Exploring the mouse mitocondrial complexome using novel data-independent quantitative proteomics approaches

Abstract

Mitochondria are the key sensors and effectors organelles within the cell. Through its highly regulated dynamics, they are able to adapt their shape and function to meet the physiological needs of each type of cell on a precise moment and/or context. The main mitochondrial function is to produce energy in form of ATP through the Oxidative Phosphorylation system (OXPHOS). This system is formed by the respiratory electron transport chain (Respiratory Complexes I to IV); and the ATP synthase, which is able to synthetize ATP from ADP thanks to the proton-motrix force generated by the transport of electrons. Apart from its role in energy production, the respiratory chain is thought to be a major source of Reactive Oxygen Species (ROS), rendering the mitochondrion an important target of ROSinduced damaged. Respiratory complexes do not escape from the mitochondrial dynamism. Located to the mitochondrial cristae in an equilibrium between free and super-complexed structures, their composition and structural arrangement have become a highly active area of research in the last years. The combination on Blue Native electrophoresis together with Mass Spectrometry (MS) analysis has been the method of choice to study the cellular or subcellular complexomes, and it has been named as “Complexome Profiling”. In this thesis we present an improved complexome profiling method named Blue-DiS, which overcomes the limitations inherent to conventional Data-Dependent Acquisition (DDA) mass spectrometry by using an improved Data-Independent Acquisition (DIA) strategy named DiS (Data Independent Scanning). The systematic peptide-fragmentation performed by Dis allows comprehensive monitorization of every peptide present in the sample along the complete chromatographic run, so that accurate protein quantification can be performed. This approach has enabled us to demonstrate that complexes have a constant core composition which remains unaltered across the different tissues analysed. However, we also demonstrate that these core complexes form superassembled structures of various sizes, generating tissuespecific patterns of migration along the blue native gel. In addition, we have been able to model the equilibrium between OXPHOS complexes, estimate their stoichiometry and identify interaction partners. We validated previously proposed interactions like those between Qil and MICOS, Ndufa4 and CI and Cox7a2l and CIII+CIV, and also identified a novel putative CI interactor, Extl1, which is currently being validated. The outstanding sensitivity achieved by the Blue-DiS technique has allowed us to deepen the Cox7a2l (SCAF1)-mediated assembly mechanism between CIII and CIV. We showed that SCAF1 is always required for the interaction between CIII and CIV, which acts as a bridge between both complexes. For the interaction between SCAF1 and CIV, the correct orientation of a specific histidine residue from the aminoacid sequence of SCAF1 is needed. Some tissues harbor a short isoform of SCAF1, in which the orientation of this residue is altered, explaining why there are no structures containing CIII+IV in the tissues expressing this short isoform. Finally, we have studied oxidative damage undergone by CI upon hypoxiareoxygenation. The oxidation of key cysteine residues within this complex, mainly from ionsulphur clusters, triggers complex destabilization and results in CI degradation. All these results provide insights into the mitochondrial complexome, which will help in the understanding of mitochondrial function and its implications in disease.