Post-Translational Modification Analysis of VDAC1 in ALS-SOD1 Model Cells Reveals Specific Asparagine and Glutamine Deamidation

Abstract

Mitochondria from affected tissues of amyotrophic lateral sclerosis (ALS) patients show morphological and biochemical abnormalities. Mitochondrial dysfunction causes oxidative damage and the accumulation of ROS, and represents one of the major triggers of selective death of motor neurons in ALS. We aimed to assess whether oxidative stress in ALS induces post-translational modifications (PTMs) in VDAC1, the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS. In this work, specific PTMs of the VDAC1 protein purified by hydroxyapatite from mitochondria of a NSC34 cell line expressing human SOD1G93A, a suitable ALS motor neuron model, were analyzed by tryptic and chymotryptic proteolysis and UHPLC/High-Resolution ESI-MS/MS. We found selective deamidations of asparagine and glutamine of VDAC1 in ALS-related NSC34-SOD1G93A cells but not in NSC34-SOD1WT or NSC34 cells. In addition, we identified differences in the over-oxidation of methionine and cysteines between VDAC1 purified from ALS model or non-ALS NSC34 cells. The specific range of PTMs identified exclusively in VDAC1 from NSC34-SOD1G93A cells but not from NSC34 control lines, suggests the appearance of important changes to the structure of the VDAC1 channel and therefore to the bioenergetics metabolism of ALS motor neurons. Data are available via ProteomeXchange with identifier <PXD022598>.

Small Hexokinase 1 Peptide against Toxic SOD1 G93A Mitochondrial Accumulation in ALS Rescues the ATP-Related Respiration

“…About 44% of those high-affinity poses located the NHK1 mod between VDAC β-strands 4 and 8, in the proximity of E73 and at only 14Å away from C127, the only cysteine residue exposed outside the barrel (Figure 6B). We have recently determined that in NSC34-SOD1 G93A cells, the C127 of VDAC1 is mostly found in a sulphonic acid over-oxidized form while a small proportion is present in a reduced form [60]. The unusual reduced form of C127 is thought to be a consequence of the destabilization of the VDAC1 structure due to deamidation of specific Asn and Gln residues found only in NSC34-SOD1 G93A cells [60].…”

Renaissance of VDAC: New Insights on a Protein Family at the Interface between Mitochondria and Cytosol

“…Most of the cysteines of VDAC2 and VDAC3 indeed are exposed to the aqueous environment and in certain situations are close enough to each other to suggest that they may engage in the formation of disulphide bridges. Taken together, the finding of cysteine oxidative post-translational modifications [ 52 , 53 , 54 , 55 ] indicate that each VDAC sulphur amino acid has a preferential sensitivity to oxidation. Indeed, some cysteines oscillate between different oxidative states (from reduced to sulfinic acid), others are always irreversibly oxidized (sulfonic acid), while many others are always reduced (for a detailed review see [ 55 ]).…”

“…The sample was then subjected to proteolytic cleavage and the peptide mixture loaded on UP-nanoLC and then analysed by a highly sensitive Orbitrap Fusion Tribrid (Q-OT-qIT) mass spectrometer. This modified procedure is all the more delicate and important to develop when considering that the main PTM studied was the oxidation of -SH [ 53 , 54 ].…”

“…A very rare and unique post-translational modification, the deamidation of specific asparagine and glutamine was found in cultured NSC34 cells transformed to express the SOD1G93A variant: this cell line is the most used cell model of ALS. It is tempting to speculate that this modification might be associated with the pathology [ 54 ].…”

Mitochondrial Oxidative and Nitrosative Stress as a Therapeutic Target in Diseases

“…The study by Pittalà et al [ 2 ] exemplifies the involvement of mitochondrial oxidative stress as a significant inducer of selective death of motor neurons in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects nerve cells in the brain and spinal cord, causing loss of muscle control. The study analyses whether oxidative stress in ALS can promote post-translational modifications in VDAC1, the main outer mitochondrial membrane protein known to interact with ALS-related SOD1 mutants.…”