Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.     

Zinc binding modulates the entire folding free energy surface of human Cu,Zn superoxide dismutase

Over 100 amino acid replacements in human Cu,Zn superoxide dismutase (SOD) are known to cause amyotrophic lateral sclerosis, a gain-of-function neurodegenerative disease that destroys motor neurons. Supposing that aggregates of partially folded states are primarily responsible for toxicity, we determined the role of the structurally important zinc ion in defining the folding free energy surface of dimeric SOD by comparing the thermodynamic and kinetic folding properties of the zinc-free and zinc-bound forms of the protein. The presence of zinc was found to decrease the free energies of a peptide model of the unfolded monomer, a stable variant of the folded monomeric intermediate, and the folded dimeric species. The unfolded state binds zinc weakly with a micromolar dissociation constant, and the folded monomeric intermediate and the native dimeric form both bind zinc tightly, with subnanomolar dissociation constants. Coupled with the strong driving force for the subunit association reaction, the shift in the populations toward more well-folded states in the presence of zinc decreases the steady-state populations of higher-energy states in SOD under expected in vivo zinc concentrations (approximately nanomolar). The significant decrease in the population of partially folded states is expected to diminish their potential for aggregation and account for the known protective effect of zinc. The ∼ 100-fold increase in the rate of folding of SOD in the presence of micromolar concentrations of zinc demonstrates a significant role for a preorganized zinc-binding loop in the transition-state ensemble for the rate-limiting monomer folding reaction in this β-barrel protein.     

Mapping the folding free energy surface for metal-free human Cu,Zn superoxide dismutase

Mutations at many different sites in the gene encoding human Cu,Zn superoxide dismutase (SOD) are known to be causative agents in amyotrophic lateral sclerosis (ALS). One explanation for the molecular basis of this pathology is the aggregation of marginally soluble, partially structured states whose populations are enhanced in the protein variants. As a benchmark for testing this hypothesis, the equilibrium and kinetic properties of the reversible folding reaction of a metal-free variant of SOD were investigated. Reversibility was achieved by replacing the two non-essential cysteine residues with non-oxidizable analogs, C6A/C111S, to produce apo-AS-SOD. The metal-free pseudo-wild-type protein is folded and dimeric in the absence of chemical denaturants, and its equilibrium folding behavior is well described by an apparent two-state mechanism involving the unfolded monomer and the native dimer. The apparent free energy of folding in the absence of denaturant and at standard state is -20.37(+/- 1.04) kcal (mol dimer)(-1). A global analysis of circular dichroism kinetic traces for both unfolding and refolding reactions, combined with results from small angle X-ray scattering and time-resolved fluorescence anisotropy measurements, supports a sequential mechanism involving the unfolded monomer, a folded monomeric intermediate, and the native dimer. The rate-limiting monomer folding reaction is followed by a near diffusion-limited self-association reaction to form the native dimer. The relative population of the folded monomeric intermediate is predicted not to exceed 0.5% at micromolar concentrations of protein under equilibrium and both strongly unfolding and refolding conditions for metal-free pseudo-wild-type SOD.     

The Folding Free-Energy Surface of HIV-1 Protease: Insights into the Thermodynamic Basis for Resistance to Inhibitors

Spontaneous mutations at numerous sites distant from the active site of human immunodeficiency virus type 1 protease enable resistance to inhibitors while retaining enzymatic activity. As a benchmark for probing the effects of these mutations on the conformational adaptability of this dimeric β-barrel protein, the folding free-energy surface of a pseudo-wild-type variant, HIV-PR^?, was determined by a combination of equilibrium and kinetic experiments on the urea-induced unfolding/refolding reactions. The equilibrium unfolding reaction was well described by a two-state model involving only the native dimeric form and the unfolded monomer. The global analysis of the kinetic folding mechanism reveals the presence of a fully folded monomeric intermediate that associates to form the native dimeric structure. Independent analysis of a stable monomeric version of the protease demonstrated that a small-amplitude fluorescence phase in refolding and unfolding, not included in the global analysis of the dimeric protein, reflects the presence of a transient intermediate in the monomer folding reaction. The partially folded and fully folded monomers are only marginally stable with respect to the unfolded state, and the dimerization reaction provides a modest driving force at micromolar concentrations of protein. The thermodynamic properties of this system are such that mutations can readily shift the equilibrium from the dimeric native state towards weakly folded states that have a lower affinity for inhibitors but that could be induced to bind to their target proteolytic sites. Presumably, subsequent secondary mutations increase the stability of the native dimeric state in these variants and, thereby, optimize the catalytic properties of the resistant human immunodeficiency virus type 1 protease.     

Friction-Limited Folding of Disulfide-Reduced Monomeric SOD1

The folding reaction of a stable monomeric variant of Cu/Zn superoxide dismutase (mSOD1), an enzyme responsible for the conversion of superoxide free radicals into hydrogen peroxide and oxygen, is known to be among the slowest folding processes that adhere to two-state behavior. The long lifetime, ∼10 s, of the unfolded state presents ample opportunities for the polypeptide chain to transiently sample nonnative structures before the formation of the productive folding transition state. We recently observed the formation of a nonnative structure in a peptide model of the C-terminus of SOD1, a sequence that might serve as a potential source of internal chain friction-limited folding. To test for friction-limited folding, we performed a comprehensive thermodynamic and kinetic analysis of the folding mechanism of mSOD1 in the presence of the viscogens glycerol and glucose. Using a, to our knowledge, novel analysis of the folding reactions, we found the disulfide-reduced form of the protein that exposes the C-terminal sequence, but not its disulfide-oxidized counterpart that protects it, experiences internal chain friction during folding. The sensitivity of the internal friction to the disulfide bond status suggests that one or both of the cross-linked regions play a critical role in driving the friction-limited folding. We speculate that the molecular mechanisms giving rise to the internal friction of disulfide-reduced mSOD1 might play a role in the amyotrophic lateral sclerosis-linked aggregation of SOD1.     

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.     

Molecular dynamics of a far positioned SOD1 mutant V14M reveals pathogenic misfolding behavior

Human superoxide dismutase (Cu/Zn SOD1) is a homodimeric enzyme. Mutations in Cu/Zn SOD1 causes a familial form of amyotrophic lateral sclerosis (fALS), and aggregation of mutant SOD1 has been proposed to play a role in neurodegeneration. Though a majority of the mutations are point substitutions, there are a few changes that result in amino acid deletions or truncations of the polypeptide. These pathogenic mutations are scattered throughout the three-dimensional structure of the dimeric enzyme, which creates a puzzling pattern to investigate the molecular determinants of fALS. The most common hypothesis proposed that the misfolding of SOD1 mutants are primarily triggered by decreased affinity for metal ions. However, this hypothesis is challenging, as a significant number of disease-causing mutations are located far away from the metal-binding site and dimer interface. So in the present study, we have investigated the influence of such a far positioned pathogenic mutation, V14M, in altering the stability and folding of the Cu/Zn SOD1. Though the location of Val14 is far positioned, it has a vital role in the stability of SOD1 by preserving its hydrophobic cluster at one end of the β barrel domain. We have performed MD simulations of the V14M mutant for 80 ns timescale. The results reveal the fact that irrespective of its location, V14M mutation triggers a conformational change that is more similar to that of the metal-deficient holo form and could resemble an intermediate state in the folding reaction which results in protein misfolding and aggregation.     

Reversible denaturation of the gene V protein of bacteriophage f1

The guanidine hydrochloride (GuHCl)-induced denaturation of the gene V protein of bacteriophage f1 has been studied, using the chemical reactivity of a cysteine residue that is buried in the folded protein and the circular dichroism (CD) at 211 and 229 nm as measures of the fraction of polypeptide chains in the folded form. It is found that this dimeric protein unfolds in a single cooperative transition from a folded dimer to two unfolded monomers. A folded, monomeric form of the gene V protein was not detected at equilibrium. The kinetics of unfolding of the gene V protein in 3 M GuHCl and the refolding in 2 M GuHCl are also consistent with a transition between a folded dimer and two unfolded monomers. The GuHCl concentration dependence of the rates of folding and unfolding suggests that the transition state for folding is near the folded conformation.     

The Disulfide Bond,but Not Zinc or Dimerization,Controls Initiation and Seeded Growth in Amyotrophic Lateral Sclerosis-linked Cu,Zn Superoxide Dismutase (SOD1) Fibrillation

Aggregation of copper-zinc superoxide dismutase (SOD1) is a defining feature of familial ALS caused by inherited mutations in the sod1 gene, and misfolded and aggregated forms of wild-type SOD1 are found in both sporadic and familial ALS cases. Mature SOD1 owes its exceptional stability to a number of post-translational modifications as follows: formation of the intramolecular disulfide bond, binding of copper and zinc, and dimerization. Loss of stability due to the failure to acquire one or more of these modifications is proposed to lead to aggregation in vivo. Previously, we showed that the presence of apo-, disulfide-reduced SOD1, the most immature form of SOD1, results in initiation of fibrillation of more mature forms that have an intact Cys-57–Cys-146 disulfide bond and are partially metallated. In this study, we examine the ability of each of the above post-translational modifications to modulate fibril initiation and seeded growth. Cobalt or zinc binding, despite conferring great structural stability, neither inhibits the initiation propensity of disulfide-reduced SOD1 nor consistently protects disulfide-oxidized SOD1 from being recruited into growing fibrils across wild-type and a number of ALS mutants. In contrast, reduction of the disulfide bond, known to be necessary for fibril initiation, also allows for faster recruitment during seeded amyloid growth. These results identify separate factors that differently influence seeded growth and initiation and indicate a lack of correlation between the overall thermodynamic stability of partially mature SOD1 states and their ability to initiate fibrillation or be recruited by a growing fibril.     

HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.     

Local unfolding of Cu, Zn superoxide dismutase monomer determines the morphology of fibrillar aggregates

Aggregation of Cu, Zn superoxide dismutase (SOD1) is often found in amyotrophic lateral sclerosis patients. The fibrillar aggregates formed by wild type and various disease-associated mutants have recently been found to have distinct cores and morphologies. Previous computational and experimental studies of wild-type SOD1 suggest that the apo-monomer, highly aggregation prone, displays substantial local unfolding dynamics. The residual folded structure of locally unfolded apoSOD1 corresponds to peptide segments forming the aggregation core as identified by a combination of proteolysis and mass spectroscopy. Therefore, we hypothesize that the destabilization of apoSOD1 caused by various mutations leads to distinct local unfolding dynamics. The partially unfolded structure, exposing the hydrophobic core and backbone hydrogen bond donors and acceptors, is prone to aggregate. The peptide segments in the residual folded structures form the "building block" for aggregation, which in turn determines the morphology of the aggregates. To test this hypothesis, we apply a multiscale simulation approach to study the aggregation of three typical SOD1 variants: wild type, G37R, and I149T. Each of these SOD1 variants has distinct peptide segments forming the core structure and features different aggregate morphologies. We perform atomistic molecular dynamics simulations to study the conformational dynamics of apoSOD1 monomer and coarse-grained molecular dynamics simulations to study the aggregation of partially unfolded SOD1 monomers. Our computational studies of monomer local unfolding and the aggregation of different SOD1 variants are consistent with experiments, supporting the hypothesis of the formation of aggregation "building blocks" via apo-monomer local unfolding as the mechanism of SOD1 fibrillar aggregation.     

Alterations in local stability and dynamics of A4V SOD1 in the presence of trifluoroethanol

Alterations in the local dynamics of Cu/Zn Superoxide dismutase (SOD1) due to mutations affect the protein folding, stability, and function leading to misfolding and aggregation seen in amyotrophic lateral sclerosis (ALS). Here, we study the structure and dynamics of the most devastating ALS mutation, A4V SOD1 in aqueous trifluoroethanol (TFE) through experiments and simulation. Far‐UV circular dichroism (CD) studies shows that TFE at intermediate concentrations (～15% ‐ 30%) induce partially unfolded β‐sheet‐rich extended conformations in A4V SOD1 which subsequently aggregates. Molecular dynamics (MD) simulation results shows that A4V SOD1 increases local dynamics in the active site loops that leads to the destabilization of the β‐barrel and loss of hydrophobic contacts, thus stipulating a basis for aggregation. Free energy landscape (FEL) and essential dynamics (ED) analysis demonstrates the conformational heterogeneity in A4V SOD1. Our results thus shed light on the role of local unfolding and conformational dynamics in aggregation of SOD1.     

Backbone resonance assignments of monomeric SOD1 in dilute and crowded environments

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that leads to movement disorders. In motor neurons of ALS patients, intracellular aggregates of superoxide dismutase 1 (SOD1) have often been observed. To elucidate the aggregation mechanism, it is important to analyze the folding equilibrium of SOD1 between folded and aggregation-prone unfolded states. However, in most cases, this folding equilibrium has been studied in dilute solution even though the aggregate formation occurs in a highly crowded intracellular environment. Indeed, a recent study reported that the folding stability of SOD1 decreased in an environment containing protein crowder molecules. To understand such a destabilization effect due to protein crowders, it is necessary to obtain more precise structural information on SOD1 in the presence of protein crowders. Here, we report the ¹H, ¹³C, and ¹⁵N backbone resonance assignments of monomeric SOD1 in the absence and presence of the protein crowder lysozyme. The chemical shift differences caused by addition of lysozyme suggest that SOD1 associated with lysozyme via negatively charged surfaces. Based on the assigned chemical shifts, the presence of lysozyme has a limited influence on the secondary structure of SOD1. We anticipate that our assignments will provide an important basis for elucidation of the crowding-induced folding destabilization of SOD1.     

Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo- and reduced form of SOD1, leading to unfolding and oxidative aggregation

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 (Tm>70 degrees 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 (Tm<37 degrees 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.     

Polyanion binding accelerates the formation of stable and low‐toxic aggregates of ALS‐linked SOD1 mutant A4V

The toxic property thus far shared by both ALS‐linked SOD1 variants and wild‐type SOD1 is an increased propensity to aggregation. However, whether SOD1 oligomers or aggregates are toxic to cells remains to be well defined. Moreover, how the toxic SOD1 species are removed from intra‐ and extracellular environments also needs to be further explored. The DNA binding has been shown to be capable of accelerating the aggregatio\n of wild‐type and oxidized SOD1 forms under acidic and neutral conditions. In this study, we explore the binding of DNA and heparin, two types of essential life polyanions, to A4V, an ALS‐linked SOD1 mutant, under acidic conditions, and its consequences. The polyanion binding alters the A4V conformation, neutralizes its local positive charges, and increases its local concentrations along the polyanion chain, which are sufficient to lead to acceleration of the pH‐dependent A4V aggregation. The accelerated aggregation, which is ascribed to the polyanion binding‐mediated removal or shortening of the lag phase in aggregation, contributes to the formation of amorphous A4V nanoparticles. The prolonged incubation with polyanions not only results in the complete conversion of likely soluble toxic A4V oligomers into non‐ and low‐toxic SDS‐resistant aggregates, but also increases their stability. Although this is only an initial step toward reducing the toxicity of SOD1 mutants, the accelerating role of polyanions in protein aggregation might become one of the rapid pathways that remove toxic forms of SOD1 mutants from intra‐ and extracellular environments. Proteins 2014; 82:3356–3372. © 2014 Wiley Periodicals, Inc.     

Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 μM, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.     

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.     

Mutational Studies Uncover Non-Native Structure in the Dimeric Kinetic Intermediate of the H2A-H2B Heterodimer

The folding pathway of the histone H2A-H2B heterodimer minimally includes an on-pathway, dimeric, burst-phase intermediate, I₂. The partially folded H2A and H2B monomers populated at equilibrium were characterized as potential monomeric kinetic intermediates. Folding kinetics were compared for initiation from isolated, folded monomers and the heterodimer unfolded in 4 M urea. The observed rates were virtually identical above 0.4 M urea, exhibiting a log-linear relationship on the final denaturant concentration. Below ∼ 0.4 M urea (concentrations inaccessible from the  4-M urea unfolded state), a rollover in the rates was observed; this suggests that a component of the I₂ ensemble contains non-native structure that rearranges/isomerizes to a more native-like species. The contribution of helix propensity to the stability of the I₂ ensemble was assessed with a set of H2A-H2B mutants containing Ala and Gly replacements at nine sites, focusing mainly on the long, central α2 helix. Equilibrium and kinetic folding/unfolding data were collected to determine the effects of the mutations on the stability of I₂ and the transition state between I₂ and N₂. This limited mutational study indicated that residues in the α2 helices of H2A and H2B as well as α1 of H2B and both the C-terminus of α3 and the short αC helix of H2A contribute to the stability of the I₂ burst-phase species. Interestingly, at least eight of the nine targeted residues stabilize I₂ by interactions that are non-native to some extent. Given that destabilizing I₂ and these non-native interactions does not accelerate folding, it is concluded that the native and non-native structures present in the I₂ ensemble enable efficient folding of H2A-H2B.     

Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis

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 ( approximately 40 microm) 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.