement of the mitochondrial membrane gap proteome.
Figure 2. Labeling the mitochondrial matrix proteomein living cells. (Rhee HW, et al. 2013)
Properties and Applications of Mitochondrial Proteomics
With the development of mitochondrial proteome extraction, isolation and identification technology, a relatively complete "catalog" of mitochondrial proteins has been established. More than 1,000 human mitochondrial proteins have been detected, and the abundance of mitochondrial proteins has been found to vary greatly. The most abundant inner and outer membrane proteins are ANT1 and VDAC, respectively. The following describes the number of proteins, abundance, localization, post-translational modification, tissue distribution, biochemical pathways and applications of mitochondrial proteome. In terms of mitochondrial protein localization, some proteins are only present in mitochondria, while other mitochondrial proteins can be "dually" localized, that is, proteins encoded by the same gene are located in cytoplasm or other organelles in addition to mitochondria. For example, under the stimulation of apoptotic signals, tBID is translocated to the outer mitochondrial membrane after being cleaved by apoptotic protease-8; mitochondrial membrane interstitial cytochrome C is translocated to the cytoplasm through the channels formed by the apoptotic protein Bax and the like. In terms of post-translational modification of mitochondrial proteins, three main approaches are used: acetylation, phosphorylation, and hydroxylation. In summary, the development of mitochondrial proteomics technologies and methods has promoted the study of dynamic and reversible post-translational modifications of proteins.
Figure 3. Protein biogenesis pathways of mitochondria. The large majority of mitochondrial proteins are synthesized in the cytosol and imported by the translocase of the outer membrane. Figure 4. TOM cooperates with downstream transport machineries. (Straub S P, et al. 2016).
The development of mitochondrial proteomics has played a positive role in the research of mitochondrial-related diseases. For example, blocked mitochondrial oxidative phosphorylation will lead to respiratory chain disease (RCD) with a prevalence of about 0.02%. Clinical features include skeletal muscle disease, cardiomyopathy, epilepsy, stroke, blindness and endocrine disorders. Scientific research has found 92 protein mutations associated with RCD. In terms of degenerative diseases, mitochondrial proteomics studies have found that complex I dysfunction is related to Parkinson's disease (PD), which is involved in protein folding and apoptosis, which has promoted the study of PD pathogenesis. Cancer cell line mitochondrial proteome research has facilitated the discovery of tumor cell mitochondrial biomarkers. In terms of cardiovascular disease, mitochondrial proteomics studies can elucidate the pathogenesis of stress-induced heart failure, ischemic heart failure, and chronic hypoxic diseases. In terms of liver disease, Eccleston et al. Found that high-fat diets lead to changes in the mitochondrial proteome, which are related to steatosis, NO synthesis and metabolism. In terms of aging, Alves et al. Used electrophoresis and mass spectrometry to study the mitochondrial proteome of skeletal muscle with and without exercise, indicating that exercise is an important regulator of improving aging and maintaining mitochondrial function.