Superoxide Dismutases

The side chains of His 30 and Tyr 166 from adjacent subunits in the homotetramer human manganese superoxide dismutase (Mn-SOD) form a hydrogen bond across the dimer interface and participate in a hydrogenbonded network that extends to the active site. Compared with wild-type Mn-SOD, the site-specific mutants H30N, Y166F, and the corresponding double mutant showed 10-fold decreases in steady-state constants for catalysis measured by pulse radiolysis. The observation of no additional effect upon the second mutation is an example of cooperatively interacting residues. A similar effect was observed in the thermal stability of these enzymes; the double mutant did not reduce the major unfolding transition to an extent greater than either single mutant. The crystal structures of these site-specific mutants each have unique conformational changes, but each has lost the hydrogen bond across the dimer interface, which results in a decrease in catalysis. These same mutations caused an enhancement of the dissociation of the product-inhibited complex. That is, His

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Amino Acid Substitution at the Dimeric Interface of Human Manganese Superoxide Dismutase

Hearn

Фан

Lepock

et al. 2004

“…2 a major intracellular antioxidant enzyme, metabolizes superoxide radicals to molecular oxygen and hydrogen peroxide (1,2). Because mutations in SOD1 linked to familial amyotrophic lateral sclerosis (ALS) were first identified (3), more than 100 mutations at over 70 residues in the 153-amino acid SOD1 protein have been reported (4).…”

mentioning

confidence: 99%

Disulfide Bond Mediates Aggregation, Toxicity, and Ubiquitylation of Familial Amyotrophic Lateral Sclerosis-linked Mutant SOD1

Niwa

Yamada

Ishigaki

et al. 2007

160

135

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys 6 -and Cys 111 -disulfide cross-linked form and targeted it for proteasomal degradation.Cu/Zn superoxide dismutase (SOD1), 2 a major intracellular antioxidant enzyme, metabolizes superoxide radicals to molecular oxygen and hydrogen peroxide (1, 2). Because mutations in SOD1 linked to familial amyotrophic lateral sclerosis (ALS) were first identified (3), more than 100 mutations at over 70 residues in the 153-amino acid SOD1 protein have been reported (4). Most mutations are missense mutations, with a few causing early termination or frame shifts near the carboxyl terminus of the protein. SOD1 mutations account for ϳ20% of familial ALS, which is characterized by selective degeneration of motor neurons. SOD1 is primarily a cytosolic protein (5), and the active enzyme is a homodimer of two subunits (6). Each subunit contains four cysteine (Cys) residues at positions 6, 57, 111, and 146. An intramolecular disulfide bond between Cys 57 and Cys 146 of each subunit facilitates its correct folding and stabilizes the active homodimeric structure (7, 8), but it is not known how the disulfide is formed in the reducing environment of the cytosol. Although the endoplasmic reticulum is the specialized site for oxidative folding (9), there is no SOD1 localization to the endoplasmic reticulum (10). Most familial ALS-linked mutations render SOD1 more susceptible to intramolecular disulfide bond reduction (11) and accelerate the rate of protein turnover (12, 13). Recent lines of evidence implicate the disulfide-reduced monomer as the common aggregation-prone, neurotoxic intermediate of mutant SOD1 proteins (8,11,[14][15][16], and a significant fraction of the insoluble SOD1 aggregates in the spinal cord of mutant SOD1 transgenic mice contains high molecular weight species cross-linked via intermolecular disulfide bonds (17). H...

“…Each subunit of the mature homodimeric SOD1 enzyme binds one atom of copper and one atom of zinc and contains a single oxidized disulfide bond between Cys-57 and Cys-146 (11)(12)(13). Because copper can participate in many types of potentially deleterious reactions, the role of the copper cofactor of SOD1 in the toxicity associated with mutant protein has been intensely studied (for review see Refs.…”

mentioning

confidence: 99%

Disease-associated Mutations at Copper Ligand Histidine Residues of Superoxide Dismutase 1 Diminish the Binding of Copper and Compromise Dimer Stability

Wang

Caruano-Yzermans

Rodriguez

et al. 2007

A subset of superoxide dismutase 1 (Cu/Zn-SOD1) mutants that cause familial amyotrophic lateral sclerosis (FALS) have heightened reactivity with ؊ ONOO and H 2 O 2 in vitro. This reactivity requires a copper ion bound in the active site and is a suggested mechanism of motor neuron injury. However, we have found that transgenic mice that express SOD1-H46R/ H48Q, which combines natural FALS mutations at ligands for copper and which is inactive, develop motor neuron disease. Using a direct radioactive copper incorporation assay in transfected cells and the established tools of single crystal x-ray diffraction, we now demonstrate that this variant does not stably bind copper. We find that single mutations at copper ligands, including H46R, H48Q, and a quadruple mutant H46R/H48Q/ H63G/H120G, also diminish the binding of radioactive copper. Further, using native polyacrylamide gel electrophoresis and a yeast two-hybrid assay, the binding of copper was found to be related to the formation of the stable dimeric enzyme. Collectively, our data demonstrate a relationship between copper and assembly of SOD1 into stable dimers and also define diseasecausing SOD1 mutants that are unlikely to robustly produce toxic radicals via copper-mediated chemistry. Amyotrophic lateral sclerosis (ALS),3 which is characterized by progressive muscle weakness and motor neuron loss, presents as both sporadic and familial (FALS) illness. A subset of FALS cases is caused by missense mutations in the superoxide scavenging enzyme, Cu/Zn-superoxide dismutase 1 (SOD1) (1, 2). To date, over 100 different point mutations, and Ͼ5 early termination mutations have been linked to FALS (www. alsod.org) (for reviews see Refs. 2-4). Early studies of FALS-SOD1 enzymes demonstrated that some mutants retain high levels of activity and relatively long half lives (5). Moreover, mutant proteins that are inactive or short-lived do not exhibit evidence of dominant negative action with regard to the superoxide-scavenging activity of enzyme derived from the normal allele (6). In transgenic mice, the hyperexpression of the G93A and G37R variants of FALS-SOD1 increases superoxide scavenging activity, kills motor neurons, and causes paralysis (7,8). SOD1 knock-out mice do not develop ALS-like phenotypes but do show sensory and motor neuropathy (9). Together, these studies establish that SOD1 mutations cause ALS through a gained toxic property. Although expressed ubiquitously (8, 10), mutant SOD1 selectively damages motor neurons, by mechanisms yet to be fully understood.