Structural characterization of zinc-deficient human superoxide dismutase and implications for ALS

Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.     

Decreased Zinc Affinity of Amyotrophic Lateral Sclerosis-Associated Superoxide Dismutase Mutants Leads to Enhanced Catalysis of Tyrosine Nitration by Peroxynitrite

Abstract: Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS-associated SOD mutants was decreased up to 30-fold compared to wild-type SOD but that both mutants and wild-type SOD retained copper with similar affinity. Neurofilament-L (NF-L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild-type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild-type SOD approximately doubled its efficiency for catalyzing peroxynitrite-mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein-bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS-associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high-capacity zinc sink, NF-L could foster the formation of zinc-deficient SOD within motor neurons.     

ALS: a disease of motor neurons and their nonneuronal neighbors

Amyotrophic lateral sclerosis is a late-onset progressive neurodegenerative disease affecting motor neurons. The etiology of most ALS cases remains unknown, but 2% of instances are due to mutations in Cu/Zn superoxide dismutase (SOD1). Since sporadic and familial ALS affects the same neurons with similar pathology, it is hoped that therapies effective in mutant SOD1 models will translate to sporadic ALS. Mutant SOD1 induces non-cell-autonomous motor neuron killing by an unknown gain of toxicity. Selective vulnerability of motor neurons likely arises from a combination of several mechanisms, including protein misfolding, mitochondrial dysfunction, oxidative damage, defective axonal transport, excitotoxicity, insufficient growth factor signaling, and inflammation. Damage within motor neurons is enhanced by damage incurred by nonneuronal neighboring cells, via an inflammatory response that accelerates disease progression. These findings validate therapeutic approaches aimed at nonneuronal cells.     

Oxidation-induced misfolding and aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis

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.     

Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cerebral cortex, brainstem, and spinal cord. The cytopathological hallmark in the remaining motor neurons of ALS is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. In this paper we report that Dorfin, a RING finger-type E3 ubiquitin ligase, is predominantly localized in the inclusion bodies of familial ALS with a copper/zinc superoxide dismutase (SOD1) mutation as well as sporadic ALS. Dorfin physically bound and ubiquitylated various SOD1 mutants derived from familial ALS patients and enhanced their degradation, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Our results indicate that Dorfin protects neurons by recognizing and then ubiquitylating mutant SOD1 proteins followed by targeting them for proteasomal degradation.     

Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder affecting motor neurons. Although most cases of ALS are sporadic, approximately 10% are inherited as an autosomal dominant trait. Mutations in the Cu/Zn superoxide dismutase gene (SOD 1) are responsible for a fraction of familial ALS (FALS). Screening our FALS kindreds by SSCP, we have identified mutations in 15 families, of which 9 have not been previously reported. Two of the new mutations alter amino acids that have never been implicated in FALS. One of them affects a highly conserved amino acid involved in dimer contact, and the other one affects the active-site loop of the enzyme. These two mutations reduce significantly SOD 1 enzyme activity in lymphoblasts. Our results suggest that SOD 1 mutations are responsible for > or = 13% of FALS cases.     

Structural Analysis of Peroxide-Soaked MnSOD Crystals Reveals Side-On Binding of Peroxide to Active-Site Manganese

The superoxide dismutase (SOD) enzymes are important antioxidant agents that protect cells from reactive oxygen species. The SOD family is responsible for catalyzing the disproportionation of superoxide radical to oxygen and hydrogen peroxide. Manganese- and iron-containing SOD exhibit product inhibition whereas Cu/ZnSOD does not. Here, we report the crystal structure of Escherichia coli MnSOD with hydrogen peroxide cryotrapped in the active site. Crystallographic refinement to 1.55 Å and close inspection revealed electron density for hydrogen peroxide in three of the four active sites in the asymmetric unit. The hydrogen peroxide molecules are in the position opposite His26 that is normally assumed by water in the trigonal bipyramidal resting state of the enzyme. Hydrogen peroxide is present in active sites B, C, and D and is side-on coordinated to the active-site manganese. In chains B and D, the peroxide is oriented in the plane formed by manganese and ligands Asp167 and His26. In chain C, the peroxide is bound, making a 70° angle to the plane. Comparison of the peroxide-bound active site with the hydroxide-bound octahedral form shows a shifting of residue Tyr34 towards the active site when peroxide is bound. Comparison with peroxide-soaked Cu/ZnSOD indicates end-on binding of peroxide when the SOD does not exhibit inhibition by peroxide and side-on binding of peroxide in the product-inhibited state of MnSOD.     

Amyotrophic lateral sclerosis: a novel hypothesis involving a gained 'loss of function' in the JNK/SAPK pathway

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that mainly affects motor neurons. Despite intensive research efforts inspired by the mile-stone discovery linking the Cu/Zn superoxide dismutase 1 (SOD1) gene to a subset of familial cases, the mechanisms underlying disease pathogenesis are still largely unknown. Nonetheless, the recent finding of a second gene associated with familial form of the disease, ALS2, is likely to be of great help in elucidating the key pathways involved in motor neuron degeneration. Here, we provide evidence that the JNK/SAPK pathway plays a critical neuroprotective role in susceptible motor neurons in ALS. The involvement of the JNK/SAPK pathway integrates our knowledge about these two known genetic factors into a single pathogenic pathway involved in both sporadic and familial ALS.     

Molecular cloning,characterization and expression analysis of cytoplasmic Cu/Zn-superoxid dismutase (SOD) from pearl oyster Pinctada fucata

Because of its capacity to rapidly convert superoxide to hydrogen peroxide, superoxide dismutase (SOD) is crucial in both intracellular signalling and regulation of oxidative stress. In this paper we report the cloning of a Cu/Zn SOD (designated as pfSOD) from the pearl oyster (Pinctada fucata) using rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA of this Cu/Zn SOD contains an open reading frame (ORF) of 471 bp coding for 156 amino acids. No signal peptide was identified at the N-terminal amino acid sequence of Cu/Zn SOD indicating that this pfSOD encodes a cytoplasmic Cu/Zn SOD. This is supported by the presence of conserved amino acids required for binding copper and zinc. Semi-quantitative analysis in adult tissues showed that the pfSOD mRNA was abundantly expressed in haemocytes and gill and scarcely expressed in other tissues tested. After challenge with lipopolysaccharide (LPS), expression of pfSOD mRNA in haemocytes was increased, reaching the highest level at 8 h, then dropping to basal levels at 36 h. These results suggest that Cu/Zn SOD might be used as a bioindicator of the aquatic environmental pollution and cellular stress in pearl oyster.     

Electron transfer facilitated by superoxide dismutase: a model for membrane redox systems?

Membranes, which are an amalgam of proteins and lipids, effect electron transfer through largely unknown mechanisms. Using albumin with bound fatty acids as a model, we have investigated the possible role of these two membrane constituents in electron transfer. In the presence of albumin: fatty acid, there is substantial enhancement of the reduction of ferricytochrome C by ferrous iron. To assess the possible role of free superoxide in cytochrome C reduction, we added mammalian copper/zinc containing superoxide dismutase (Cu/Zn SOD), which catalyzes the transfer of electrons between superoxide anion radicals, forming oxygen and hydrogen peroxide. Surprisingly, in the presence of either albumin or fatty acid free albumin, Cu/Zn SOD actually accelerates electron transfer from ferrous iron to ferricytochrome C. By contrast, neither inactive Cu/Zn SOD nor active manganese SOD facilitates the ferrous iron-dependent reduction of cytochrome C. These results suggest that, in some circumstances, Cu/Zn SOD may transfer electrons to alternative acceptors and that such transfer depends upon the unique reduction/oxidation reaction mechanism of Cu/Zn SOD. If so, this ubiquitous enzyme could be involved in regulating cellular electron transfer reactions as well as acting as a superoxide 'detoxify-ing' agent.     

Liposome-delivered superoxide dismutase prevents nitric oxide-dependent motor neuron death induced by trophic factor withdrawal

Inhibition of nitric oxide synthesis prevents rat embryonic motor neurons from undergoing apoptosis when initially cultured without brain-derived neurotrophic factor. Using an improved cell culture medium, we found that the partial withdrawal of trophic support even weeks after motor neurons had differentiated into a mature phenotype still induced apoptosis through a process dependent upon nitric oxide. However, nitric oxide itself was not directly toxic to motor neurons. To investigate whether intracellular superoxide contributed to nitric oxide-dependent apoptosis, we developed a novel method using pH-sensitive liposomes to deliver Cu, Zn superoxide dismutase intracellularly into motor neurons. Intracellular superoxide dismutase prevented motor neuron apoptosis from trophic factor withdrawal, whereas empty liposomes, inactivated superoxide dismutase in liposomes or extracellular superoxide dismutase did not. Neither hydrogen peroxide nor nitrite added separately or in combination affected motor neuron survival. Our results suggest that a partial reduction in trophic support induced motor neuron apoptosis by a process requiring the endogenous production of both nitric oxide and superoxide, irrespective of the extent of motor neuron maturation in culture.     

人铜锌超氧化物歧化酶cDNA的克隆,测序及表达

用逆转录聚合酶链反应（ＲＴＰＣＲ）,以人胎肝组织总ＲＮＡ为模板,扩增了人铜锌超氧化物歧化酶（ｈＣｕ,ＺｎＳＯＤ）的ｃＤＮＡ,并进行序列分析,将该ｈＣｕ,ＺｎＳＯＤｃＤＮＡ重组到Ｔ７启动子控制下的分泌型表达载体ｐＥＴ２２ｂ（＋）中,构建表达质粒ｐＥＴＳＯＤ,并转化大肠杆菌ＢＬ２１（ＤＥ３）。ＳＤＳＰＡＧＥ及蛋白质印迹分析表明,经１ｍｍｏｌ／Ｌ异丙基硫代βＤ半乳糖苷（ＩＰＴＧ）诱导后,可高效表达一分子量为１９ｋＤ的蛋白质,与抗人ＳＯＤ多抗有特异的免疫反应,表达量约为菌体总蛋白质的３０％,具有特异性ＳＯＤ酶活性,酶活力可达１７９７ｕ／ｍｌ培基。     

Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis

Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.     

ALS-causing SOD1 mutations promote production of copper-deficient misfolded species

Point mutations scattered throughout the sequence of Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis (ALS) cases. SOD1 is a homodimer in which each subunit binds one copper atom and one zinc atom. Inclusions containing misfolded SOD1 are seen in motor neurons of SOD1-associated ALS cases. The mechanism by which these diverse mutations cause misfolding and converge on the same disease is still not well understood. Previously, we developed several time-resolved techniques to monitor structural changes in SOD1 as it unfolds in guanidine hydrochloride. By measuring the rates of Cu and Zn release using an absorbance-based assay, dimer dissociation through chemical cross-linking, and β-barrel conformation changes by tryptophan fluorescence, we established that wild-type SOD1 unfolds by a branched pathway involving a Zn-deficient monomer as the dominant intermediate of the major pathway, and with various metal-loaded and Cu-deficient dimers populated along the minor pathway. We have now compared the unfolding pathway of wild-type SOD1 with those of A4V, G37R, G85R, G93A, and I113T ALS-associated mutant SOD1. The kinetics of unfolding of the mutants were generally much faster than those of wild type. However, all of the mutants utilize the minority pathway to a greater extent than the wild-type protein, leading to greater populations of Cu-deficient intermediates and decreases in Zn-deficient intermediates relative to the wild-type protein. The greater propensity of the mutants to populate Cu-deficient states potentially implicates these species as a pathogenic form of SOD1 in SOD1-associated ALS and provides a novel target for therapeutic intervention.     

Mitochondrial dysfunction in familial amyotrophic lateral sclerosis

A growing body of evidence suggests that mitochondrial dysfunctions play a crucial role in the pathogenesis of various neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting both upper and lower motor neurons. Although ALS is predominantly a sporadic disease, approximately 10% of cases are familial. The most frequent familial form is caused by mutations in the gene encoding Cu/Zn superoxide dismutase 1 (SOD1). A dominant toxic gain of function of mutant SOD1 has been considered as the cause of the disease and mitochondria are thought to be key players in the pathogenesis. However, the exact nature of the link between mutant SOD1 and mitochondrial dysfunctions remains to be established. Here, we briefly review the evidence for mitochondrial dysfunctions in familial ALS and discuss a possible link between mutant SOD1 and mitochondrial dysfunction.     

Molecular Analyses of the Cu/Zn Superoxide Dismutase Gene in Patients with Familial Amyotrophic Lateral Sclerosis (ALS) in Japan

1. Amyotrophic lateral sclerosis (ALS) is a degenerative disorder characterized by selective damage to the neural system that mediates voluntary movement. Although the pathophysiologic process of ALS remains unknown, about 5 to 10% of cases are familial. According to genetic linkage studies, the familial ALS (FALS) gene has been mapped on chromosome 21 in some families and recent work identified some different missense mutations in the Cu/Zn superoxide dismutase gene in FALS families.2. We recently identified five mutations in six FALS families. The mutations identified in our FALS families are H46R, L84V, I104F, S134N, and V148I. The H46R mutation that locates in the active site of Cu/Zn SOD gene is associated with two Japanese families with very slow progression of ALS. On the other hand, the L84V mutation associated with a rapidly progressive loss of motor function with predominant lower motor neuron manifestations.3. In the family with the V148I, the phenotype of the patient varied very much among the affected members. One case had weakness of the lower extremities at first and died without bulbar paresis. The second case first noticed wasting of the upper limbs with bulbar symptoms, but the third had weakness of upper extremities without developing dysarthria nor dysphagia until death. These mutations account for 50% of all FALS families screened, although Cu/Zn SOD gene mutations are responsible for less than about 13–21% in the Western population.4. Our results indicate that the progression of disease with mutations of Cu/Zn SOD is well correlated with each mutation. The exact mechanism by which the abnormal Cu/Zn SOD molecules selectively affect the function of motor neurons is still unknown.     

Structural instability and Cu-dependent pro-oxidant activity acquired by the apo form of mutant SOD1 associated with amyotrophic lateral sclerosis

Cu,Zn-superoxide dismutase (SOD1) is a cytosolic antioxidant enzyme, and its mutation has been implicated in amyotrophic lateral sclerosis (ALS), a disease causing a progressive loss of motor neurons. Although the pathogenic mechanism of ALS remains unclear, it is hypothesized that some toxic properties acquired by mutant SOD1 play a role in the development of ALS. We have examined the structural and catalytic properties of an ALS-linked mutant of human SOD1, His43Arg (H43R), which is characterized by rapid disease progression. As revealed by circular dichroism spectroscopy, H43R assumes a stable β-barrel structure in the Cu(2+),Zn(2+)-bound holo form, but its metal-depleted apo form is highly unstable and readily unfolds or misfolds into an irregular structure at physiological temperature. The conformational change occurs as a two-state transition from a nativelike apo form to a denatured apo form with a half-life of ～0.5 h. At the same time as the denaturation, the apo form of H43R acquires pro-oxidant potential, which is fully expressed in the presence of Cu(2+) and H(2)O(2), as monitored with a fluorogenic probe for detecting pro-oxidant activity. Comparison of d-d absorption bands suggests that the Cu(2+) binding mode of the denatured apo form is different from that of the native holo form. The denatured apo form of H43R is likely to provide non-native Cu(2+) binding sites where the Cu(2+) ion is activated to catalyze harmful oxidation reactions. This study raises the possibility that the structural instability and the resultant Cu-dependent pro-oxidant activity of the apo form of mutant SOD1 may be one of the pathogenic mechanisms of ALS.     

铜锌超氧化物歧化酶(SOD)研究进展——从基因到功能

超氧化物歧化酶(SOD,EC 1.15.1.1),己经在多种组织中发现,它能将O2.-催化生成H2O2及O2.迄今为止,已经从哺乳动物体内分离出三种SOD:CuZnSOD(SOD1)、MnSOD(SOD2)TLEC-SOD(胞外超氧化物歧化酶,SOD3),各自具有不同的生化及分子特性.CuZnSOD(SOD1),是一类含有Cu及Zn原子的二聚体,存在于特定细胞的基质内,约占SOD总量的90%.在胞质及周质中,SOD以二聚体形式存在,而在线粒体及质外,则以四聚体形式存在.在保护脑、肺及其它组织的氧化应激中,CuZnSOD被认为起着保护作用.运动神经元肌萎缩侧索硬化症(ALS),据称也与同源二聚体CuZnSOD的错误折叠有关,己经报导,有多个CuZnSOD基因位点突变与ALS有关.本文将从基因的结构、表达、调节及蛋白的结构与功能等方面,对CuZnSOD进行简要论述.