Amyotrophic lateral sclerosis-associated copper/zinc superoxide dismutase mutations preferentially reduce the repulsive charge of the proteins

We provide bioinformatical evidence that protein charge plays a key role in the disease mechanism of amyotrophic lateral sclerosis (ALS). Analysis of 100 ALS-associated mutations in copper/zinc superoxide dismutase (SOD1) shows that these are site-selective with a preference to decrease the proteins' net repulsive charge. For each SOD1 monomer this charge is normally -6. Because biomolecules as a rule maintain net negative charge to assure solubility in the cellular interior, the result lends support to the hypothesis of protein aggregation as an initiating event in the ALS pathogenesis. The strength of the preferential reduction of repulsive charge is higher in SOD1-associated ALS than in other inherited protein disorders.     

Cutting off functional loops from homodimeric enzyme superoxide dismutase 1 (SOD1) leaves monomeric β-barrels

Demetallation of the homodimeric enzyme Cu/Zn-superoxide dismutase (SOD1) is known to unleash pronounced dynamic motions in the long active-site loops that comprise almost a third of the folded structure. The resulting apo species, which shows increased propensity to aggregate, stands out as the prime disease precursor in amyotrophic lateral sclerosis (ALS). Even so, the detailed structural properties of the apoSOD1 framework have remained elusive and controversial. In this study, we examine the structural interplay between the central apoSOD1 barrel and the active-site loops by simply cutting them off; loops IV and VII were substituted with short Gly-Ala-Gly linkers. The results show that loop removal breaks the dimer interface and leads to soluble, monomeric β-barrels with high structural integrity. NMR-detected nuclear Overhauser effects are found between all of the constituent β-strands, confirming ordered interactions across the whole barrel. Moreover, the breathing motions of the SOD1 barrel are overall insensitive to loop removal and yield hydrogen/deuterium protection factors typical for cooperatively folded proteins (i.e. the active-site loops act as a "bolt-on" domain with little dynamic influence on its structural foundation). The sole exceptions are the relatively low protection factors in β-strand 5 and the turn around Gly-93, a hot spot for ALS-provoking mutations, which decrease even further upon loop removal. Taken together, these data suggest that the cytotoxic function of apoSOD1 does not emerge from its folded ground state but from a high energy intermediate or even from the denatured ensemble.     

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.     

A copper chaperone–mimetic polytherapy for SOD1-associated amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease in which motor neurons progressively and rapidly degenerate, eventually leading to death. The first protein found to contain ALS-associated mutations was copper/zinc superoxide dismutase 1 (SOD1), which is conformationally stable when it contains its metal ligands and has formed its native intramolecular disulfide. Mutations in SOD1 reduce protein folding stability via disruption of metal binding and/or disulfide formation, resulting in misfolding, aggregation, and ultimately cellular toxicity. A great deal of effort has focused on preventing the misfolding and aggregation of SOD1 as a potential therapy for ALS; however, the results have been mixed. Here, we utilize a small-molecule polytherapy of diacetylbis(N(4)-methylthiosemicarbazonato)copper(II) (CuATSM) and ebselen to mimic the metal delivery and disulfide bond promoting activity of the cellular chaperone of SOD1, the “copper chaperone for SOD1.” Using microscopy with automated image analysis, we find that polytherapy using CuATSM and ebselen is highly effective and acts in synergy to reduce inclusion formation in a cell model of SOD1 aggregation for multiple ALS-associated mutants. Polytherapy reduces mutant SOD1-associated cell death, as measured by live-cell microscopy. Measuring dismutase activity via zymography and immunoblotting for disulfide formation showed that polytherapy promoted more effective maturation of transfected SOD1 variants beyond either compound alone. Our data suggest that a polytherapy of CuATSM and ebselen may merit more study as an effective method of treating SOD1-associated ALS.     

Solid-state NMR studies of metal-free SOD1 fibrillar structures

Copper–zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90 %) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSODΔIV–VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central β-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.     

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.     

Genotype-Property Patient-Phenotype Relations Suggest that Proteome Exhaustion Can Cause Amyotrophic Lateral Sclerosis

Late-onset neurodegenerative diseases remain poorly understood as search continues for the perceived pathogenic protein species. Previously, variants in Superoxide Dismutase 1 (SOD1) causing Amyotrophic Lateral Sclerosis (ALS) were found to destabilize and reduce net charge, suggesting a pathogenic aggregation mechanism. This paper reports analysis of compiled patient data and experimental and computed protein properties for variants of human SOD1, a major risk factor of ALS. Both stability and reduced net charge correlate significantly with disease, with larger significance than previously observed. Using two independent methods and two data sets, a probability < 3% (t-statistical test) is found that ALS-causing mutations share average stability with all possible 2907 SOD1 mutations. Most importantly, un-weighted patient survival times correlate strongly with the misfolded/unfolded protein copy number, expressed as an exponential function of the experimental stabilities (R ² = 0.31, p = 0.002), and this phenotype is further aggravated by charge (R ² = 0.51, p = 1.8 x 10−5). This finding suggests that disease relates to the copy number of misfolded proteins. Exhaustion of motor neurons due to expensive protein turnover of misfolded protein copies is consistent with the data but can further explain e.g. the expression-dependence of SOD1 pathogenicity, the lack of identification of a molecular toxic mode, elevated SOD1 mRNA levels in sporadic ALS, bioenergetic effects and increased resting energy expenditure in ALS patients, genetic risk factors affecting RNA metabolism, and recent findings that a SOD1 mutant becomes toxic when proteasome activity is recovered after washout of a proteasome inhibitor. Proteome exhaustion is also consistent with energy-producing mitochondria accumulating at the neuromuscular junctions where ALS often initiates. If true, this exhaustion mechanism implies a complete change of focus in treatment of ALS towards actively nursing the energy state and protein turnover of the motor neurons.     

Using yeast models to probe the molecular basis of amyotrophic lateral sclerosis

ALS (amyotrophic lateral sclerosis) is a fatal neurodegenerative disease attributable to the death of motor neurons. Associated with ALS are mutations in the genes encoding SOD1 (superoxide dismutase 1), FUS (fused in Sarcoma) protein and TDP-43 (TAR DNA-binding protein-43) each of which leads to aggregation of the respective protein. For example, the ALS-associated mutations in the hSOD1 (human SOD1) gene typically destabilize the native SOD homodimer, leading to misfolding, aggregation and degradation of SOD1. The ALS-associated pathology is not a consequence of the functional inactivation of SOD1 itself, but is rather due to a toxic gain-of-function triggered by mutant SOD1. Recently, the molecular basis of a number of human neurodegenerative diseases resulting from protein misfolding and aggregation, including fALS (familial ALS), was probed by using the baker's yeast, Saccharomyces cerevisiae, as a highly tractable model. Such studies have, for example, identified novel mutant SOD1-specific interactions and demonstrated that mutant SOD1 disrupts mitochondrial homoeostasis. Features of ALS associated with TDP-43 aggregation have also been recapitulated in S. cerevisiae including the identification of modulators of the toxicity of TDP-43. In this paper, we review recent studies of ALS pathogenesis using S. cerevisiae as a model organism and summarize the potential mechanisms involved in ALS progression.     

Salting the charged surface: pH and salt dependence of protein G B1 stability

This study shows significant effects of protein surface charges on stability and these effects are not eliminated by salt screening. The stability for a variant of protein G B1 domain was studied in the pH-range of 1.5-11 at low, 0.15 M, and 2 M salt. The variant has three mutations, T2Q, N8D, and N37D, to guarantee an intact covalent chain at all pH values. The stability of the protein shows distinct pH dependence with the highest stability close to the isoelectric point. The stability is pH-dependent at all three NaCl concentrations, indicating that interactions involving charged residues are important at all three conditions. We find that 2 M salt stabilizes the protein at low pH (protein net charge is +6 and total number of charges is 6) but not at high pH (net charge is or=18). Furthermore, 0.15 M salt slightly decreases the stability of the protein over the pH range. The results show that a net charge of the protein is destabilizing and indicate that proteins contain charges for reasons other than improved stability. Salt seems to reduce the electrostatic contributions to stability under conditions with few total charges, but cannot eliminate electrostatic effects in highly charged systems.     

Nonamyloid aggregates arising from mature copper/zinc superoxide dismutases resemble those observed in amyotrophic lateral sclerosis

Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS) where aggregation of copper/zinc superoxide dismutase (SOD1) is implicated in pathogenesis. We report here that fully metallated (holo) SOD1 under physiologically relevant solution conditions can undergo changes in metallation and/or dimerization over time and form aggregates that do not exhibit classical characteristics of amyloid. The relevance of the observed aggregation to disease is demonstrated by structural and tinctorial analyses, including the novel observation of binding of an anti-SOD1 antibody that specifically recognizes aggregates in ALS patients and mice models. ALS-associated SOD1 mutations can promote aggregation but are not essential. The SOD1 aggregation is characterized by a lag phase, which is diminished by self- or cross-seeding and by heterogeneous nucleation. We interpret these findings in terms of an expanded aggregation mechanism consistent with other in vitro and in vivo findings that point to multiple pathways for the formation of toxic aggregates by different forms of SOD1.     

Disulfide Scrambling in Superoxide Dismutase 1 Reduces Its Cytotoxic Effect in Cultured Cells and Promotes Protein Aggregation

Mutations in the gene coding for superoxide dismutase 1 (SOD1) are associated with familiar forms of the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These mutations are believed to result in a “gain of toxic function”, leading to neuronal degeneration. The exact mechanism is still unknown, but misfolding/aggregation events are generally acknowledged as important pathological events in this process. Recently, we observed that demetallated apoSOD1, with cysteine 6 and 111 substituted for alanine, is toxic to cultured neuroblastoma cells. This toxicity depended on an intact, high affinity Zn²⁺ site. It was therefor contradictory to discover that wild-type apoSOD1 was not toxic, despite of its high affinity for Zn²⁺. This inconsistency was hypothesized to originate from erroneous disulfide formation involving C6 and C111. Using high resolution non-reducing SDS-PAGE, we have in this study demonstrated that the inability of wild-type apoSOD1 to cause cell death stems from formation of non-native intra-molecular disulfides. Moreover, monomeric apoSOD1 variants capable of such disulfide scrambling aggregated into ThT positive oligomers under physiological conditions without agitation. The oligomers were stabilized by inter-molecular disulfides and morphologically resembled what has in other neurodegenerative diseases been termed protofibrils. Disulfide scrambling thus appears to be an important event for misfolding and aggregation of SOD1, but may also be significant for protein function involving cysteines, e.g. mitochondrial import and copper loading.     

A double point mutation of SOD1 targeting net charge promotes aggregation under destabilizing conditions: Correlation of charge distribution and ALS-provoking mutation

A progressive neurodegenerative illness such as amyotrophic lateral sclerosis (ALS) is characterized by the misfolding and aggregation of human CuZn superoxide dismutase (hSOD1) into amyloid aggregates. Thus, designing strategies for the choice of WT-SOD1 and double mutant (G12D/G138E) with an increased net negative charge can be a good idea to elucidate the pathological mechanism of SOD1 in ALS under some destabilizing conditions. Consequently, we show evidence that protein charge, together with other destabilizing conditions, plays an important role in ALS disease. To achieve this purpose, we use methods, such as spectroscopy and transmission electron microscopy (TEM) to monitor the formation of amyloid aggregation. The specific activity of WT-SOD1 was approximately 1.72 times higher than that of the double mutant. Under amyloidogenic circumstances, structural properties such as local, secondary, oligomeric, and fibrillar structures were explored. The double mutant's far-UV CD spectra displayed a broad minimum peak in the region 213 to 218 nm, suggesting the production of β-rich amyloid fibrils. FTIR spectra of the double mutant samples at different incubation times showed a low-frequency peak around 1630–1640 cm^-1, attributed to a parallel β-sheet. Moreover, CR-binding assay and TEM analysis revealed and confirmed that mutation with an increased repulsive charge promotes the formation of fibrous aggregates. Consequently, ALS mutations with a higher repulsive charge are the apparent exceptions that validate the rule. This findings revealed that the double mutant increases protein aggregation through a novel mechanism, likely involving destabilization of structure and a change in the net negative charge.     

Effect of Metal Loading and Subcellular pH on Net Charge of Superoxide Dismutase-1

The net charge of a folded protein is hypothesized to influence myriad biochemical processes (e.g., protein misfolding, electron transfer, molecular recognition); however, few tools exist for measuring net charge and this elusive property remains undetermined—at any pH—for nearly all proteins. This study used lysine-acetyl “protein charge ladders” and capillary electrophoresis to measure the net charge of superoxide dismutase-1 (SOD1)—whose aggregation causes amyotrophic lateral sclerosis (ALS)—as a function of coordinated metal ions and pH. The net negative charge of apo-SOD1 was similar to predicted values; however, the binding of a single Zn^2 + or Cu^2 + ion reduced the net negative charge by a greater magnitude than predicted (i.e., ~ 4 units, instead of 2), whereas the SOD1 protein underwent charge regulation upon binding 2–4 metal ions. From pH5 to pH8 (i.e., a range consistent with the multiple subcellular loci of SOD1), the holo-SOD1 protein underwent smaller fluctuations in net negative charge than predicted (i.e., ~ 3 units, instead of ~ 14) and did not undergo charge inversion at its isoelectric point (pI = 5.3) but remained anionic. The regulation of SOD1 net charge along its pathways of metal binding, and across solvent pH, provides insight into its metal-induced maturation and enzymatic activity (which remains diffusion-limited across pH5–8). The anionic nature of holo-SOD1 across subcellular pH suggests that ~ 45 different ALS-linked mutations to SOD1 will reduce its net negative charge regardless of subcellular localization.     

Dimerization,Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.     

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.     

Disease-associated mutations in SOD1 are impervious to dominant positive or negative effects

The familial form of amyotrophic lateral sclerosis is caused by mutations in the SOD1 gene encoding the cytosolic antioxidant enzyme Cu,Zn superoxide dismutase. Although there is no clear correlation between disease and dismutating catalytic activity among the various disease-associated SOD1 alleles, all of the known missense mutations significantly alter the half-life of the encoded polypeptides. Using transient transfection studies in mammalian cells, it was demonstrated that a frameshift mutation in SOD1 which results in a truncated polypeptide is similarly destabilized. Using an epitope-tagging strategy to discriminate between mutant and wild-type SOD1 polypeptides, no evidence for dominant effects on polypeptide stability was detected, including that of a positive effect of the wild-type on mutant SOD1 polypeptides or that of a negative effect of mutant on wild-type SOD1 polypeptides. These experiments thus favor a non-catalytic role of mutant forms of SOD1 in disease progression.     

Cytotoxicity of superoxide dismutase 1 in cultured cells is linked to Zn2+ chelation

Neurodegeneration in protein-misfolding disease is generally assigned to toxic function of small, soluble protein aggregates. Largely, these assignments are based on observations of cultured neural cells where the suspect protein material is titrated directly into the growth medium. In the present study, we use this approach to shed light on the cytotoxic action of the metalloenzyme Cu/Zn superoxide dismutase 1 (SOD1), associated with misfolding and aggregation in amyotrophic lateral sclerosis (ALS). The results show, somewhat unexpectedly, that the toxic species of SOD1 in this type of experimental setting is not an aggregate, as typically observed for proteins implicated in other neuro-degenerative diseases, but the folded and fully soluble apo protein. Moreover, we demonstrate that the toxic action of apoSOD1 relies on the protein's ability to chelate Zn(2+) ions from the growth medium. The decreased cell viability that accompanies this extraction is presumably based on disturbed Zn(2+) homeostasis. Consistently, mutations that cause global unfolding of the apoSOD1 molecule or otherwise reduce its Zn(2+) affinity abolish completely the cytotoxic response. So does the addition of surplus Zn(2+). Taken together, these observations point at a case where the toxic response of cultured cells might not be related to human pathology but stems from the intrinsic limitations of a simplified cell model. There are several ways proteins can kill cultured neural cells but all of these need not to be relevant for neurodegenerative disease.     

Decreased Zinc Affinity of Amyotrophic Lateral Sclerosis-Associated Superoxide Dismutase Mutants Leads to Enhanced Catalysis of Tyrosine Nitration by Peroxynitrite

Abstract: Mutations to Cu/Zn superoxide dismutase (SOD) linked to familial amyotrophic lateral sclerosis (ALS) enhance an unknown toxic reaction that leads to the selective degeneration of motor neurons. However, the question of how >50 different missense mutations produce a common toxic phenotype remains perplexing. We found that the zinc affinity of four ALS-associated SOD mutants was decreased up to 30-fold compared to wild-type SOD but that both mutants and wild-type SOD retained copper with similar affinity. Neurofilament-L (NF-L), one of the most abundant proteins in motor neurons, bound multiple zinc atoms with sufficient affinity to potentially remove zinc from both wild-type and mutant SOD while having a lower affinity for copper. The loss of zinc from wild-type SOD approximately doubled its efficiency for catalyzing peroxynitrite-mediated tyrosine nitration, suggesting that one gained function by SOD in ALS may be an indirect consequence of zinc loss. Nitration of protein-bound tyrosines is a permanent modification that can adversely affect protein function. Thus, the toxicity of ALS-associated SOD mutants may be related to enhanced catalysis of protein nitration subsequent to zinc loss. By acting as a high-capacity zinc sink, NF-L could foster the formation of zinc-deficient SOD within motor neurons.     

Biochemical characterization of actin assembly mechanisms with ALS-associated profilin variants

Eight separate mutations in the actin-binding protein profilin-1 have been identified as a rare cause of amyotrophic lateral sclerosis (ALS). Profilin is essential for many neuronal cell processes through its regulation of lipids, nuclear signals, and cytoskeletal dynamics, including actin filament assembly. Direct interactions between profilin and actin monomers inhibit actin filament polymerization. In contrast, profilin can also stimulate polymerization by simultaneously binding actin monomers and proline-rich tracts found in other proteins. Whether the ALS-associated mutations in profilin compromise these actin assembly functions is unclear. We performed a quantitative biochemical comparison of the direct and formin mediated impact for the eight ALS-associated profilin variants on actin assembly using classic protein-binding and single-filament microscopy assays. We determined that the binding constant of each profilin for actin monomers generally correlates with the actin nucleation strength associated with each ALS-related profilin. In the presence of formin, the A20T, R136W, Q139L, and C71G variants failed to activate the elongation phase of actin assembly. This diverse range of formin-activities is not fully explained through profilin-poly-L-proline (PLP) interactions, as all ALS-associated variants bind a formin-derived PLP peptide with similar affinities. However, chemical denaturation experiments suggest that the folding stability of these profilins impact some of these effects on actin assembly. Thus, changes in profilin protein stability and alterations in actin filament polymerization may both contribute to the profilin-mediated actin disruptions in ALS.     

Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in the familial form of amyotrophic lateral sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) cause a familial form of amyotrophic lateral sclerosis (fALS), and aggregation of mutant SOD1 has been proposed to play a role in neurodegeneration. A growing body of evidence suggests that fALS-causing mutations destabilize the native structure of SOD1, leading to aberrant protein interactions for aggregation. SOD1 becomes stabilized and enzymatically active after copper and zinc binding and intramolecular disulfide formation, but it remains unknown which step(s) in the SOD1 maturation process is important in the pathological aggregation. In this study we have shown that apoSOD1 without disulfide is the most facile state for formation of amyloid-like fibrillar aggregates. fALS mutations impair either zinc binding, disulfide formation, or both, leading to accumulation of the aggregation-prone, apo, and disulfide-reduced SOD1. Moreover, we have found that the copper chaperone for SOD1 (CCS) facilitates maturation of SOD1 and that CCS overexpression ameliorates intracellular aggregation of mutant SOD1 in vivo. Based on our in vivo and in vitro results, we propose that facilitation of post-translational modifications is a promising strategy to reduce SOD1 aggregation in the cell.