TY - JOUR
T1 - High resolution mass spectrometry characterization of the oxidation pattern of methionine and cysteine residues in rat liver mitochondria voltage-dependent anion selective channel 3 (VDAC3)
AU - Saletti, Rosaria
AU - Reina, Simona
AU - Pittalà, Maria G.G.
AU - Belfiore, Ramona
AU - Cunsolo, Vincenzo
AU - Messina, Angela
AU - De Pinto, Vito
AU - Foti, Salvatore
N1 - Publisher Copyright:
© 2016 Elsevier B.V.
PY - 2017/3/1
Y1 - 2017/3/1
N2 - Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122 could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.
AB - Voltage-dependent anion selective channels (VDACs) are integral membrane proteins found in the mitochondrial outer membrane. In comparison with the most abundant isoform VDAC1, there is little knowledge about the functional role of VDAC3. Unlikely VDAC1, cysteine residues are particularly abundant in VDAC3. Since the mitochondrial intermembrane space (IMS) has an oxidative potential we questioned whether the redox state of VDAC3 can be modified. By means of SDS-PAGE separation, tryptic and chymotryptic proteolysis and UHPLC/High Resolution ESI-MS/MS analysis we investigated the oxidation state of cysteine and methionine residues of rat liver VDAC3. Our results demonstrate that the mitochondrial VDAC3, in physiological state, contains methionines oxidized to methionine sulfoxide. Furthermore, cysteine residues 36, 65, and 165 are oxidized to a remarkable extend to sulfonic acid. Cysteines 2 and 8 are observed exclusively in the carboxyamidomethylated form. Cys229 is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys122 could not be determined because peptides containing this residue were not detected. Control experiments ruled out the possibility that over-oxidation of cysteines might be due to artefactual reasons. The peculiar behavior of Met and Cys residues of VDAC3 may be related with the accessibility of the protein to a strongly oxidizing environment and may be connected with the regulation of the activity of this trans-membrane pore protein.
KW - Amino acid redox state
KW - Cysteine
KW - Mitochondrial intermembrane space
KW - Mitochondrial outer membrane
KW - Orbitrap tribrid mass spectrometer
KW - Voltage dependent anion selective channel isoform 3 (VDAC3)
UR - https://www.scopus.com/pages/publications/85006862909
U2 - 10.1016/j.bbamem.2016.12.003
DO - 10.1016/j.bbamem.2016.12.003
M3 - Article
C2 - 27989743
AN - SCOPUS:85006862909
SN - 0005-2736
VL - 1859
SP - 301
EP - 311
JO - Biochimica et Biophysica Acta - Biomembranes
JF - Biochimica et Biophysica Acta - Biomembranes
IS - 3
ER -