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. 2022 Dec 13;23(24):15853.
doi: 10.3390/ijms232415853.

Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

Maria Gaetana Giovanna Pittalà  1 , Simona Reina  2 , Stefano Conti Nibali  2 , Annamaria Cucina  1 , Salvatore Antonio Maria Cubisino  2 , Vincenzo Cunsolo  1 , Giuseppe Federico Amodeo  3 , Salvatore Foti  1 , Vito De Pinto  2 , Rosaria Saletti  1 , Angela Messina  4
Affiliations

Specific Post-Translational Modifications of VDAC3 in ALS-SOD1 Model Cells Identified by High-Resolution Mass Spectrometry

Maria Gaetana Giovanna Pittalà et al. Int J Mol Sci. .

Abstract

Damage induced by oxidative stress is a key driver of the selective motor neuron death in amyotrophic lateral sclerosis (ALS). Mitochondria are among the main producers of ROS, but they also suffer particularly from their harmful effects. Voltage-dependent anion-selective channels (VDACs) are the most represented proteins of the outer mitochondrial membrane where they form pores controlling the permeation of metabolites responsible for mitochondrial functions. For these reasons, VDACs contribute to mitochondrial quality control and the entire energy metabolism of the cell. In this work we assessed in an ALS cell model whether disease-related oxidative stress induces post-translational modifications (PTMs) in VDAC3, a member of the VDAC family of outer mitochondrial membrane channel proteins, known for its role in redox signaling. At this end, protein samples enriched in VDACs were prepared from mitochondria of an ALS model cell line, NSC34 expressing human SOD1G93A, and analyzed by nUHPLC/High-Resolution nESI-MS/MS. Specific over-oxidation, deamidation, succination events were found in VDAC3 from ALS-related NSC34-SOD1G93A but not in non-ALS cell lines. Additionally, we report evidence that some PTMs may affect VDAC3 functionality. In particular, deamidation of Asn215 alone alters single channel behavior in artificial membranes. Overall, our results suggest modifications of VDAC3 that can impact its protective role against ROS, which is particularly important in the ALS context. Data are available via ProteomeXchange with identifier PXD036728.

Keywords: SOD1; amyotrophic lateral sclerosis; deamidation; high-resolution mass spectrometry; mitochondria; neurodegeneration; over-oxidation; post-translational modifications; succination; voltage dependent anion channel.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Sequence coverage map of VDAC3 from NSC34, NSC34-SOD1WT and NSC34-SOD1G93A cell lines obtained by tryptic and chymotryptic digestion. The solid line indicates the sequence that was obtained from tryptic peptides; dotted lines: sequence obtained from chymotryptic peptides. Unique tryptic (indicated in bold and blue) and chymotryptic (indicated in bold and black) peptides originating from missed cleavages were used to distinguish and cover the sequences shared by isoforms. Sequences shared by multiple isoforms are shown in red. Sequence numbering considered the starting methionine residue, which is eliminated during protein maturation.
Figure 2
Figure 2
MS/MS spectrum of the doubly charged molecular ion at m/z 660.2664 (calculated 660.2663) of the N-terminal tryptic peptide of VDAC3 from NSC34-SOD1WT cell line with cysteine residue 2 in the form of sulfonic acid and cysteine residue 8 in the carboxyamidomethylated form. The inset shows the full scan mass spectrum of the molecular ion. Fragment ions originating from the neutral loss of H2O are indicated by an asterisk. The fragment ion originating from the neutral loss of NH3 is indicated by two asterisks.
Figure 3
Figure 3
MS/MS spectrum of the doubly charged molecular ion at m/z 924.9586 (calculated 924.9582) of the VDAC3 tryptic peptide from NSC34-SOD1G93A cell line containing the asparagine residue 215 in the deamidated form. The inset shows the full scan mass spectrum of molecular ion. Fragment ions originated from the neutral loss of H2O are indicated by an asterisk. Fragment ions originated from the neutral loss of NH3 are indicated by two asterisks.
Figure 4
Figure 4
(A) Structural predictions of hVDAC3 N215D. Side view (on the left) and top view (on the right) were obtained by VMD software. In both figures, the top side represent the cytosolic environment. (B) Structural predictions of hVDAC3 wild type. Side view (on the left) and top view (on the right) were obtained by VMD software. In both figures, the top side represent the cytosolic environment. (C) Structural predictions of hVDAC3 N215D showing the localization of N167 and Cys65 residues. Side view (on the left) and top view (on the right) were obtained by VMD software. In both figures, the top side represent the cytosolic environment.
Figure 5
Figure 5
Amplitude distribution of VDAC3 wt and N215D channels in non-reducing conditions. (A) Representative current trace of VDAC3 wt in 1 M KCl at + 10 mV applied. (B) Amplitude histograms of single channel recording data of VDAC3 wt. (C) Representative current trace of VDAC3 N215D in 1 M KCl at +10 mV applied. (D) Amplitude histograms of single channel recording data of VDAC3 N215D.
Figure 6
Figure 6
Voltage dependence analysis of VDAC3 wt and N215D in non-reducing conditions. (A) Voltage ramp from 0 to ± 50 mVA of VDAC3 wt. (B) Current vs. voltage (I–V) plot of VDAC3 wt. (C) Voltage ramp from 0 to ± 50 mVA of N215D VDAC3. (D) Current vs. voltage (I–V) plot of of N215D VDAC3. (E) Voltage dependence graph of wt and mutated VDAC3. The normalized average conductance G/G0 was plotted as a function of applied voltage (Vm).
Figure 7
Figure 7
Voltage dependence analysis of VDAC3 wt and N215D preincubated with 5 mM DTT. (A) Voltage ramp from 0 to ± 50 mVA of VDAC3 wt. (B) Current vs. voltage (I–V) plot of VDAC3 wt. (C) Voltage ramp from 0 to ± 50 mVA of N215D VDAC3. (D) Current vs. voltage (I–V) plot of N215D VDAC3. (E) Voltage dependence graph of wt and mutated VDAC3. The normalized average conductance G/G0 was plotted as a function of applied voltage (Vm).
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