Tryptophan 32 potentiates aggregation and cytotoxicity of a copper/zinc superoxide dismutase mutant associated with familial amyotrophic lateral sclerosis (original) (raw)
Related papers
2002
The presence of intracellular aggregates that contain Cu/Zn superoxide dismutase (SOD1) in spinal cord motor neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although SOD1 is abundant in all cells, its half-life in motor neurons far exceeds that in any other cell type. On the basis of the premise that the long half-life of the protein increases the potential for oxidative damage, we investigated the effects of oxidation on misfolding/aggregation of SOD1 and ALS-associated SOD1 mutants. Zinc-deficient wild-type SOD1 and SOD1 mutants were extremely prone to form visible aggregates upon oxidation as compared with wild-type holo-protein. Oxidation of select histidine residues that bind metals in the active site mediates SOD1 aggregation. Our results provide a plausible model to explain the accumulation of SOD1 aggregates in motor neurons affected in ALS.
Biochemical Society Transactions, 1998
Superoxide dismutase (SODl) is a copper-and zinc-dependent enzyme, which is highly conserved and ubiquitously distributed in nature. It transforms the superoxide anion free radical to hydrogen peroxide and oxygen. If coupled to the action of catalase or glutathione peroxidase, HzOZ can then be converted to water. Otherwise the Fenton reaction will convert Hz02 to hydroxyl free radical, a highly reactive species capable of bringing about damaging effects on lipids, proteins and DNA. Superoxide may also react with NO to form peroxynitrite which is also toxic largely through its breakdown to yield hydroxyl free radicals. SODl contains 153 amino acids and the active enzyme exists as a homodimer. The crystallographic structure shows that the core is a flattened Greek key /?-barrel motif of eight antiparallel /?-strands joined by seven loops. Two loops form an electrostatic channel at the active site and, at the base of this channel, the Cu ion is liganded to the surface of the /?-barrel by four histidine residues. The strong dimer interaction is due to hydrophobic interactions and contributes to the high stability of SOD1. Subunits cannot be separated without inactivating the enzyme. The SODl gene spans 11 kb, is localized to chromosome 21 (21q22.1) and was first implicated in diseases of the nervous system due to its over-representation in Down's syndrome where trisomy of chromosome 21 occurs. However, the greatest impact on unravelling the neurobiology of SODl was brought about from the discovery of mutations of SODl which are associated with familial motor neurone disease [ 11. Abbreviations used: SODl, superoxide dismutase; ALS, amyotrophic lateral sclerosis; FALS, familial ALS; ICE, interleukin-1-fl converting enzyme; IEG, immediate early gene; DMPO, 5,s dimethyl-l-pyrroline N-oxide.
2004
Proteinacious intracellular aggregates in motor neurons are a key feature of both sporadic and familial amyotrophic lateral sclerosis (ALS). These inclusion bodies are often immunoreactive for Cu,Zn-superoxide dismutase (SOD1) and are implicated in the pathology of ALS. On the basis of this and a similar clinical presentation of symptoms in the familial (fALS) and sporadic forms of ALS, we sought to investigate the possibility that there exists a common disease-related aggregation pathway for fALS-associated mutant SODs and wild type SOD1. We have previously shown that oxidation of fALS-associated mutant SODs produces aggregates that have the same morphological, structural, and tinctorial features as those found in SOD1 inclusion bodies in ALS. Here, we show that oxidative damage of wild type SOD at physiological concentrations (ϳ40 M) results in destabilization and aggregation in vitro. Oxidation of either mutant or wild type SOD1 causes the enzyme to dissociate to monomers prior to aggregation. Only small changes in secondary and tertiary structure are associated with monomer formation. These results indicate a common aggregation prone monomeric intermediate for wild type and fALS-associated mutant SODs and provides a link between sporadic and familial ALS.
Proceedings of the National Academy of Sciences, 2003
Mutations in Cu/Zn superoxide dismutase (SOD) are associated with the fatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS). There is considerable evidence that mutant SOD has a gain of toxic function; however, the mechanism of this toxicity is not known. We report here that purified SOD forms aggregates in vitro under destabilizing solution conditions by a process involving a transition from small amorphous species to fibrils. The assembly process and the tinctorial and structural properties of the in vitro aggregates resemble those for aggregates observed in vivo . Furthermore, the familial ALS SOD mutations A4V, G93A, G93R, and E100G decrease protein stability, which correlates with an increase in the propensity of the mutants to form aggregates. These mutations also increase the rate of protein unfolding. Our results suggest three possible mechanisms for the increase in aggregation: ( i ) an increase in the equilibrium population of unfolded or of partially unfold...
Journal of Biological Chemistry, 2014
Background: Copper/zinc superoxide dismutase (SOD1) genetic mutants are associated with familial amyotrophic lateral sclerosis (ALS). Mutant proteins form abnormal aggregates. Results: We used imaging of live cells to observe SOD1 proteins harboring mutations associated with ALS. Conclusion: SOD1 mutations impair its dimerization, leading to subsequent aggregation. Significance: Analysis of the SOD1 quaternary structure in living human cells correlates with previous biochemical data. More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins. ALS is a progressive neurodegenerative disorder caused by the degeneration of motor neurons. Most cases of ALS are sporadic, but ϳ10% are familial (fALS). 3 One-quarter of fALS cases are inherited because of mutations in the sod1 gene, which encodes an enzyme responsible for scavenging free radicals (1). The fALS disorder is primarily a heterozygous genetic condition. More than 140 point mutations have been found in the
Journal of Biological Chemistry, 2000
The presence of the copper ion at the active site of human wild type copper-zinc superoxide dismutase (CuZnSOD) is essential to its ability to catalyze the disproportionation of superoxide into dioxygen and hydrogen peroxide. Wild type CuZnSOD and several of the mutants associated with familial amyotrophic lateral sclerosis (FALS) (Ala 4 3 Val, Gly 93 3 Ala, and Leu 38 3 Val) were expressed in Saccharomyces cerevisiae. Purified metal-free (apoproteins) and various remetallated derivatives were analyzed by metal titrations monitored by UV-visible spectroscopy, histidine modification studies using diethylpyrocarbonate, and enzymatic activity measurements using pulse radiolysis. From these studies it was concluded that the FALS mutant CuZnSOD apoproteins, in direct contrast to the human wild type apoprotein, have lost their ability to partition and bind copper and zinc ions in their proper locations in vitro. Similar studies of the wild type and FALS mutant CuZn-SOD holoenzymes in the "as isolated" metallation state showed abnormally low copper-to-zinc ratios, although all of the copper acquired was located at the native copper binding sites. Thus, the copper ions are properly directed to their native binding sites in vivo, presumably as a result of the action of the yeast copper chaperone Lys7p (yeast CCS). The loss of metal ion binding specificity of FALS mutant CuZnSODs in vitro may be related to their role in ALS. Copper-zinc superoxide dismutase (CuZnSOD) 1,2 is an abundant cytosolic eukaryotic enzyme that catalyzes the disproportionation of superoxide anion to dioxygen and hydrogen peroxide (Reaction 1) (1-4). 2O 2 Ϫ ϩ 2H ϩ O ¡ CuZnSOD O 2 ϩ H 2 O 2 REACTION 1
Archives of Biochemistry and Biophysics, 1998
Inactivation of copper- and zinc-containing superoxide dismutase (Cu,ZnSOD) by H2O2 is the consequence of several sequential reactions: reduction of the active site Cu(II) to Cu(I) by H2O2; oxidation of the Cu(I) by a second H2O2, thus generating a powerful oxidant, which may be Cu(I)O or Cu(II)OH or Cu(III); and finally oxidation of one of the histidines in the ligand field, causing loss of SOD activity. Three familial amyotrophic lateral sclerosis (FALS)-associated mutant Cu,ZnSODs, i.e., E100G, G93A, and G93R, did not differ from the control enzyme in susceptibility to inactivation by H2O2. It thus appears that an increased peroxidase activity of the FALS-associated Cu,ZnSOD variants might not be a factor in the development of this disease. This leaves the loss of Zn, and the consequent increase in peroxidase activity, or in nitration activity, as a viable explanation (J. P. Crow et al., 1997, J. Neurochem. 69, 1936-1944), among other possibilities.
Proceedings of the National Academy of Sciences, 2010
Amyotrophic lateral sclerosis (ALS) is a disorder characterized by the death of both upper and lower motor neurons and by 3-to 5-yr median survival postdiagnosis. The only US Food and Drug Administration-approved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life; therefore innovative approaches are needed to combat neurodegenerative disease. Some familial forms of ALS (fALS) have been linked to mutations in the Cu/Zn superoxide dismutase (SOD1). The dominant inheritance of mutant SOD1 and lack of symptoms in knockout mice suggest a "gain of toxic function" as opposed to a loss of function. A prevailing hypothesis for the mechanism of the toxicity of fALS-SOD1 variants, or the gain of toxic function, involves dimer destabilization and dissociation as an early step in SOD1 aggregation. Therefore, stabilizing the SOD1 dimer, thus preventing aggregation, is a potential therapeutic strategy. Here, we report a strategy in which we chemically cross-link the SOD1 dimer using two adjacent cysteine residues on each respective monomer (Cys111). Stabilization, measured as an increase in melting temperature, of ∼20°C and ∼45°C was observed for two mutants, G93A and G85R, respectively. This stabilization is the largest for SOD1, and to the best of our knowledge, for any disease-related protein. In addition, chemical cross-linking conferred activity upon G85R, an otherwise inactive mutant. These results demonstrate that targeting these cysteine residues is an important new strategy for development of ALS therapies. mass spectrometry | thiol-disulfide I nnovative approaches are needed to combat neurodegenerative disease, among the most serious of which is amyotrophic lateral sclerosis (ALS), a disorder characterized by the death of both upper and lower motor neurons and by 3-to-5-yr median survival postdiagnosis. The only US Food and Drug Administrationapproved drug for the treatment of ALS, Riluzole, has at best, moderate effect on patient survival and quality of life (1-3). Although the causes of sporadic neurodegenerative diseases remain a mystery, mutations causing familial forms of many of these diseases (e.g., Alzheimer's, Parkinson, and ALS) are known. For example, mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) are responsible for ∼20% of the familial ALS cases (fALS) and 2% of all ALS (4, 5). Two such mutations are G93A, which maintains wild-type-like enzymatic activity, and the metal-deficient G85R, which is essentially inactive. Posttranslational modifications of proteins involved in familial diseases have been invoked in the etiology of the corresponding sporadic diseases, for example, alpha-synuclein (6) and Parkin (7) modification in Parkinson, Abeta (8) and tau (9) modification in Alzheimer's, and TDP43 (10) and SOD1 (11-14) modification in ALS. The hope, therefore, is that strategies for treating familial diseases may translate to at least a subset of sporadic diseases. Both dominant inheritance of mutant SOD1 (15) and lack of symptoms in knockout mice (16) suggest a "gain of toxic function" as opposed to a loss of function (16-22). Aggregation propensity and loss of stability of SOD1 are synergistic risk fac
Journal of Biological …, 2008
Converging evidence indicates that aberrant aggregation of mutant Cu,Zn-superoxide dismutase (mutSOD1) is strongly implicated in familial amyotrophic lateral sclerosis (FALS). MutSOD1 forms high molecular weight oligomers, which disappear under reducing conditions, both in neural tissues of FALS transgenic mice and in transfected cultured cells, indicating a role for aberrant intermolecular disulfide cross-linking in the oligomerization and aggregation process. To study the contribution of specific cysteines in the mechanism of aggregation, we mutated human SOD1 in each of its four cysteine residues and, using a cell transfection assay, analyzed the solubility and aggregation of those SOD1s. Our results suggest that the formation of mutSOD1 aggregates are the consequence of covalent disulfide cross-linking and non-covalent interactions. In particular, we found that the removal of Cys-111 strongly reduces the ability of a range of different FALS-associated mutSOD1s to form aggregates and impair cell viability in cultured NSC-34 cells. Moreover, the removal of Cys-111 impairs the ability of mutSOD1s to form disulfide cross-linking. Treatments that deplete the cellular pool of GSH exacerbate mutSOD1s insolubility, whereas an overload of intracellular GSH or overexpression of glutaredoxin-1, which specifically catalyzes the reduction of protein-SSG-mixed disulfides, significantly rescues mutSOD1s solubility. These data are consistent with the view that the redox environment influences the oligomerization/aggregation pathway of mutSOD1 and point to Cys-111 as a key mediator of this process. More than 100 different mutations in the gene encoding Cu,Zn-superoxide dismutase (SOD1) 2 have been causally linked to familial amyotrophic lateral sclerosis (FALS), and * This work was supported by SISAL and the Telethon Foundation (Grant GGP07018) and by Ministero Salute Progetto Finalizzato Approcci Neuroprotettivi nel Danno da Deprivazione Energetica.