Quantitative Proteomics of Cysteine Redox Post-Translational Modifications

Cardiovascular disease is the leading cause of death worldwide with a mortality rate of 17 million people per annum. Ischemic heart disease is the largest contributor to this mortality rate. Ischemia is the loss of an adequate supply of oxygenated blood to the heart. This severely impairs heart function. During ischemia / reperfusion (I/R) the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is sufficient to cause cellular damage. ROS/RNS can alter proteins via posttranslational modification (PTM) of Cys residues. Redox PTM of Cys can be broken down into two groups. Those that are considered biologically ‘reversible’ and those that are considered biologically ‘irreversible’. Irreversible modifications are associated with protein dysfunction and proteolytic degradation. Current techniques to measure Cys PTMs are limited to few methods. To overcome this, we applied an enrichment technique for irreversibly modified Cys peptides to profile sites of irreversible Cys redox PTM in rat hearts subjected to ex vivo Langendorff perfusion and I/R injury. Quantitation was achieved using a novel parallel reaction monitoring mass spectrometry (PRM-MS) strategy on Cys peptides. We correlated changes to specific Cys peptide redox status with a targeted metabolomics approach. We next applied this approach to investigate ROS/RNS imbalance in the diabetic heart. Increased oxidative stress via ROS/RNS has been implicated in the aetiology of the diabetic heart, however, the targets of ROS/RNS are poorly defined. We utilised a rodent model of T2DM to examine hearts by Langendorff perfusion. Irreversibly modified Cys were largely increased in abundance in diabetic hearts under baseline conditions. This thesis has developed methods suitable for the routine identification and quantification of sites of irreversible Cys redox PTMs. Identification of such sites provides targets for specific antioxidant therapy to preserve enzyme functions during pathogenesis

Altered SOD1 maturation and post-translational modification in amyotrophic lateral sclerosis spinal cord

International audienceAberrant self-assembly and toxicity of wild-type and mutant superoxide dismutase 1 (SOD1) has been widely examined in silico, in vitro and in transgenic animal models of amyotrophic lateral sclerosis. Detailed examination of the protein in disease-affected tissues from amyotrophic lateral sclerosis patients, however, remains scarce.We used histological, biochemical and analytical techniques to profile alterations to SOD1 protein deposition, subcellular localization, maturation and post-translational modification in post-mortem spinal cord tissues from amyotrophic lateral sclerosis cases and controls. Tissues were dissected into ventral and dorsal spinal cord grey matter to assess the specificity of alterations within regions of motor neuron degeneration.We provide evidence of the mislocalization and accumulation of structurally disordered, immature SOD1 protein conformers in spinal cord motor neurons of SOD1-linked and non-SOD1-linked familial amyotrophic lateral sclerosis cases, and sporadic amyotrophic lateral sclerosis cases, compared with control motor neurons. These changes were collectively associated with instability and mismetallation of enzymatically active SOD1 dimers, as well as alterations to SOD1 post-translational modifications and molecular chaperones governing SOD1 maturation. Atypical changes to SOD1 protein were largely restricted to regions of neurodegeneration in amyotrophic lateral sclerosis cases, and clearly differentiated all forms of amyotrophic lateral sclerosis from controls. Substantial heterogeneity in the presence of these changes was also observed between amyotrophic lateral sclerosis cases.Our data demonstrate that varying forms of SOD1 proteinopathy are a common feature of all forms of amyotrophic lateral sclerosis, and support the presence of one or more convergent biochemical pathways leading to SOD1 proteinopathy in amyotrophic lateral sclerosis. Most of these alterations are specific to regions of neurodegeneration, and may therefore constitute valid targets for therapeutic development