High Resolution Mass Spectrometry Characterization of Cysteine Residues Oxidation in Rat Liver Mitochondria Voltage-Dependent Anion selective Channel 1 (VDAC1) Rosaria Salettia*, Simona Reinab, Maria Pittalàc, Angela Messinab, Vincenzo Cunsoloa, Vito De Pintoc, Salvatore Fotiaa) Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Catania, Italy;b) Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology University of Catania, Catania, Italy;c) Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane [1,2]. They are a small family of pore-forming proteins (30-35 kDa) that allows the flow of hydrophilic molecules through the membrane. VDAC isoforms show sequence homology and a similar β-barrel structure. The VDAC1 protein is the predominant form expressed in mitochondria of every tissue. Since the mitochondrial intermembrane space (IMS) has an oxidative potential, we have investigated the oxidation state of cysteine residues, as performed for the isoform VDAC3 [3]. To accomplish this purpose, VDAC1 was enriched with an hydroxyapatite (HTP) chromatography of Triton X-100 solubilized rat liver mitochondria. Next, the eluate was separated by SDS-PAGE, stained and the resulting peptide mixture obtained from in-gel enzymatic digestion was analyzed by nano UPLC/nano ESI MSMS. Our results demonstrate that the mitochondrial VDAC1, in physiological state, contains cysteines in the oxidized form of sulfonic acid. Ratios of peptides containing oxidized cysteine residues and “normal” peptides (containing carboxyamidomethylated cysteines) were estimated from the absolute intensities of the respective molecular ions. The results obtained demonstrate that the oxidation state of cysteine residues detected in the rVDAC1 sequence is a consequence of the ROS level in the IMS.The structural features elucidated by the present work may be helpful for a better understanding of the functional role of VDAC1.* Corresponding author: Dr. Rosaria Saletti. Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.Tel.: +39 0957385026;E-mail address: rsaletti@unict.itReferences: [1] Varda Shoshan-Barmatz, Vito De Pinto, Markus Zweckstetter, Ziv Raviv, Nurit Keinan, Nir Arbel (2010) VDAC, a multi- functional mitochondrial protein regulating cell life and death. Mol. Aspects Med. 31: 227-285. [2] Roland Benz (1994) Permeation of hydrophilic solutes through mitochondrial outer membranes - review on mitochondrial porins. Biochim Biophys Acta 1197: 167-196. [3] 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, Vito De Pinto (2016) VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications. Oncotarget 7(3): 2249-2268.

High Resolution Mass Spectrometry Characterization of Cysteine Residues Oxidation in Rat Liver Mitochondria Voltage-Dependent Anion selective Channel 1 (VDAC1)

SALETTI, Rosaria;Reina S;Pittalà M;Messina A;CUNSOLO, VINCENZO;DE PINTO, Vito Nicola;FOTI, Salvatore
2016

Abstract

High Resolution Mass Spectrometry Characterization of Cysteine Residues Oxidation in Rat Liver Mitochondria Voltage-Dependent Anion selective Channel 1 (VDAC1) Rosaria Salettia*, Simona Reinab, Maria Pittalàc, Angela Messinab, Vincenzo Cunsoloa, Vito De Pintoc, Salvatore Fotiaa) Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Catania, Italy;b) Department of Biological, Geological and Environmental Sciences, Section of Molecular Biology University of Catania, Catania, Italy;c) Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy.Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane [1,2]. They are a small family of pore-forming proteins (30-35 kDa) that allows the flow of hydrophilic molecules through the membrane. VDAC isoforms show sequence homology and a similar β-barrel structure. The VDAC1 protein is the predominant form expressed in mitochondria of every tissue. Since the mitochondrial intermembrane space (IMS) has an oxidative potential, we have investigated the oxidation state of cysteine residues, as performed for the isoform VDAC3 [3]. To accomplish this purpose, VDAC1 was enriched with an hydroxyapatite (HTP) chromatography of Triton X-100 solubilized rat liver mitochondria. Next, the eluate was separated by SDS-PAGE, stained and the resulting peptide mixture obtained from in-gel enzymatic digestion was analyzed by nano UPLC/nano ESI MSMS. Our results demonstrate that the mitochondrial VDAC1, in physiological state, contains cysteines in the oxidized form of sulfonic acid. Ratios of peptides containing oxidized cysteine residues and “normal” peptides (containing carboxyamidomethylated cysteines) were estimated from the absolute intensities of the respective molecular ions. The results obtained demonstrate that the oxidation state of cysteine residues detected in the rVDAC1 sequence is a consequence of the ROS level in the IMS.The structural features elucidated by the present work may be helpful for a better understanding of the functional role of VDAC1.* Corresponding author: Dr. Rosaria Saletti. Department of Chemical Sciences, Organic Mass Spectrometry Laboratory, University of Catania, Viale A. Doria 6, 95125 Catania, Italy.Tel.: +39 0957385026;E-mail address: rsaletti@unict.itReferences: [1] Varda Shoshan-Barmatz, Vito De Pinto, Markus Zweckstetter, Ziv Raviv, Nurit Keinan, Nir Arbel (2010) VDAC, a multi- functional mitochondrial protein regulating cell life and death. Mol. Aspects Med. 31: 227-285. [2] Roland Benz (1994) Permeation of hydrophilic solutes through mitochondrial outer membranes - review on mitochondrial porins. Biochim Biophys Acta 1197: 167-196. [3] 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, Vito De Pinto (2016) VDAC3 as a sensor of oxidative state of the intermembrane space of mitochondria: the putative role of cysteine residue modifications. Oncotarget 7(3): 2249-2268.
File in questo prodotto:
Non ci sono file associati a questo prodotto.

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11769/112576
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus ND
  • ???jsp.display-item.citation.isi??? ND
social impact