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)

Abstract

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. Cys is detected exclusively in the oxidized form of sulfonic acid, whereas the oxidation state of Cys 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.

Helix–strand interaction regulates stability and aggregation of the human mitochondrial membrane protein channel VDAC3

“…Cysteines respond to mitochondrial oxidative stress through oxidative modifications of their side chain (Guardiani et al, 2016; Reina et al, 2016a,b; Saletti et al, 2017, 2018; Sorrentino et al, 2017; Iadanza et al, 2018). In particular, hV3 is known to oligomerize and aggregate upon oxidation of its cysteines to S–OH, S–O 2 H, and S–O 3 H (Okazaki et al, 2015; De Pinto et al, 2016; Guardiani et al, 2016; Reina et al, 2016a,b; Saletti et al, 2017). Our recent reports (Reina et al, 2016a) also identify the susceptibility of hVDAC3 to aggregation under oxidative stress.…”

“…The other cysteines (C36 and C122) are present in large hydrophobic segments. It has not been possible to map these peptides even in prior MS studies of hV3, owing to the extremely hydrophobic nature and poor ionization efficiency of these segments (Saletti et al, 2017). MS fingerprinting using other proteases such as pepsin also did not yield the peptide fragments corresponding to these cysteines (not depicted).…”

“…Recent findings have revealed the importance of VDAC2 and VDAC3 cysteines in sensing and responding to mitochondrial oxidative stress by way of cysteine modifications (Guardiani et al, 2016; Reina et al, 2016a,b; Iadanza et al, 2018; Saletti et al, 2018). Furthermore, human VDAC3 (hV3) cysteines are oxidized to S–OH, S–O 2 H, and S–O 3 H when reactive oxygen species accumulate in the cell (Okazaki et al, 2015; De Pinto et al, 2016; Reina et al, 2016a,b; Saletti et al, 2017), causing hV3 to aggregate. Such aggregates can lead to cellular toxicity (Boulbrima et al, 2016).…”

A lower affinity to cytosolic proteins reveals VDAC3 isoform-specific role in mitochondrial biology

“…Similarly, the oxidation state of cysteines was found to be important for the insertion and activity of VDAC3 samples (Reina et al, 2016b). It must be pointed out that in these previous works, any reducing agent was avoided during purification and reconstitution steps, with the purpose of not altering the naturally occurring oxidation state of cysteines (Reina et al, 2016a; Saletti et al, 2017). In the present study, all VDAC3 samples tested showed transitions between an open state to a variety of closed states, with very similar gating characteristics to VDAC1 (Fig.…”

Porphyrin-polymer nanocompartments: singlet oxygen generation and antimicrobial activity

“…The substrate chosen for the photocatalytic oxidation investigation was the amino acid, l -methionine [ 40 ]. Its selective oxidation to the sulfoxide represents a model for the oxidation of methionine residues in proteins (see for example: 41 – 44 ]). An aqueous solution of l -methionine containing 0.06 mol% of TPyCP was irradiated with red light ( λ exc = 660 nm) and the reaction mixture was monitored by 1 H NMR spectroscopy.…”