Folding of Cu, Zn superoxide dismutase and familial amyotrophic lateral sclerosis

Cu, Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.     

Unfolding and folding kinetics of amyotrophic lateral sclerosis-associated mutant Cu,Zn superoxide dismutases

More than 110 mutations in dimeric, Cu,Zn superoxide dismutase (SOD) have been linked to the fatal neurodegenerative disease, amyotrophic lateral sclerosis (ALS). In both human patients and mouse model studies, protein misfolding has been implicated in disease pathogenesis. A central step in understanding the misfolding/aggregation mechanism of this protein is the elucidation of the folding pathway of SOD. Here we report a systematic analyses of unfolding and folding kinetics using single- and double-jump experiments as well as measurements as a function of guanidium chloride, protein, and metal concentration for fully metallated (holo) pseudo wild-type and ALS-associated mutant (E100G, G93R, G93A, and metal binding mutants G85R and H46R) SODs. The kinetic mechanism for holo SODs involves native dimer, monomer intermediate, and unfolded monomer, with variable metal dissociation from the monomeric states depending on solution conditions. The effects of the ALS mutations on the kinetics of the holoproteins in guanidium chloride are markedly different from those observed previously for acid-induced unfolding and for the unmetallated (apo) forms of the proteins. The mutations decrease the stability of holo SOD mainly by increasing unfolding rates, which is particularly pronounced for the metal-binding mutants, and have relatively smaller effects on the observed folding kinetics. Mutations also seem to favour increased formation of a Zn-free monomer intermediate, which has been implicated in the formation of toxic aggregates. The results reveal the kinetic basis for the extremely high stability of wild-type holo SOD and the possible consequences of kinetic changes for disease.     

Structural and thermodynamic effects of post-translational modifications in mutant and wild type Cu, Zn superoxide dismutase

Aggregation of Cu,Zn superoxide dismutase (SOD1) is implicated in amyotrophic lateral sclerosis. Glutathionylation and phosphorylation of SOD1 is omnipresent in the human body, even in healthy individuals, and has been shown to increase SOD1 dimer dissociation, which is the first step on the pathway toward SOD1 aggregation. We found that post-translational modification of SOD1, especially glutathionylation, promotes dimer dissociation. We discovered an intermediate state in the pathway to dissociation, a conformational change that involves a “loosening” of the β-barrels and a loss or shift of dimer interface interactions. In modified SOD1, this intermediate state is stabilized as compared to unmodified SOD1. The presence of post-translational modifications could explain the environmental factors involved in the speed of disease progression. Because post-translational modifications such as glutathionylation are often induced by oxidative stress, post-translational modification of SOD1 could be a factor in the occurrence of sporadic cases of amyotrophic lateral sclerosis, which represent 90% of all cases of the disease.     

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.     

Oxidative inactivation of calcineurin by Cu,Zn superoxide dismutase G93A, a mutant typical of familial amyotrophic lateral sclerosis

Calcineurin is a serine/threonine phosphatase involved in a wide range of cellular responses to calcium mobilizing signals. Previous evidence supports the notion of the existence of a redox regulation of this enzyme, which might be relevant for neurodegenerative processes, where an imbalance between generation and removal of reactive oxygen species could occur. In a recent work, we have observed that calcineurin activity is depressed in two models for familial amyotrophic lateral sclerosis (FALS) associated with mutations of the antioxidant enzyme Cu,Zn superoxide dismutase (SOD1), namely in neuroblastoma cells expressing either SOD1 mutant G93A or mutant H46R and in brain areas from G93A transgenic mice. In this work we report that while wild-type SOD1 has a protective effect, calcineurin is oxidatively inactivated by mutant SOD1s in vitro; this inactivation is mediated by reactive oxygen species and can be reverted by addition of reducing agents. Furthermore, we show that calcineurin is sensitive to oxidation only when it is in an 'open', calcium-activated conformation, and that G93A-SOD1 must have its redox-active copper site available to substrates in order to exert its pro-oxidant properties on calcineurin. These findings demonstrate that both wild-type and mutant SOD1s can interfere directly with calcineurin activity and further support the possibility of a relevant role for calcineurin-regulated biochemical pathways in the pathogenesis of FALS.     

A misfolded dimer of Cu/Zn‐superoxide dismutase leading to pathological oligomerization in amyotrophic lateral sclerosis

Misfolding of mutant Cu/Zn‐superoxide dismutase (SOD1) is a pathological hallmark in a familial form of amyotrophic lateral sclerosis. Pathogenic mutations have been proposed to monomerize SOD1 normally adopting a homodimeric configuration and then trigger abnormal oligomerization of SOD1 proteins. Despite this, a misfolded conformation of SOD1 leading to the oligomerization at physiological conditions still remains ambiguous. Here, we show that, around the body temperature (～37°C), mutant SOD1 maintains a dimeric configuration but lacks most of its secondary structures. Also, such an abnormal SOD1 dimer with significant structural disorder was prone to irreversibly forming the oligomers crosslinked via disulfide bonds. The disulfide‐crosslinked oligomers of SOD1 were detected in the spinal cords of the diseased mice expressing mutant SOD1. We hence propose an alternative pathway of mutant SOD1 misfolding that is responsible for oligomerization in the pathologies of the disease.     

Differential role of superoxide and glutathione in S‐nitrosoglutathione-mediated apoptosis: a rationale for mild forms of familial amyotrophic lateral sclerosis associated with less active Cu,Zn superoxide dismutase mutants

SH-SY5Y cells transfected with the enzymatically inactive Cu,Zn superoxide dismutase mutant H46R were more resistant to S-nitrosoglutathione (GSNO)-induced apoptosis. Cytochrome c release from mitochondria, caspase 3 activation, p53 up-regulation, p21 cleavage and Bcl-2 modulation, all involved in the apoptotic process, were significantly less altered with respect to untransfected cells. The H46R resistance to NO was associated with a higher content of reduced glutathione (GSH) and was abolished by blockage of glutathione synthesis. On the other hand, H46R cells were as sensitive as SH-SY5Y cells to puromycin-induced apoptosis; furthermore, they were more susceptible to apoptosis elicited by the superoxide-generating drug paraquat and to cell necrosis provoked by t-butyl hydroperoxide. These results confirm that the level of superoxide dismutase activity is fundamental for protecting cells against oxygen free radical challenge. Its impairment is not detrimental to cells exposed to NO, as long as the overall reducing power represented by GSH is assured. These results are relevant to explain a milder progression of the familial amyotrophic lateral sclerosis disease when associated with the H46R mutation.     

An RNAi strategy for treatment of amyotrophic lateral sclerosis caused by mutant Cu,Zn superoxide dismutase

Amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease) is a neurodegenerative disease characterized by motor neuron degeneration, paralysis and death. One cause of this disease is mutations in the Cu,Zn superoxide dismutase (SOD1) gene. As mutant SOD1 acquires a toxic property that kills motor neurons, by reducing the mutant protein the disease progression may be slowed or prevented. While mutant SOD1 is toxic, the wild-type SOD1 is indispensable for motor neuron health. Therefore, the ideal therapeutic strategy would be to inhibit selectively the mutant protein expression. Previously we have demonstrated that RNA interference (RNAi) can selectively inhibit some mutant SOD1 expression. However, more than 100 SOD1 mutants can cause ALS and all mutants cannot be inhibited selectively by RNAi. To overcome this obstacle, we have designed a replacement RNAi strategy. Using this strategy, all mutants and wild-type genes are inhibited by RNAi. The wild-type SOD1 function is then replaced by designed wild-type SOD1 genes that are resistant to the RNAi. Here we demonstrate the concept of this strategy.     

Denaturational stress induces formation of zinc-deficient monomers of Cu,Zn superoxide dismutase: implications for pathogenesis in amyotrophic lateral sclerosis

Mutations in the Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis cases. SOD1 is a homodimer in which each monomer binds one copper atom and one zinc atom. Mutation is believed to increase the conformational flexibility of SOD1, giving rise to a misfolded SOD1 population with novel cytotoxic properties. While SOD1's metal ligands affect its stability greatly, little is known about the role these metals play in the folding, unfolding, and misfolding processes. Here, we present a method by which we were able to measure the rates of metal release during SOD1 unfolding in guanidine hydrochloride. Rates of dimer dissociation, measured by a time-resolved cross-linking assay, and conformational changes in SOD1's β-barrel core, monitored by tryptophan fluorescence intensity, were compared with the rates of copper release and zinc release. Correlations were observed across a range of denaturant concentrations, giving rise to a more detailed model of the SOD1 unfolding process than was previously available. According to this model, the major unfolding pathway involves simultaneous dimer dissociation and zinc release as an early step that is followed by a slow conformational change in the protein's core, which, in turn, is followed by rapid copper release. This model establishes a zinc-deficient, copper-loaded SOD1 monomer as a well-populated SOD1 unfolding intermediate and a species likely to be populated under conditions of denaturational stress. Because the cytotoxicity of zinc-deficient SOD1 has been demonstrated previously, this species is a good candidate for the cytotoxic species in SOD1-associated amyotrophic lateral sclerosis.     

Selective loss of neurofilament expression in Cu/Zn superoxide dismutase (SOD1) linked amyotrophic lateral sclerosis

Neurofilament pathology is a hallmark of sporadic and familial amyotrophic lateral sclerosis (SALS and FALS). The disease mechanisms underlying this pathology are presently unclear, but recent evidence in SALS patients suggest that reductions in neurofilament light subunit (NFL) mRNA may contribute to the death of motor neurones. Mutations in the gene encoding Cu-Zn superoxide dismutase (SOD1) represent the best-studied cause of FALS, and a number of laboratory models of SOD1-mediated disease exist. Here we have used microdissected lumbar spinal cord motor neurones from human SOD1 FALS patients as well as G93A SOD1 transgenic mice and demonstrated that reduced NFL mRNA levels are seen in both. To probe the molecular mechanisms underpinning these observations, we generated NSC34 motor neurone-like cell lines expressing wild-type and mutant SOD1. NSC34 cells expressing G37R or G93A SOD1 showed selective reductions in NFL and NFM mRNA and protein. These data suggest that NFL mRNA reductions are common to SALS and FALS patients, and that cells and mice expressing mutant SOD1 may enable us to characterize the molecular mechanism(s) responsible for the loss of neurofilament mRNA.     

Sequence and structural determinants of Cu, Zn superoxide dismutase aggregation

Diverse point mutations in the enzyme Cu, Zn superoxide dismutase (SOD1) are linked to its aggregation in the familial form of the disease amyotrophic lateral sclerosis. The disease-associated mutations are known to destabilize the protein, but the structural basis of the aggregation of the destabilized protein and the structure of aggregates are not well understood. Here, we investigate in silico the sequence and structural determinants of SOD1 aggregation: (1) We identify sequence fragments in SOD1 that have a high aggregation propensity, using only the sequence of SOD1, and (2) we perform molecular dynamics simulations of the SOD1 dimer folding and misfolding. In both cases, we identify identical regions of the protein as having high propensity to form intermolecular interactions. These regions correspond to the N- and C-termini, and two crossover loops and two beta-strands in the Greek-key native fold of SOD1. Our results suggest that the high aggregation propensity of mutant SOD1 may result from a synergy of two factors: the presence of highly amyloidogenic sequence fragments ("hot spots"), and the presence of these fragments in regions of the protein that are structurally most likely to form intermolecular contacts under destabilizing conditions. Therefore, we postulate that the balance between the self-association of aggregation-prone sequences and the specific structural context of these sequences in the native state determines the aggregation propensity of proteins.     

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.     

The coupling between disulphide status, metallation and dimer interface strength in Cu/Zn superoxide dismutase

The gain of neurotoxic function in amyotrophic lateral sclerosis (ALS) has been linked to misfolding of the homodimeric enzyme Cu/Zn superoxide dismutase (SOD). Here, we present the crystal structure of fully cysteine-depleted human SOD (SOD(CallA)), representing a reduced, marginally stable intermediate on the folding pathway in vivo that has also been implicated as neurotoxic precursor state. A hallmark of this species is that it fails to dimerize and becomes trapped as a monomer in the absence of the active-site metals. The crystallographic data show that removal of the C57-C146 disulphide bond sets free the interface loop IV in the apo protein, whereas the same loop remains unaffected in the holo protein. Thus, the low dimerisation propensity of disulphide-reduced apoSOD seems to be of entropic origin due to increased loop flexibility in the monomeric state: in the disulphide-reduced holo protein this gain in configurational entropy upon splitting of the dimer interface is reduced by the metal coordination.     

Effect of overexpression of wild-type and mutant Cu/Zn-superoxide dismutases on oxidative damage and antioxidant defences: relevance to Down's syndrome and familial amyotrophic lateral sclerosis

Patients with Down's syndrome (DS) show elevated levels of copper, zinc-containing superoxide dismutase (SOD1) and appear to have increased lipid peroxidation and oxidative damage to DNA as well as elevated glutathione peroxidase activity. Increasing SOD1 levels by gene transfection in NT-2 and SK-N-MC cell lines also led to a rise in glutathione peroxidase activity, but this was nevertheless accompanied by decreased proliferation rates, increased lipid peroxidation and protein carbonyls, and a trend to a rise in 8-hydroxyguanine and protein-bound 3-nitrotyrosine. Transfection of these cell lines with DNA encoding two mutant SOD1 enzymes (G37R and G85R) associated with familial amyotrophic lateral sclerosis (FALS), produced similar, but more severe changes, i.e. even lower growth rates, higher lipid peroxidation, 3-nitrotyrosine and protein carbonyl levels, decreased GSH levels, raised GSSG levels and higher glutathione peroxidase activities. Since G85R has little SOD activity, these changes cannot be related to increased O(2)(-) scavenging. In no case was SOD2 (mitochondrial Mn-SOD) level altered. Our cellular systems reproduce many of the biochemical changes observed in patients with DS or ALS, and in transgenic mice overexpressing mutant SOD1. They also show the potentially deleterious effects of SOD1 overexpression on cellular proliferation, which may be relevant to abnormal development in DS.     

Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS

A dominant mutation in the gene for copper-zinc superoxide dismutase (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis. Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Exploiting both affinity purification and mass spectrometry, we identified a novel interaction between heat-shock protein 105 (Hsp105) and mutant SOD1. We detected this interaction both in spinal cord extracts of mutant SOD1(G93A) transgenic mice and in cultured neuroblastoma cells. Expression of Hsp105, which is found in mouse motor neurons, was depressed in the spinal cords of SOD1(G93A) mice as disease progressed, while levels of expression of two other heat-shock proteins, Hsp70 and Hsp27, were elevated. Moreover, Hsp105 suppressed the formation of mutant SOD1-containing aggregates in cultured cells. These results suggest that techniques that raise levels of Hsp105 might be promising tools for alleviation of the mutant SOD1 toxicity.     

No correlation between aggregates of Cu/Zn superoxide dismutase and cell death in familial amyotrophic lateral sclerosis

Aggregates of Cu/Zn superoxide dismutase (SOD) have been demonstrated in familial amyotrophic lateral sclerosis (FALS) and other neurodegenerative diseases; however, their role in disease pathogenesis is unclear. In this study, we investigated the presence of SOD aggregates in nerve growth factor (NGF)-differentiated PC12 cells and cell viability following: (i) transduction with replication-deficient recombinant adenoviruses (AdVs) expressing wild-type SOD (SODWT) or mutant SOD (SODMT, V148G or A4V); (ii) transfection of yellow fluorescent protein-tagged SODWT (SODWT-YFP) or SODMT (SODA4V-YFP, SODV148G-YFP). SOD aggregates were more prominent in cells following transduction of AdSODMT than AdSODWT and following treatment with H2O2, suggesting that mutant SOD leads to oxidation of cellular components. In addition, cells expressing SODMT-YFP yielded SOD aggregates that were significantly larger and more frequent than SOD aggregates in cells expressing SODWT-YFP. Proteasome inhibitors, but not cathepsin B inhibitors, increased aggregate formation but did not increase cell death. In addition, treatments that increased cell viability did not significantly decrease SOD aggregates. Taken together, our data demonstrate that there is no association between SOD aggregates and cell death in FALS.     

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values

This article utilized “protein charge ladders”—chemical derivatives of proteins with similar structure, but systematically altered net charge—to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn²⁺ stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude—by up to 7.4 units per dimer at lysosomal pH—than values predicted from standard pK_a values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn²⁺ to mutant SOD1 lowered its net charge by an additional +2.33 ± 0.01 to +3.18 ± 0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn²⁺ (ΔZ < 0.44 ± 0.07 per additional Zn²⁺). Both metalated and apo-SOD1 regulated net charge across subcellular pH, without inverting from negative to positive at the theoretical pI. Differential scanning calorimetry, hydrogen-deuterium exchange, and inductively coupled plasma mass spectrometry confirmed that the structure, stability, and metal content of mutant proteins were not significantly affected by lysine acetylation. Measured values of net charge should be used when correlating the biophysical properties of a specific ALS-variant SOD1 protein with its observed aggregation propensity or clinical phenotype.     

Dorfin prevents cell death by reducing mitochondrial localizing mutant superoxide dismutase 1 in a neuronal cell model of familial amyotrophic lateral sclerosis

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.     

Prion-like activity of Cu/Zn superoxide dismutase

Neurodegenerative diseases belong to a larger group of protein misfolding disorders, known as proteinopathies. There is increasing experimental evidence implicating prion-like mechanisms in many common neurodegenerative disorders, including Alzheimer disease, Parkinson disease, the tauopathies, and amyotrophic lateral sclerosis (ALS), all of which feature the aberrant misfolding and aggregation of specific proteins. The prion paradigm provides a mechanism by which a mutant or wild-type protein can dominate pathogenesis through the initiation of self-propagating protein misfolding. ALS, a lethal disease characterized by progressive degeneration of motor neurons is understood as a classical proteinopathy; the disease is typified by the formation of inclusions consisting of aggregated protein within and around motor neurons that can contribute to neurotoxicity. It is well established that misfolded/oxidized SOD1 protein is highly toxic to motor neurons and plays a prominent role in the pathology of ALS. Recent work has identified propagated protein misfolding properties in both mutant and wild-type SOD1, which may provide the molecular basis for the clinically observed contiguous spread of the disease through the neuroaxis. In this review we examine the current state of knowledge regarding the prion-like properties of SOD1 and comment on its proposed mechanisms of intercellular transmission.