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

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.

Intramolecular Disulfide Bridges in Voltage-Dependent Anion Channel 2 (VDAC2) Protein from Rattus norvegicus Revealed by High-Resolution Mass Spectrometry

“…13,15,16 The preferred redox state of homologous cysteines, in general, was also found to be conserved between human, rat and mouse VDAC proteins. 13,15,16,38,39 As an extension of the previous study, the existence of intramolecular disulfide bridges was investigated in the present work, and the topology of six disulfide bridges in rVDAC2 was unequivocally determined, including a disulfide bridge between the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14 and the alternative disulfide bridges between cysteine 48 and cysteines 77 and 104. Furthermore, a disulfide bridge, strongly resistant to reduction, linking cysteines 134 and 139 was identified.…”

“…From these considerations stems the importance of a definitive characterization of the relevant structural constraints related to the functional VDAC pores. In recent years, the oxidation state of cysteines in VDACs has been characterized by nUHPLC/HR nESI-MS/MS analysis which demonstrated that such residues can be subjected to different degree of oxidation, ranging from the disulfide bond to the most oxidized, the sulfonic acid, an irreversible and permanent protein modification. ,, The preferred redox state of homologous cysteines, in general, was also found to be conserved between human, rat and mouse VDAC proteins. ,,,, As an extension of the previous study, the existence of intramolecular disulfide bridges was investigated in the present work, and the topology of six disulfide bridges in rVDAC2 was unequivocally determined, including a disulfide bridge between the two adjacent cysteines 4 and 5, a disulfide bridge linking cysteines 9 and 14 and the alternative disulfide bridges between cysteine 48 and cysteines 77 and 104. Furthermore, a disulfide bridge, strongly resistant to reduction, linking cysteines 134 and 139 was identified.…”

Orchestration of Mitochondrial Function and Remodeling by Post-Translational Modifications Provide Insight into Mechanisms of Viral Infection

“…VDAC dysfunction has been reported in various neurodegenerative diseases such as Alzheimer's disease [171,172], Down's syndrome [172], and familial amyotrophic lateral sclerosis (ALS) [173,174]. Recent studies have shown that specific PTMs, such as VDAC1 and VDAC3 deamidations, are enhanced and can regulate channel behavior in ALS cell lines [175,176]. Moreover, VDAC1 phosphorylation is dynamically regulated in a variety of human cancers, suggesting a role in tumorigenesis [177].…”