Characterization of disease genes and proteins

The physiological role of many components of the mitochondrial proteome, including many gene products identified through disease, is unknown or poorly understood. Our investigation is focused on some disease-associated proteins, we have identified in the context of specific mitochondrial disease phenotypes.

Mass spectroscopy, including SILAC-based approaches, and bioinformatics tools are used to identify the partners interacting with the disease-associated proteins in recombinant cell systems, mutant cell lines from patients, mouse embryonic fibroblasts from knockout mouse models, and, whenever possible, yeast models.

Some of these proteins, for instance MPV17 and FBXL4 are broadly linked to the maintenance of mtDNA metabolism, since mutations in the corresponding genes cause tissue-specific (MPV17), or a generalized (FBXL4) reduction of the mtDNA copy number. However, no mechanistic information exists for either protein, although both seem to interact with unknown partner proteins, the identification of which can shed light on their function. A second group of mutant genes encode putative assembly factors of individual respiratory chain complexes. Examples include SURF1, TTC19, SDHAF1 and ACAD9, which play a role in the formation of complex IV, III, II and I, respectively.

One cell line is cultured in regular or light-isotope media whereas the other cell line is cultured in ‘heavy isotope’ media (containing 15N-labelled Arginine and Lysine). Equal quantities of protein from light-labeled and heavy-labeled cells are mixed and, after isolation of mitoplasts or mitochondria, affinity purification of protein complexes is performed. Eluted fractions are then run on SDS-PAGE, gels are sliced, peptides are digested and eventually analysed by LC-MS/MS

Additional information can be provided by the creation and study of in-vivo mutant models, particularly mouse recombinant models. A unique panel of recombinant models is now available, and we are currently implementing the CRISPR/Cas9 technique to produce genomic modifications in embryos, bypassing the time consuming processes of constructing recombinant vectors and screening ES clones. Multidisciplinary approaches are used to characterize the phenotype of the mouse strains both in-vivo and in-vitro, and thus obtain insight into the molecular pathogenesis of the corresponding human disease. In vivo phenotypization includes indirect calorimetry, treadmill endurance assays, spontaneous motor activity (in an activity cage), rotarod and Y-maze tests; in-vitro characterization will be based on histological, ultrastructural, biochemical, and molecular analyses