Modifications of Superoxide Dismutase (SOD1) in Human Erythrocytes

Abstract

Over 100 mutations in Cu/Zn-superoxide dismutase (SOD1) result in familial amyotrophic lateral sclerosis. Dimer dissociation is the first step in SOD1 aggregation, and studies suggest nearly every amino acid residue in SOD1 is dynamically connected to the dimer interface. Post-translational modifications of SOD1 residues might be expected to have similar effects to mutations, but few modifications have been identified. Here we show, using SOD1 isolated from human erythrocytes, that human SOD1 is phosphorylated at threonine 2 and glutathionylated at cysteine 111. A second SOD1 phosphorylation was observed and mapped to either Thr-58 or Ser-59. Cysteine 111 glutathionylation promotes SOD1 monomer formation, a necessary initiating step in SOD1 aggregation, by causing a 2-fold increase in the K d . This change in the dimer stability is expected to result in a 67% increase in monomer concentration, 315 nM rather than 212 nM at physiological SOD1 concentrations. Because protein glutathionylation is associated with redox regulation, our finding that glutathionylation promotes SOD1 monomer formation supports a model in which increased oxidative stress promotes SOD1 aggregation. Familial amyotrophic lateral sclerosis (FALS)4 is the hereditary form of amyotrophic lateral sclerosis, a fatal disease characterized by progressive motor neuron loss (1). A subset of FALS is caused by mutations in the gene encoding homodimeric Cu/Zn-superoxide dismutase (SOD1), which forms intraneuronal aggregates (2). Although SOD1 aggregation is involved in SOD1-mediated FALS, it is generally believed that the functional properties of the enzyme are not related to the toxic gain of function imparted by mutations in SOD1 (3). However, the discovery of roles for SOD1 in the regulation of the cellular phosphorylation balance (4) and redox state (5) provides additional avenues for connecting the cellular role of SOD1 to FALS. The classical studies of SOD1 were generally performed using bovine erythrocyte SOD1 or recombinant human SOD1. Although recombinant methods are widely used to produce SOD1 mutants, a disadvantage of studying recombinant SOD1 is the absence of potentially important post-translational modifications present in human tissues. The initial SOD1 crystal structure was solved using bovine erythrocyte SOD1 (6) and no structure of human erythrocyte SOD1 is available. Here we report results using human erythrocyte SOD1 rather than the recombinant enzyme and find that the native enzyme features a consistent pattern of post-translational modifications. Using a combination of "bottom-up" and "top-down" mass spectrometry (MS) approaches, we show that SOD1 isolated from human erythrocytes is post-translationally phosphorylated and glutathionylated. These modifications occur near the SOD1 dimer interface. Because monomer formation is thought to be the first intermediate leading to SOD1 aggregation (7, 8), we tested the dimer stability of modified SOD1 found, as expected, that glutathionylation promotes the formation of SOD1 monomer. EXPERIMEN...

Study of the effect of varicocelectomy on sperm proteins expression in patients with varicocele and poor sperm quality by using two-dimensional gel electrophoresis

“…SOD1 is responsible for destroying free superoxide radicals in the body [14]. Our data suggested that down-regulation of SOD1 in patients with varicocele and abnormal spermogram can elevate DNA fragmentation and apoptosis, but after varicocelectomy, SOD1 activity increases and it can be resulted in improving DNA damage.…”

“…Our data suggested that down-regulation of SOD1 in patients with varicocele and abnormal spermogram can elevate DNA fragmentation and apoptosis, but after varicocelectomy, SOD1 activity increases and it can be resulted in improving DNA damage. Several studies have demonstrated that the total seminal plasma antioxidant levels in patients with varicocele are decreased and DNA damage in these patients was increased [12][13][14][15][16][17][18]. Moreover, some studies have shown that varicocelectomy can improve DNA fragmentation and total antioxidant capacity [19,20].…”

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

“…For determination of surface-accessible sites in the SOD1 trimer in limited proteolysis studies, we isolated and purified SOD1 from human erythrocytes, as previously described (26). To generate SOD1 mutants for aggregation time courses using SEC, and WT SOD1 for comparison of comparable species, we performed cloning, expression, and purification of human recombinant SOD1 in Saccharomyces cerevisiae as described elsewhere (15,26,38). We determined the concentration of purified SOD1 species by measuring the A at 280 nm with an extinction coefficient of 10,800 M −1 ·cm −1 .…”

“…We reconstructed the four-bead centroid structure obtained from the DMD REX simulations described above to an all-atom model (29) and performed structural minimization using Chiron (50) to remove clashes introduced by reconstruction. To equilibrate our structure in a physical force field, we first performed low-temperature (below the melting transition) allatom REX simulations using 26 We selected the ideal number of replicas and spread of replica temperatures such that exchange of replicas occurs with an acceptance rate between 0.2 and 0.7, with exchange attempted every 1,000 time steps. We performed simulations for 10 6 time steps.…”

“…After purification (26), aggregation, and isolation of the SOD1 trimer (15), we conducted limited proteolysis, followed by MS, to identify unstructured, solvent-accessible regions (27). Several proteolytic cuts are located in regions that are buried in the dimer interface or contain secondary structure elements in the SOD1 dimer ( Fig.…”