Biomedical subjects

Joan Selverstone Valentine

Publications and source records attributed to Joan Selverstone Valentine.

At least 19 recordsLinked to original sources

PubMed2007

How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein?

More than 100 different mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS)--a fatal neurodegenerative disease in which aggregation of the SOD1 protein is considered to be the primary mode of pathogenesis. Recent results show that these mutations have remarkably diverse and unexpected effects on the structure, activity and native state stability of SOD1. Intriguingly, many mutations seem to have no measurable effect on the biophysical and biochemical properties of SOD1, except for decreasing the net charge of the protein. Thus, it seems likely that different ALS-associated mutations promote SOD1 aggregation by fundamentally distinct mechanisms. Understanding this complexity has implications for drug development and treatment of the disease.

Amyotrophic Lateral Sclerosis↗

PubMed2006

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials.

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die.

Amyotrophic Lateral Sclerosis↗

PubMed2006

Superoxide dismutase 1 modulates expression of transferrin receptor.

Copper-zinc superoxide dismutase (SOD1) plays a protective role against the toxicity of superoxide, and studies in Saccharomyces cerevisiae and in Drosophila have suggested an additional role for SOD1 in iron metabolism. We have studied the effect of the modulation of SOD1 levels on iron metabolism in a cultured human glial cell line and in a mouse motoneuronal cell line. We observed that levels of the transferrin receptor and the iron regulatory protein 1 were modulated in response to altered intracellular levels of superoxide dismutase activity, carried either by wild-type SOD1 or by an SOD-active amyotrophic lateral sclerosis (ALS) mutant enzyme, G93A-SOD1, but not by a superoxide dismutase inactive ALS mutant, H46R-SOD1. Ferritin expression was also increased by wild-type SOD1 overexpression, but not by mutant SOD1s. We propose that changes in superoxide levels due to alteration of SOD1 activity affect iron metabolism in glial and neuronal cells from higher eukaryotes and that this may be relevant to diseases of the nervous system.

Actins↗

PubMed2006

Local unfolding in a destabilized, pathogenic variant of superoxide dismutase 1 observed with H/D exchange and mass spectrometry.

Hydrogen exchange monitored by mass spectrometry has been used to study the structural behavior of the pathogenic A4V variant of superoxide dismutase 1 (SOD1) in the metal-free (apo) form. Mass spectrometric data revealed that in the disulfide-intact (S-S) form, the A4V variant is destabilized at residues 50-53, in the disulfide subloop of the dimer interface, but many other regions of the A4V protein exhibited hydrogen exchange properties identical to that of the wild type protein. Additionally, mass spectrometry revealed that A4V apoSOD1(S-S) undergoes slow localized unfolding in a large segment of the beta-barrel that included beta3, beta4, and loops II and III. In the disulfide-reduced form, A4V apoSOD1 exchanged like a "random coil" polypeptide at 20 degrees C and began to populate folded states at 4 degrees C. These local and global unfolding events could facilitate intermolecular protein-protein interactions that cause the aggregation or neurotoxicity of A4V SOD1.

Cold Temperature↗

PubMed2005

Variable metallation of human superoxide dismutase: atomic resolution crystal structures of Cu-Zn, Zn-Zn and as-isolated wild-type enzymes.

Human Cu-Zn superoxide dismutase (SOD1) protects cells from the effects of oxidative stress. Mutations in SOD1 are linked to the familial form of amyotrophic lateral sclerosis. Several hypotheses for their toxicity involve the mis-metallation of the enzyme. We present atomic-resolution crystal structures and biophysical data for human SOD1 in three metallation states: Zn-Zn, Cu-Zn and as-isolated. These data represent the first atomic-resolution structures for human SOD1, the first structure of a reduced SOD1, and the first structure of a fully Zn-substituted SOD1 enzyme. Recombinantly expressed as-isolated SOD1 contains a mixture of Zn and Cu at the Cu-binding site. The Zn-Zn structure appears to be at least as stable as the correctly (Cu-Zn) metallated enzyme. These data raise the possibility that in a cellular environment with low availability of free copper, Zn-Zn may be the preferred metallation state of SOD1 prior to its interaction with the copper chaperone.

Animals↗

PubMed2005

Exogenous manganous ion at millimolar levels rescues all known dioxygen-sensitive phenotypes of yeast lacking CuZnSOD.

Yeasts lacking copper-zinc superoxide dismutase (sod1Delta) exhibit a broad range of phenotypes, many of which can be rescued by growth in the presence of high levels of ionic manganese. We undertook a comprehensive survey of the effects of manganese on wild-type and sod1Delta yeasts and found that 5 mM Mn2+ rescued all known growth-related phenotypes, such as slow growth in air, temperature sensitivity, specific amino acid auxotrophies, no growth in high oxygen, poor growth in nonfermentable carbon sources, and decreased stationary-phase survival. Iron-related phenotypes-elevated electron paramagnetic resonance detectable ("free") iron, decreased aconitase activity, and fragmenting vacuoles-as well as zinc sensitivity were also rescued. The activity of manganese superoxide dismutase remained constant or was reduced when the yeasts were grown in the presence of MnCl2, indicating that induction of this alternative superoxide dismutase is not the explanation. In contrast to MnCl2 treatment, addition of two manganese-containing superoxide dismutase mimetic compounds to the growth medium did not provide any rescue of sod1Delta yeast growth but rather had an sod1Delta-selective inhibitory effect at micromolar concentrations. Mechanisms by which ionic manganese can effect this rescue, while the mimetic compounds do not, are discussed.

Electron Spin Resonance Spectroscopy↗

PubMed2005

Fully metallated S134N Cu,Zn-superoxide dismutase displays abnormal mobility and intermolecular contacts in solution.

S134N copper-zinc superoxide dismutase (SOD1) is one of the many mutant SOD1 proteins known to cause familial amyotrophic lateral sclerosis. Earlier studies demonstrated that partially metal-deficient S134N SOD1 crystallized in filament-like arrays with abnormal contacts between the individual protein molecules. Because protein aggregation is implicated in SOD1-linked familial amyotrophic lateral sclerosis, abnormal intermolecular interactions between mutant SOD1 proteins could be relevant to the mechanism of pathogenesis in the disease. We have therefore applied NMR methods to ascertain whether abnormal contacts also form between S134N SOD1 molecules in solution and whether Cys-6 or Cys-111 plays any role in the aggregation. Our studies demonstrate that the behavior of fully metallated S134N SOD1 is dramatically different from that of fully metallated wild type SOD1 with a region of subnanosecond mobility located close to the site of the mutation. Such a high degree of mobility is usually seen only in the apo form of wild type SOD1, because binding of zinc to the zinc site normally immobilizes that region. In addition, concentration-dependent chemical shift differences were observed for S134N SOD1 that were not observed for wild type SOD1, indicative of abnormal intermolecular contacts in solution. We have here also established that the two free cysteines (6 and 111) do not play a role in this behavior.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis.

The relative stabilities and structural properties of a representative set of 20 ALS-mutant Cu,Zn-superoxide dismutase apoproteins were examined by using differential scanning calorimetry and hydrogen-deuterium (H/D) exchange followed by MS. Contrary to recent reports from other laboratories, we found that ALS-mutant apoproteins are not universally destabilized by the disease-causing mutations. For example, several of the apoproteins with substitutions at or near the metal binding region (MBR) (MBR mutants) exhibited melting temperatures (Tm) in the range 51.6 degrees C to 56.2 degrees C, i.e., similar to or higher than that of the WT apoprotein (Tm = 52.5 degrees C). The apoproteins with substitutions remote from the MBR (WT-like mutants) showed a wide range of Tms, 40.0 degrees C to 52.4 degrees C. The H/D exchange properties of the mutants were also wide-ranging: the MBR mutant apoproteins exhibited H/D exchange kinetics similar to the WT apoprotein, as did some of the more stable WT-like mutant apoproteins, whereas the less stable apoproteins exhibited significantly less protection from H/D exchange than the WT apoprotein. Most striking were the three mutant apoproteins, D101N, E100K, and N139K, which have apparently normal metallation properties, and differ little from the WT apoprotein in either thermal stability or H/D exchange kinetics. Thus, the ALS mutant Cu,Zn-superoxide dismutase apoproteins do not all share reduced global stability, and additional properties must be identified and understood to explain the toxicity of all of the mutant proteins.

Amino Acid Substitution↗

PubMed2005

Induction of phenotypes resembling CuZn-superoxide dismutase deletion in wild-type yeast cells: an in vivo assay for the role of superoxide in the toxicity of redox-cycling compounds.

Yeast (Saccharomyces cerevisiae) lacking the enzyme CuZn-superoxide dismutase (sod1delta) display a large number of dioxygen sensitive phenotypes, such as amino acid auxotrophies, sensitivity to elevated temperatures, and sensitivity to 100% dioxygen, which are attributed to superoxide stress. Such cells are exquisitely sensitive to small amounts of the herbicide paraquat (methyl viologen), which is known to produce high fluxes of superoxide in vivo via a redox-cycling mechanism. We report that dioxygen sensitive phenotypes similar to those seen in sod1delta cells can be induced in wild-type cells by treatment with moderate concentrations of paraquat or diquat, another bipyridyl herbicide, providing strong evidence that the mechanism of toxicity for both of these compounds is attributable to superoxide stress. Certain redox-cycling quinone compounds (e.g., menadione and plumbagin) are also far more toxic toward sod1delta than to wild type. However, treatment of wild-type yeast with menadione or plumbagin did not induce sod1delta-like phenotypes, although toxicity was evident. Thus, their toxicity in wild type cells is predominantly, but not exclusively, due to mechanisms unrelated to superoxide production. Further evidence for a different basis of toxicity toward wild-type yeast in these two classes of redox-cycling compounds includes the observations that (i) growth in low oxygen alleviated the effects of paraquat and diquat but not those of menadione or plumbagin and (ii) activity of the superoxide sensitive enzyme aconitase is affected by very low concentrations of paraquat but only by higher, growth inhibitory concentrations of menadione. These results provide the basis for an easy qualitative assay of the contribution of redox-cycling to the toxicity of a test compound. Using this method, we analyzed the Parkinsonism-inducing compound 1-methyl-4-phenylpyridinium and found that redox cycling and superoxide toxicity are not the predominant factor in its toxic mechanism.

1-Methyl-4-phenylpyridinium↗

PubMed2005

Structural consequences of the familial amyotrophic lateral sclerosis SOD1 mutant His46Arg.

The His46Arg (H46R) mutant of human copper-zinc superoxide dismutase (SOD1) is associated with an unusual, slowly progressing form of familial amyotrophic lateral sclerosis (FALS). Here we describe in detail the crystal structures of pathogenic H46R SOD1 in the Zn-loaded (Zn-H46R) and metal-free (apo-H46R) forms. The Zn-H46R structure demonstrates a novel zinc coordination that involves only three of the usual four liganding residues, His 63, His 80, and Asp 83 together with a water molecule. In addition, the Asp 124 "secondary bridge" between the copper- and zinc-binding sites is disrupted, and the "electrostatic loop" and "zinc loop" elements are largely disordered. The apo-H46R structure exhibits partial disorder in the electrostatic and zinc loop elements in three of the four dimers in the asymmetric unit, while the fourth has ordered loops due to crystal packing interactions. In both structures, nonnative SOD1-SOD1 interactions lead to the formation of higher-order filamentous arrays. The disordered loop elements may increase the likelihood of protein aggregation in vivo, either with other H46R molecules or with other critical cellular components. Importantly, the binding of zinc is not sufficient to prevent the formation of nonnative interactions between pathogenic H46R molecules. The increased tendency to aggregate, even in the presence of Zn, arising from the loss of the secondary bridge is consistent with the observation of an increased abundance of hyaline inclusions in spinal motor neurons and supporting cells in H46R SOD1 transgenic rats.

Amyotrophic Lateral Sclerosis↗

PubMed2005

Copper-zinc superoxide dismutase and amyotrophic lateral sclerosis.

Copper-zinc superoxide dismutase (CuZnSOD, SOD1 protein) is an abundant copper- and zinc-containing protein that is present in the cytosol, nucleus, peroxisomes, and mitochondrial intermembrane space of human cells. Its primary function is to act as an antioxidant enzyme, lowering the steady-state concentration of superoxide, but when mutated, it can also cause disease. Over 100 different mutations have been identified in the sod1 genes of patients diagnosed with the familial form of amyotrophic lateral sclerosis (fALS). These mutations result in a highly diverse group of mutant proteins, some of them very similar to and others enormously different from wild-type SOD1. Despite their differences in properties, each member of this diverse set of mutant proteins causes the same clinical disease, presenting a challenge in formulating hypotheses as to what causes SOD1-associated fALS. In this review, we draw together and summarize information from many laboratories about the characteristics of the individual mutant SOD1 proteins in vivo and in vitro in the hope that it will aid investigators in their search for the cause(s) of SOD1-associated fALS.

Amyotrophic Lateral Sclerosis↗

PubMed2004

Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability.

The dissociation of apo- and metal-bound human copper-zinc superoxide dismutase (SOD1) dimers induced by the chaotrope guanidine hydrochloride (GdnHCl) or the reductant Tris(2-carboxyethyl)phosphine (TCEP) has been analyzed using analytical ultracentrifugation. Global fitting of sedimentation equilibrium data under native solution conditions (without GdnHCl or TCEP) demonstrate that both the apo- and metal-bound forms of SOD1 are stable dimers. Sedimentation velocity experiments show that apo-SOD1 dimers dissociate cooperatively over the range 0.5-1.0 M GdnHCl. In contrast, metal-bound SOD1 dimers possess a more compact shape and dissociate at significantly higher GdnHCl concentrations (2.0-3.0 M). Reduction of the intrasubunit disulfide bond within each SOD1 subunit by 5-10 mM TCEP promotes dissociation of apo-SOD1 dimers, whereas the metal-bound enzyme remains a stable dimer under these conditions. The Cys-57 --> Ser mutant of SOD1, a protein incapable of forming the intrasubunit disulfide bond, sediments as a monomer in the absence of metal ions and as a dimer when metals are bound. Taken together, these data indicate that the stability imparted to the human SOD1 dimer by metal binding and the formation of the intrasubunit disulfide bond are mediated by independent molecular mechanisms. By combining the sedimentation data with previous crystallographic results, a molecular explanation is provided for the existence of different SOD1 macromolecular shapes and multiple SOD1 dimeric species with different stabilities.

Chemical Phenomena↗

PubMed2004

Superoxide inhibits 4Fe-4S cluster enzymes involved in amino acid biosynthesis. Cross-compartment protection by CuZn-superoxide dismutase.

Among the phenotypes of Saccharomyces cerevisiae mutants lacking CuZn-superoxide dismutase (Sod1p) is an aerobic lysine auxotrophy; in the current work we show an additional leaky auxotrophy for leucine. The lysine and leucine biosynthetic pathways each contain a 4Fe-4S cluster enzyme homologous to aconitase and likely to be superoxide-sensitive, homoaconitase (Lys4p) and isopropylmalate dehydratase (Leu1p), respectively. We present evidence that direct aerobic inactivation of these enzymes in sod1 Delta yeast results in the auxotrophies. Located in the cytosol and intermembrane space of the mitochondria, Sod1p likely provides direct protection of the cytosolic enzyme Leu1p. Surprisingly, Lys4p does not share a compartment with Sod1p but is located in the mitochondrial matrix. The activity of a second matrix protein, the tricarboxylic acid cycle enzyme aconitase, was similarly lowered in sod1 Delta mutants. We measured only slight changes in total mitochondrial iron and found no detectable difference in mitochondrial "free" (EPR-detectable) iron making it unlikely that a gross defect in mitochondrial iron metabolism is the cause of the decreased enzyme activities. Thus, we conclude that when Sod1p is absent a lysine auxotrophy is induced because Lys4p is inactivated in the matrix by superoxide that originates in the intermembrane space and diffuses across the inner membrane.

Amino Acids↗

PubMed2004

Mutations in Saccharomyces cerevisiae iron-sulfur cluster assembly genes and oxidative stress relevant to Cu,Zn superoxide dismutase.

Saccharomyces cerevisiae lacking Cu,Zn superoxide dismutase (SOD1) show several metabolic defects including aerobic blockages in methionine and lysine biosynthesis. We have previously shown that mutations in genes implicated in the formation of iron-sulfur clusters, designated seo (suppressors of endogenous oxidation), reverse the oxygen-dependent methionine and lysine auxotrophies of a sod1Delta strain. We now report the surprising finding that seo mutants do not reduce oxidative damage as shown by the lack of reduction of EPR-detectable "free" iron, which is characteristic of sod1Delta mutants. In fact, they exhibit increased oxidative damage as evidenced by increased accumulation of protein carbonyls. The seo class of mutants overaccumulates mitochondrial iron, and this iron accumulation is critical for suppression of the sod1Delta biosynthetic defects. Blocking overaccumulation of mitochondrial iron abolished the ability of the seo mutants to suppress the sod1Delta auxotrophies. By contrast, increasing the mitochondrial iron content of sod1Delta yeast using high copy MMT1, which encodes a mitochondrial iron transporter, was sufficient to mimic the seo mutants. Our studies indicated that suppression of the sod1Delta methionine auxotrophy was dependent on the pentose phosphate pathway, which is a major source of NADPH production. By comparison, the sod1Delta lysine auxotrophy appears to be reversed in the seo mutants by increased expression of genes in the lysine biosynthetic pathway, perhaps through sensing of mitochondrial damage by the retrograde response.

Carbon↗

PubMed2004

Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone.

The Cu- and Zn-containing superoxide dismutase 1 (SOD1) largely obtains Cu in vivo by means of the action of the Cu chaperone CCS. Yet, in the case of mammalian SOD1, a secondary pathway of activation is apparent. Specifically, when human SOD1 is expressed in either yeast or mammalian cells that are null for CCS, the SOD1 enzyme retains a certain degree of activity. This CCS-independent activity is evident with both wild-type and mutant variants of SOD1 that have been associated with familial amyotrophic lateral sclerosis. We demonstrate here that the CCS-independent activation of mammalian SOD1 involves glutathione, particularly the reduced form, or GSH. A role for glutathione in CCS-independent activation was seen with human SOD1 molecules that were expressed in either yeast cells or immortalized fibroblasts. Compared with mammalian SOD1, the Saccharomyces cerevisiae enzyme cannot obtain Cu without CCS in vivo, and this total dependence on CCS involves the presence of dual prolines near the C terminus of the SOD1 polypeptide. Indeed, the insertion of such prolines into human SOD1 rendered this molecule refractory to CCS-independent activation. The possible implications of multiple pathways for SOD1 activation are discussed in the context of SOD1 evolutionary biology and familial amyotrophic lateral sclerosis.

Animals↗

PubMed2004

Dimer destabilization in superoxide dismutase may result in disease-causing properties: structures of motor neuron disease mutants.

More than 90 point mutations in human CuZn superoxide dismutase lead to the development of familial amyotrophic lateral sclerosis, known also as motor neuron disease. A growing body of evidence suggests that a subset of mutations located close to the dimeric interface can lead to a major destabilization of the mutant enzymes. We have determined the crystal structures of the Ala4Val (A4V) and Ile113Thr (I113T) mutants to 1.9 and 1.6 A, respectively. In the A4V structure, small changes at the dimer interface result in a substantial reorientation of the two monomers. This effect is also seen in the case of the I113T crystal structure, but to a smaller extent. X-ray solution scattering data show that in the solution state, both of the mutants undergo a more pronounced conformational change compared with wild-type superoxide dismutase (SOD) than that observed in the A4V crystal structure. Shape reconstructions from the x-ray scattering data illustrate the nature of this destabilization. Comparison of these scattering data with those for bovine CuZn SOD measured at different temperatures shows that an analogous change in the scattering profile occurs for the bovine enzyme in the temperature range of 70-80 degrees C. These results demonstrate that the A4V and I113T mutants are substantially destabilized in comparison with wild-type SOD1, and it is possible that the pathogenic properties of this subset of familial amyotrophic lateral sclerosis mutants are at least in part due to this destabilization.

Amyotrophic Lateral Sclerosis↗

PubMed2003

Misfolded CuZnSOD and amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease of motor neurons. The inherited form of the disease, familial ALS, represents 5-10% of the total cases, and the best documented of these are due to lesions in SOD1, the gene encoding copper-zinc superoxide dismutase (CuZnSOD). The mechanism by which mutations in SOD1 cause familial ALS is currently unknown. Two hypotheses have dominated recent discussion of the toxicity of ALS mutant CuZnSOD proteins: the oligomerization hypothesis and the oxidative damage hypothesis. The oligomerization hypothesis maintains that mutant CuZnSOD proteins are, or become, misfolded and consequently oligomerize into increasingly high-molecular-weight species that ultimately lead to the death of motor neurons. The oxidative damage hypothesis maintains that ALS mutant CuZnSOD proteins catalyze oxidative reactions that damage substrates critical for viability of the affected cells. This perspective reviews some of the properties of both wild-type and mutant CuZnSOD proteins, suggests how these properties may be relevant to these two hypotheses, and proposes that these two hypotheses are not necessarily mutually exclusive.

Humans↗

PubMed2003

An alternative mechanism of bicarbonate-mediated peroxidation by copper-zinc superoxide dismutase: rates enhanced via proposed enzyme-associated peroxycarbonate intermediate.

Hydrogen peroxide can interact with the active site of copper-zinc superoxide dismutase (SOD1) to generate a powerful oxidant. This oxidant can either damage amino acid residues at the active site, inactivating the enzyme (the self-oxidative pathway), or oxidize substrates exogenous to the active site, preventing inactivation (the external oxidative pathway). It is well established that the presence of bicarbonate anion dramatically enhances the rate of oxidation of exogenous substrates. Here, we show that bicarbonate also substantially enhances the rate of self-inactivation of human wild type SOD1. Together, these observations suggest that the strong oxidant formed by hydrogen peroxide and SOD1 in the presence of bicarbonate arises from a pathway mechanistically distinct from that producing the oxidant in its absence. Self-inactivation rates are further enhanced in a mutant SOD1 protein (L38V) linked to the fatal neurodegenerative disorder, familial amyotrophic lateral sclerosis. The 1.4 A resolution crystal structure of pathogenic SOD1 mutant D125H reveals the mode of oxyanion binding in the active site channel and implies that phosphate anion attenuates the bicarbonate effect by competing for binding to this site. The orientation of the enzyme-associated oxyanion suggests that both the self-oxidative and external oxidative pathways can proceed through an enzyme-associated peroxycarbonate intermediate.

Amyotrophic Lateral Sclerosis↗