Oncotarget

Research Papers: Autophagy and Cell Death:

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

Simona Reina, Vanessa Checchetto, Rosaria Saletti, Ankit Gupta, Deepti Chaturvedi, Carlo Guardiani, Francesca Guarino, Mariano Andrea Scorciapino, Andrea Magrì, Salvatore Foti, Matteo Ceccarelli, Angela Anna Messina, Radhakrishnan Mahalakshmi, Ildiko Szabo and Vito De Pinto _

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Oncotarget. 2016; 7:2249-2268. https://doi.org/10.18632/oncotarget.6850

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Abstract

Simona Reina1,2*, Vanessa Checchetto3,4,5,*, Rosaria Saletti6, Ankit Gupta7,*, Deepti Chaturvedi7,*, Carlo Guardiani8, Francesca Guarino1,2, Mariano Andrea Scorciapino9, Andrea Magrì1,2, Salvatore Foti6, Matteo Ceccarelli8,10,**, Angela Anna Messina11,12,**, Radhakrishnan Mahalakshmi7,**, Ildiko Szabo3,4,** and Vito De Pinto1,2

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

10 Istituto Officina dei Materiali del Consiglio Nazionale delle Ricerche (IOM-CNR), UOS, Trieste, Italy

11 Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology, University of Catania, Catania, Italy

12 National Institute for Biomembranes and Biosystems, Section of Catania, Catania, Italy

* These authors have contributed equally to this work

** Co-corresponding author

Correspondence to:

Vito De Pinto, email:

Keywords: VDACs, cysteine oxidation, disulfide bridge, mitochondrial intermembrane space, mass spectrometry

Received: October 21, 2015 Accepted: December 23, 2015 Published: January 08, 2016

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.


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