Sensitive enzyme immunoassay for human Cu/Zn superoxide dismutase

Kurobe, Naomi; Suzuki, Fujiko; Okajima, Kazuki; Kato, Kanefusa

doi:10.1016/0009-8981(90)90257-s

Cited by 51 publications

(34 citation statements)

References 20 publications

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“…3A), a concentration within the physiological range of 10 -100 M (34,35). Consistent with our previous findings (36), either disulfide formation, zinc addition, or both leads to dimerization (Fig.…”

Section: Resultssupporting

confidence: 92%

Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo- and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation

Furukawa¹,

O’Halloran

2005

Journal of Biological Chemistry

245

290

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Mutant forms of Cu,Zn-superoxide dismutase (SOD1) that cause familial amyotrophic lateral sclerosis (ALS) exhibit toxicity that promotes the death of motor neurons. Proposals for the toxic properties typically involve aberrant catalytic activities or protein aggregation. The striking thermodynamic stability of mature forms of the ALS mutant SOD1 (T m >70°C) is not typical of protein aggregation models that involve unfolding. Over 44 states of the polypeptide are possible, depending upon metal occupancy, disulfide status, and oligomeric state; however, it is not clear which forms might be responsible for toxicity. Recently the intramolecular disulfide has been shown to be required for SOD1 activity, leading us to examine these states of several disease-causing SOD1 mutants. We find that ALS mutations have the greatest effect on the most immature form of SOD1, destabilizing the metal-free and disulfide-reduced polypeptide to the point that it is unfolded at physiological temperatures (T m < 37°C). We also find that immature states of ALS mutant (but not wild type) proteins readily form oligomers at physiological concentrations. Furthermore, these oligomers are more susceptible to mild oxidative stress, which promotes incorrect disulfide cross-links between conserved cysteines and drives aggregation. Thus it is the earliest disulfide-reduced polypeptides in the SOD1 assembly pathway that are most destabilized with respect to unfolding and oxidative aggregation by ALS-causing mutations. Amyotrophic lateral sclerosis (ALS)1 is a neurodegenerative disease caused by selective degeneration of motor neurons in the brain and spinal cord (1). About 10% of total ALS cases are of familial origin, and of these, ϳ20% are caused by point mutations in the abundant enzyme Cu,Zn-superoxide dismutase (SOD1) (2). SOD1 is a dimer of identical subunits, each of which contains a copper and a zinc ion, and catalyzes conversion of superoxide anion to oxygen and hydrogen peroxide at a dinuclear copper-zinc site (3). Studies of SOD1 knock-out mice reveal no motor neuron disease (4), indicating new toxic functions of ALS mutations. Among the proposed gain of functions is aberrant redox chemistry arising from modification of active copper and zinc sites in ALS mutants. For example, enhanced peroxidase activity (5), protein nitration (6), and superoxide production (7) have been proposed. The requirement of copper ion for toxicity has, however, been put into question, because ALS symptoms are observed in mice expressing the SOD1 mutant in which all of the copper ligands are mutated (8).Another hypothesis for a toxic function acquired by the mutant SOD1 proteins is an increased propensity for cytoplasmic aggregation, which is one of the pathological features common to familial ALS (9). ALS mutations have been considered to induce protein misfolding and/or destabilization; in fact, ALS mutations provoke a decrease of 1-6°C in the melting temperature of SOD1 (10), although wild type (WT) SOD1 remains active after treatment at 80°C (11). Reduced z...

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Section: Resultssupporting

confidence: 92%

Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo- and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation

Furukawa¹,

O’Halloran

2005

Journal of Biological Chemistry

245

290

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“…has been estimated to be present in cells at concentrations of Ϸ100 M (15)(16)(17). To mimic as closely as possible the conditions experienced by the protein in vivo, we prepared 100 M protein solutions at pH 7.0 and incubated them at 37°C.…”

Section: Sod1mentioning

confidence: 99%

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Banci

Bertini

Durazo³

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

225

262

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Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder selectively affecting motor neurons; 90% of the total cases are sporadic, but 2% are associated with mutations in the gene coding for the antioxidant enzyme copper-zinc superoxide dismutase (SOD1). The causes of motor neuron death in ALS are poorly understood in general, but for SOD1-linked familial ALS, aberrant oligomerization of SOD1 mutant proteins has been strongly implicated. In this work, we show that wild-type human SOD1, when lacking both its metal ions, forms large, stable, soluble protein oligomers with an average molecular mass of Ϸ650 kDa under physiological conditions, i.e., 37°C, pH 7.0, and 100 M protein concentration. It further is shown here that intermolecular disulfide bonds are formed during oligomerization and that Cys-6 and Cys-111 are implicated in this bonding. The formation of the soluble oligomers was monitored by their ability to enhance the fluorescence of thioflavin T, a benzothiazole dye that increases in fluorescence intensity upon binding to amyloid fibers, and by disruption of this binding upon addition of the chaotropic agent guanidine hydrochloride. Our results suggest a general, unifying picture of SOD1 aggregation that could operate when wild-type or mutant SOD1 proteins lack their metal ions. Although we cannot exclude other mechanisms in SOD1-linked familial ALS, the one proposed here has the strength of explaining how a large and diverse set of SOD1 mutant proteins all could lead to disease through the same mechanism.amyloid ͉ neurodegeneration ͉ protein aggregation ͉ amyotrophic lateral sclerosis ͉ protein misfolding P rotein oligomerization, aggregation, and formation of insoluble amyloid deposits commonly are observed in neurodegenerative diseases, but the factors initiating and modulating the abnormal protein-protein interactions that lead to oligomerization remain elusive (1, 2). Metal ions frequently have been implicated in these phenomena, but how exactly they are involved remains unclear (3). Over 114 different variants of human copper-zinc superoxide dismutase (Cu 2 Zn 2 SOD1) have been linked to the neurodegenerative disease familial ALS (FALS) by a gain-of-function mechanism (4-6). Although the exact cellular sites and mechanisms of toxicity are unknown, aberrant SOD1 protein oligomerization has been strongly implicated in disease causation (7,8). Several recent publications have presented compelling evidence that abnormal disulfide cross-linking of ALS-mutant SOD1 plays a role in this oligomerization, and disulfide-linked SOD1 multimers have been detected in neural tissues of SOD1-ALS transgenic mice that are presumed to be components of higher-molecular-weight species or intermediates in their formation (7, 9-11).Wild-type (WT) human SOD1 is an exceptionally stable protein in its holo form and, although some of the ALS-mutant SOD1 proteins are severely destabilized by their mutations, others largely retain the stability of WT SOD1 (4). In the fully demetallated (apo) states, some ...

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“…To ensure the physiological relevance of our in vitro aggregation system, we used neurophysiological concentrations of SOD1. Kurobe et al (27) found that the SOD1 content of erythrocytes is 0.95 Ϯ 0.07 g of SOD1/mg hemoglobin, which translates to a molar concentration of 10 M (based on normal values for mean corpuscular hemoglobin and volume (28)). The concentration of SOD1 is ϳ4.5 times greater in brain than in erythrocytes (27), corresponding to a concentration of 45 M. Intracellular SOD1 concentration in motor neurons may be even higher (13).…”

Section: Wild Type Sod1 Is Aggregation-prone Under Oxidativementioning

confidence: 99%

Monomeric Cu,Zn-superoxide Dismutase Is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis

Rakhit

Crow

Lepock

et al. 2004

Journal of Biological Chemistry

304

303

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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. ALS1 is a fatal neurodegenerative disease that leads to the selective loss of motor neurons. Although ALS is predominately a sporadic disease, ϳ10% of cases are inherited in an autosomal dominant manner and a subset of these fALS cases are caused by mutations in the SOD1 gene (1). The gene product of SOD1, cytoplasmic Cu,Zn-superoxide dismutase (SOD1), is a ubiquitously expressed enzyme that catalyzes the disproportionation reaction of superoxide radicals (1). There are several lines of evidence that SOD1 mutations result in a gain, rather than loss of function that causes ALS. For instance, some fALS-associated mutant SOD1s retain full enzymatic activity (2). In addition, SOD1 knock-out mice lack ALS symptoms, whereas transgenic mice expressing the fALS-associated mutant G93A SOD1 develop ALS-like symptoms despite expression of endogenous mouse SOD1 (3). Lastly, overexpression of human wild type SOD1 fails to alleviate symptoms in this transgenic mouse model for ALS (3). One hypothesis of the gain of function of SOD1 is that misfolding of the mutant alters the catalytic mechanism to allow production of oxidants such as peroxynitrite (4) and possibly hydrogen peroxide (5). These reactive nitrogen and oxygen species cause toxicity by accumulated damage to proteins, nucleic acids, and lipids. Another major hypothesis is toxicity caused by intracellular aggregation of SOD1. SOD1 inclusion bodies, which also react with anti-ubiquitin antibodies, are a common pathological finding in motor neurons and neighboring astrocytes of ALS patients (6). These two hypotheses, however, are not mutually exclusive when considering that oxidative modification of proteins may contribute to aggregation and protease resistance. Protein aggregates are a common pathological feature...

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Sensitive enzyme immunoassay for human Cu/Zn superoxide dismutase

Cited by 51 publications

References 20 publications

Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo- and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation

Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo- and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation

Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: A possible general mechanism for familial ALS

Monomeric Cu,Zn-superoxide Dismutase Is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis

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