2. Academic Validation
  3. VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications

  • Oncotarget. 2016 Jan 19;7(3):2249-68. doi: 10.18632/oncotarget.6850.
Simona Reina 1 2,  Vanessa Checchetto 3 4,  Rosaria Saletti 5,  Ankit Gupta 6,  Deepti Chaturvedi 6,  Carlo Guardiani 7,  Francesca Guarino 1,  Mariano Andrea Scorciapino 7,  Andrea Magrì 1,  Salvatore Foti 4,  Matteo Ceccarelli 6 8,  Angela Anna Messina 9,  Radhakrishnan Mahalakshmi 5,  Ildiko Szabo 2,  Vito De Pinto 1
Affiliations

Affiliations

  • 1 Department of Biomedicine and Biotechnology BIOMETEC, Section of Biology and Genetics, University of Catania, Catania, Italy.
  • 2 National Institute for Biomembranes and Biosystems, Section of Catania, Catania, Italy.
  • 3 Department of Biology, University of Padova, Padova, Italy.
  • 4 CNR Institute of Neurosciences, Padova, Italy.
  • 5 Department of Biomedical Sciences, University of Padova, Padova, Italy.
  • 6 Department of Chemical Sciences, Mass Spectrometry Unit, University of Catania, Catania, Italy.
  • 7 Molecular Biophysics Laboratory, Department of Biological Sciences, Indian Institute of Science Education and Research, Bhopal, India.
  • 8 Department of Physics, University of Cagliari, Cagliari, Italy.
  • 9 Department of Biomedical Sciences, Biochemistry Unit, University of Cagliari, Cagliari, Italy.
PMID: 26760765 DOI: 10.18632/oncotarget.6850
Abstract

Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.

Keywords

VDACs; cysteine oxidation; disulfide bridge; mass spectrometry; mitochondrial intermembrane space.

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