Structural characterization of the Voltage-Dependent Selective Anion Channels (VDACs)

Cysteine and methionine residues are unique amino acids in proteins for their property to undergo several redox reactions as part of their normal function. Mitochondria are the cell source of most chemical energy obtained as the end-product of the aerobic oxidation of glucose. Mitochondria are also the source of an abundant production of radical species and it is surprising that such a large availability of highly reactive chemicals is compatible with viable and active organelles, needed for the cell functions. In this work I show the studies of post-translational modifications (PTMs) of cysteine and methionine residues, both sulfur amino acids, in the Voltage-Dependent Selective Anion Channel Isoforms (VDACs), the most abundant proteins of the Outer Mitochondrial Membrane (OMM) where they form aqueous pores which allow the passage of ions and small molecules. VDACs are involved in complex interactions regulating organelle and cellular functions and thus contribute to homeostasis maintenance. Because of their central role in metabolism and interactions with several cytosolic enzymes and apoptotic factors, VDACs have emerged as key players in cancer and in neurodegenerative diseases such as Alzheimer’s disease (AD), Parkinson’s disease (PD) and Amyotrophic Lateral Sclerosis (ALS). This interesting protein family carries several cysteines in the loop regions exposed to the oxidative inter-membrane space (IMS) and it is reasonable to suppose that the redox state of VDACs can be modified in this peculiar environment. Through UHPLC/High Resolution nESI MS/MS and the development of a new “in solution-digestion” protocol, cysteine and methionine PTMs were precisely determined in rat and human VDAC proteins obtaining very consistent results. These analysis show that cysteine residues, in physiological state, can be subject to several oxidization steps, ranging from the permanently reduced state, that indicates the possibility of disulfide bridges formation, to the most oxidized, the sulfonic acid, one. Noteworthy, this last oxidative state is irreversible in the cells and it is an exclusive feature of VDAC proteins because it was not detectable in other mitochondrial membrane proteins, as defined by their elution at low salt concentration by a hydroxyapatite (HTP) column. Furthermore, methionine residues are detected both in normal form and oxidized to methionine sulfoxide. The large spectra of VDAC cysteine oxidations, indicate that they could have both a regulative function and a buffering capacity able to counteract excess of mitochondria ROS load. The quest for the complete identification of disulfide bridges is the next, highly challenging, from the technical point of view, goal of this research. Finally, other PTMs of cysteines such as succination or the presence of selenocysteines and phosphorylation of serine, threonine and tyrosine residues were investigated. The results of my PhD thesis are reported in two papers published on important impact factor journals.