Post-translational modification by cysteine protects Cu/Zn-superoxide dismutase from oxidative damage.

Reactive oxygen species (ROS) are cytotoxic. To remove ROS, cells have developed ROS-specific defense mechanisms, including the enzyme Cu/Zn superoxide dismutase (SOD1), which catalyzes the disproportionation of superoxide anions into molecular oxygen and hydrogen peroxide. Although hydrogen peroxide is less reactive than superoxide, it is still capable of oxidizing, unfolding, and inactivating SOD1, at least in vitro. To explore the relevance of post-translational modification (PTM) of SOD1, including peroxide-related modifications, SOD1 was purified from postmortem human nervous tissue. As much as half of all purified SOD1 protein contained non-native post-translational modifications (PTMs), the most prevalent modifications being cysteinylation and peroxide-related oxidations. Many PTMs targeted a single reactive SOD1 cysteine, Cys111. An intriguing observation was that unlike native SOD1, cysteinylated SOD1 was not oxidized. To further characterize how cysteinylation may protect SOD1 from oxidation, cysteine-modified SOD1 was prepared in vitro and exposed to peroxide. Cysteinylation conferred nearly complete protection from peroxide-induced oxidation of SOD1. Moreover, SOD1 that has been cysteinylated and peroxide oxidized in vitro comprised a set of PTMs that bear a striking resemblance to the myriad of PTMs observed in SOD1 purified from human tissue.

[1]  S. Moore,et al.  THE CONCENTRATIONS OF CYSTEINE AND CYSTINE IN HUMAN BLOOD PLASMA. , 1960, The Journal of clinical investigation.

[2]  P. Jocelyn.  The standard redox potential of cysteine-cystine from the thiol-disulphide exchange reaction with glutathione and lipoic acid. , 1967, European journal of biochemistry.

[3]  I. Fridovich,et al.  The reduction of cytochrome c by milk xanthine oxidase. , 1968, The Journal of biological chemistry.

[4]  A. Teelken,et al.  Amino acid abnormalities in cerebrospinal fluid of patients with parkinsonism and extrapyramidal disorders , 1976, Neurology.

[5]  A. Slivka,et al.  Reduced and oxidized glutathione in human and monkey brain , 1987, Neuroscience Letters.

[6]  A. Slivka,et al.  Histochemical evaluation of glutathione in brain , 1987, Brain Research.

[7]  M. Harada,et al.  Alteration of amino acid content of cerebrospinal fluid from patients with epilepsy , 1988, Acta neurologica Scandinavica.

[8]  T. Murphy,et al.  Macromolecular synthesis inhibitors prevent oxidative stress-induced apoptosis in embryonic cortical neurons by shunting cysteine from protein synthesis to glutathione , 1994, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[9]  A. Lazzarin,et al.  Cerebrospinal fluid S-adenosylmethionine (SAMe) and glutathione concentrations in HIV infection Effect of parenteral treatment with SAMe , 1995, Neurology.

[10]  J. Tainer,et al.  Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[11]  F. Polticelli,et al.  Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues. , 1996, European journal of biochemistry.

[12]  S. Sakoda,et al.  Simple and defined method to detect the SOD-1 mutants from patients with familial amyotrophic lateral sclerosis by mass spectrometry , 1998, Journal of Neuroscience Methods.

[13]  J S Valentine,et al.  Copper(2+) binding to the surface residue cysteine 111 of His46Arg human copper-zinc superoxide dismutase, a familial amyotrophic lateral sclerosis mutant. , 2000, Biochemistry.

[14]  M. Skinner,et al.  Identification and location of a cysteinyl posttranslational modification in an amyloidogenic kappa1 light chain protein by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry. , 2001, Analytical biochemistry.

[15]  M. Masu,et al.  Distribution of Cystine/Glutamate Exchange Transporter, System xc−, in the Mouse Brain , 2002, The Journal of Neuroscience.

[16]  Robert H. Brown,et al.  Decreased Metallation and Activity in Subsets of Mutant Superoxide Dismutases Associated with Familial Amyotrophic Lateral Sclerosis* 210 , 2002, The Journal of Biological Chemistry.

[17]  A. Chakrabartty,et al.  Monomeric Cu,Zn-superoxide Dismutase Is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis*[boxs] , 2004, Journal of Biological Chemistry.

[18]  I. Bertini,et al.  The Unusually Stable Quaternary Structure of Human Cu,Zn-Superoxide Dismutase 1 Is Controlled by Both Metal Occupancy and Disulfide Status* , 2004, Journal of Biological Chemistry.

[19]  A. Hilliker,et al.  Increased spontaneous DNA damage in Cu/Zn superoxide dismutase (SOD1) deficient Drosophila. , 2004, Genome.

[20]  Borries Demeler,et al.  Dissociation of Human Copper-Zinc Superoxide Dismutase Dimers Using Chaotrope and Reductant , 2004, Journal of Biological Chemistry.

[21]  G. Allmaier,et al.  Characterization of cysteinylation of pharmaceutical-grade human serum albumin by electrospray ionization mass spectrometry and low-energy collision-induced dissociation tandem mass spectrometry. , 2005, Rapid communications in mass spectrometry : RCM.

[22]  A. Levey,et al.  Oxidative Modifications and Aggregation of Cu,Zn-Superoxide Dismutase Associated with Alzheimer and Parkinson Diseases* , 2005, Journal of Biological Chemistry.

[23]  E. Keithley,et al.  Cu/Zn superoxide dismutase and age-related hearing loss , 2005, Hearing Research.

[24]  A. Shimizu,et al.  Detection and characterization of variant and modified structures of proteins in blood and tissues by mass spectrometry. , 2006, Mass spectrometry reviews.

[25]  A. Chakrabartty,et al.  An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS , 2007, Nature Medicine.

[26]  M. Hecker,et al.  S-Cysteinylation Is a General Mechanism for Thiol Protection of Bacillus subtilis Proteins after Oxidative Stress* , 2007, Journal of Biological Chemistry.

[27]  Keiichiro Suzuki,et al.  Oxidative Modification to Cysteine Sulfonic Acid of Cys111 in Human Copper-Zinc Superoxide Dismutase* , 2007, Journal of Biological Chemistry.

[28]  J. Agar,et al.  Tryptophan 32 Potentiates Aggregation and Cytotoxicity of a Copper/Zinc Superoxide Dismutase Mutant Associated with Familial Amyotrophic Lateral Sclerosis* , 2007, Journal of Biological Chemistry.

[29]  S. Marklund,et al.  The coupling between disulphide status, metallation and dimer interface strength in Cu/Zn superoxide dismutase. , 2007, Journal of molecular biology.

[30]  Dean P. Jones,et al.  A rapid LC-FTMS method for the analysis of cysteine, cystine and cysteine/cystine steady-state redox potential in human plasma. , 2008, Clinica chimica acta; international journal of clinical chemistry.

[31]  A. Yarnell.  CYSTEINE OXIDATION: NEW CHEMICAL TOOLS are poised to help scientists explore the roles oxidized cysteine residues might play in biology , 2009 .

[32]  Nikolay V Dokholyan,et al.  Modifications of Superoxide Dismutase (SOD1) in Human Erythrocytes , 2009, Journal of Biological Chemistry.

[33]  Leonid Zamdborg,et al.  The emerging process of Top Down mass spectrometry for protein analysis: biomarkers, protein-therapeutics, and achieving high throughput. , 2010, Molecular bioSystems.

[34]  Jared R. Auclair,et al.  Strategies for stabilizing superoxide dismutase (SOD1), the protein destabilized in the most common form of familial amyotrophic lateral sclerosis , 2010, Proceedings of the National Academy of Sciences.

[35]  Jeffery N Agar,et al.  Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS , 2010, Nature Neuroscience.

[36]  J. Agar,et al.  Structural characterization of intact proteins is enhanced by prevalent fragmentation pathways rarely observed for peptides , 2010, Journal of the American Society for Mass Spectrometry.

[37]  N. Dokholyan,et al.  Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers. , 2011, Biochemistry.