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

Maria Gaetana Giovanna Pittalà,Simona Reina,Rosaria Saletti,Beatrice Formicola,Salvatore Foti,Salvatore Antonio Maria Cubisino,Annamaria Cucina,Vincenzo Cunsolo,Angela Messina

doi:10.3390/antiox9121218

Maria Gaetana Giovanna Pittalà, Simona Reina + Show 7 more

Open Access

https://doi.org/10.3390/antiox9121218

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Journal: Antioxidants	Publication Date: Dec 2, 2020
Citations: 13	License type: CC BY 4.0

Affiliation: University of Catania, University of Milano-Bicocca

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>.

Highlights

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by progressive degeneration of the motor neurons in the brainstem and spinal cord that leads patients to death by respiratory paralysis within 2–5 years of onset [1]
We aimed to assess whether oxidative stress in amyotrophic lateral sclerosis (ALS) induces post-translational modifications (PTMs) in voltage dependent anion selective channel isoform 1 (VDAC1), the main protein of the outer mitochondrial membrane and known to interact with SOD1 mutants related to ALS
Our study was mainly focused on the identification of enzymatic and non-enzymatic PTMs in the VDAC1 protein from an established ALS mouse motor neuron-like cell line, especially considering the changes induced by oxidative stress typical of neurons affected by the disease

Summary

Introduction

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease caused by progressive degeneration of the motor neurons in the brainstem and spinal cord that leads patients to death by respiratory paralysis within 2–5 years of onset [1]. Several genetic risk factors are implicated in sporadic ALS (sALS) [2]. Oxidative stress-induced damage is a major mechanism in the ALS pathophysiology and can result from an imbalance between free radical production and degradation. Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are generated from various cell sources. ROS produced through Nox enzymes have been implicated in ALS pathogenesis [5]. Free radicals cause oxidative damage to lipids, proteins, and nucleic acids contributing to trigger or amplify the pathological mechanisms related to ALS [6]. Mitochondrial dysfunction is both the main contributor to oxidative stress and its main consequence

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