DNA is a template for accelerating the aggregation of copper, zinc superoxide dismutase

The proteinaceous aggregates rich in copper, zinc superoxide dismutase (SOD1) have been shown to be involved in pathogenesis of amyotrophic lateral sclerosis (ALS). Since negatively charged species such as nucleic acids have frequently been found associated with the proteinaceous deposits in the tissues of patients with amyloid diseases, we examined here the aggregation behavior of SOD1 in the presence of DNA under acidic conditions that facilitate protein aggregation. Several forms of double-stranded DNA were tested to trigger SOD1 aggregation by light scattering, single- and double-fluorescence imaging with dyes, atomic force microscopy, and direct observations under visible light. The results reveal that DNA acts as a template for accelerating the formation of SOD1 aggregates and is incorporated into SOD1 aggregates. The spherical and ellipsoidal SOD1 aggregates were characterized in both hydrated and dried states and have morphology similar to those identified in the diseased neurons. Light scattering experiments indicate that the aggregation first undergoes a rapid phase where the aggregates with average diameters of 40-80 nm rapidly form in <2 min, and then passes through a slow phase where the average diameters of aggregates were increased to at least 200-260 nm in 2 h. All forms of DNAs tested can lead to the aggregation of SOD1 at nanomolar levels. The association of SOD1 with DNA, driven by electrostatic interactions between both, can restrict the orientation of SOD1 molecules and increase a SOD1 population along DNA strands. This facilitates the hydrophobic interactions between SOD1 molecules, as indicated by hydrophobic probe binding and chemical denaturant treatment experiments. Demonstration of the DNA-accelerated aggregation of SOD1 might establish a possible role of DNA in the pathogenesis of some diseases because of the ubiquitous expression of SOD1 and the coexistence of SOD1 and DNA in the crowded molecular environment of a cell.     

Mutation-dependent Polymorphism of Cu,Zn-Superoxide Dismutase Aggregates in the Familial Form of Amyotrophic Lateral Sclerosis

More than 100 different mutations in Cu,Zn-superoxide dismutase (SOD1) are linked to a familial form of amyotrophic lateral sclerosis (fALS). Pathogenic mutations facilitate fibrillar aggregation of SOD1, upon which significant structural changes of SOD1 have been assumed; in general, however, a structure of protein aggregate remains obscure. Here, we have identified a protease-resistant core in wild-type as well as fALS-causing mutant SOD1 aggregates. Three different regions within an SOD1 sequence are found as building blocks for the formation of an aggregate core, and fALS-causing mutations modulate interactions among these three regions to form a distinct core, namely SOD1 aggregates exhibit mutation-dependent structural polymorphism, which further regulates biochemical properties of aggregates such as solubility. Based upon these results, we propose a new pathomechanism of fALS in which mutation-dependent structural polymorphism of SOD1 aggregates can affect disease phenotypes.     

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.     

DNA-Triggered Aggregation of Copper,Zinc Superoxide Dismutase in the Presence of Ascorbate

The oxidative damage hypothesis proposed for the function gain of copper, zinc superoxide dismutase (SOD1) maintains that both mutant and wild-type (WT) SOD1 catalyze reactions with abnormal substrates that damage cellular components critical for viability of the affected cells. However, whether the oxidative damage of SOD1 is involved in the formation of aggregates rich in SOD1 or not remains elusive. Here, we sought to explore the oxidative aggregation of WT SOD1 exposed to environments containing both ascorbate (Asc) and DNA under neutral conditions. The results showed that the WT SOD1 protein was oxidized in the presence of Asc. The oxidation results in the higher affinity of the modified protein for DNA than that of the unmodified protein. The oxidized SOD1 was observed to be more prone to aggregation than the WT SOD1, and the addition of DNA can significantly accelerate the oxidative aggregation. Moreover, a reasonable relationship can be found between the oxidation, increased hydrophobicity, and aggregation of SOD1 in the presence of DNA. The crucial step in aggregation is neutralization of the positive charges on some SOD1 surfaces by DNA binding. This study might be crucial for understanding molecular forces driving the protein aggregation.     

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.     

Mapping superoxide dismutase 1 domains of non-native interaction: roles of intra- and intermolecular disulfide bonding in aggregation

Superoxide dismutase 1 (SOD1) proteins harboring mutations linked to familial amyotrophic lateral sclerosis (FALS) uniformly show heightened potential to form high molecular weight structures. Here, we examine the domains of SOD1 that are involved in forming these structures (aggregates) and study the role of intra- and intermolecular disulfide bonds. An analysis of disease mutations identified to date reveals a non-random distribution with predominant occurrence at residues within highly conserved beta-strands or at highly conserved residues in loop domains. Using a cell transfection assay for aggregation, we determined that no single domain in SOD1 is indispensable in the formation of sedimentable aggregates, suggesting multiple potential motifs in the protein mediate non-native interactions. By a cell-free aggregation assay, analysis of transgenic mouse tissues, and mutagenesis approaches, we found evidence that redox conditions may modulate SOD1 aggregation; reduction of the native intramolecular disulfide bonds may predispose SOD1 to unfolding and aggregation, whereas non-native intermolecular disulfide linkages may help stabilize aggregates in vivo. The results suggest a possible mechanism for diversity in the structures formed by different SOD1 mutants, and define a potential contribution of redox conditions to SOD1 aggregation.     

A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis

One of the mechanisms by which mutations in superoxide dismutase 1 (SOD1) cause familial amyotrophic lateral sclerosis (fALS) is proposed to involve the accumulation of detergent-insoluble, disulfide-cross-linked, mutant protein. Recent studies have implicated cysteine residues at positions 6 and 111 as critical in mediating disulfide cross-linking and promoting aggregation. In the present study, we used a panel of experimental and disease-linked mutations at cysteine residues of SOD1 (positions 6, 57, 111, and 146) in cell culture assays for aggregation to demonstrate that extensive disulfide cross-linking is not required for the formation of mutant SOD1 aggregates. Experimental mutants possessing only a single cysteine residue or lacking cysteine entirely were found to retain high potential to aggregate. Furthermore we demonstrate that aggregate structures in symptomatic SOD1-G93A mice can be dissociated such that they no longer sediment upon ultracentrifugation (i.e. appear soluble) under relatively mild conditions that leave disulfide bonds intact. Similar to other recent work, we found that cysteines 6 and 111, particularly the latter, play interesting roles in modulating the aggregation of human SOD1. However, we did not find that extensive disulfide cross-linking via these residues, or any other cysteine, is critical to aggregate structure. Instead we suggest that these residues participate in other features of the protein that, in some manner, modulate aggregation.     

Aggregate formation in Cu,Zn superoxide dismutase-related proteins

Aggregation of Cu,Zn superoxide dismutase (SOD1) protein is a pathologic hallmark of familial amyotrophic lateral sclerosis linked to mutations in the SOD1 gene, although the structural motifs within mutant SOD1 that are responsible for its aggregation are unknown. Copper chaperone for SOD1 (CCS) and extracellular Cu,Zn superoxide dismutase (SOD3) have some sequence identity with SOD1, particularly in the regions of metal binding, but play no significant role in mutant SOD1-induced disease. We hypothesized that it would be possible to form CCS- or SOD3-positive aggregates by making these molecules resemble mutant SOD1 via the introduction of point mutations in codons homologous to a disease causing G85R SOD1 mutation. Using an in vitro assay system, we found that expression of wild type human CCS or a modified intracellular wild type SOD3 does not result in significant aggregate formation. In contrast, expression of G168R CCS or G146R SOD3 produced aggregates as evidenced by the presence of high molecular weight protein complexes on Western gels or inclusion bodies on immunofluorescence. CCS- and SOD3-positive inclusions appear to be ubiquitinated and localized to aggresomes. These results suggest that proteins sharing structural similarities to mutant SOD1 are also at risk for aggregate formation.     

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

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.     

Mutant SOD1 aggregates formed in vitro and in cultured cells are polymorphic and differ from those arising in the CNS

Mutations in the human Superoxide dismutase 1 (hSOD1) gene are well-established cause of the motor neuron disease ALS. Patients and transgenic (Tg) ALS model mice carrying mutant variants develop hSOD1 aggregates in the CNS. We have identified two hSOD1 aggregate strains, which both transmit spreading template-directed aggregation and premature fatal paralysis when inoculated into adult transgenic mice. This prion-like spread of aggregation could be a primary disease mechanism in SOD1-induced ALS. Human SOD1 aggregation has been studied extensively both in cultured cells and under various conditions in vitro. To determine how the structure of aggregates formed in these model systems related to disease-associated aggregates in the CNS, we used a binary epitope-mapping assay to examine aggregates of hSOD1 variants G93A, G85R, A4V, D90A, and G127X formed in vitro, in four different cell lines and in the CNS of Tg mice. We found considerable variability between replicate sets of in vitro-generated aggregates. In contrast, there was a high similarity between replicates of a given hSOD1 mutant in a given cell line, but pronounced variations between different hSOD1 mutants and different cell lines in both structures and amounts of aggregates formed. The aggregates formed in vitro or in cultured cells did not replicate the aggregate strains that arise in the CNS. Our findings suggest that the distinct aggregate morphologies in the CNS could result from a micro-environment with stringent quality control combined with second-order selection by spreading ability. Explorations of pathogenesis and development of therapeutics should be conducted in models that replicate aggregate structures forming in the CNS.

     

Agitation and High Ionic Strength Induce Amyloidogenesis of a Folded PDZ Domain in Native Conditions

Amyloid fibril formation is a distinctive hallmark of a number of degenerative diseases. In this process, protein monomers self-assemble to form insoluble structures that are generally referred to as amyloid fibrils. We have induced in vitro amyloid fibril formation of a PDZ domain by combining mechanical agitation and high ionic strength under conditions otherwise close to physiological (pH 7.0, 37°C, no added denaturants). The resulting aggregates enhance the fluorescence of the thioflavin T dye via a sigmoidal kinetic profile. Both infrared spectroscopy and circular dichroism spectroscopy detect the formation of a largely intermolecular β-sheet structure. Atomic force microscopy shows straight, rod-like fibrils that are similar in appearance and height to mature amyloid-like fibrils. Under these conditions, before aggregation, the protein domain adopts an essentially native-like structure and an even higher conformational stability (ΔG_U-F^H2O). These results show a new method for converting initially folded proteins into amyloid-like aggregates. The methodological approach used here does not require denaturing conditions; rather, it couples agitation with a high ionic strength. Such an approach offers new opportunities to investigate protein aggregation under conditions in which a globular protein is initially folded, and to elucidate the physical forces that promote amyloid fibril formation.     

Novel Mutations that Enhance or Repress the Aggregation Potential of SOD1

Mutations in SOD1 cause FALS by a gain of function likely related to protein misfolding and aggregation. SOD1 mutations encompass virtually every domain of the molecule, making it difficult to identify motifs important in SOD1 aggregation. Zinc binding to SOD1 is important for structural integrity, and is hypothesized to play a role in mutant SOD1 aggregation. To address this question, we mutated the unique zinc binding sites of SOD1 and examined whether these changes would influence SOD1 aggregation. We generated single and multiple mutations in SOD1 zinc binding residues (H71, H80 and D83) either alone or in combination with an aggregate forming mutation (A4V) known to cause disease. These SOD1 mutants were assayed for their ability to form aggregates.Using an in vitro cellular SOD1 aggregation assay, we show that combining A4V with mutations in non-zinc binding domains (G37R or G85R) increases SOD1 aggregation potential. Mutations at two zinc binding residues (H71G and D83G) also increase SOD1 aggregation potential. However, an H80G mutation at the third zinc binding residue decreases SOD1 aggregation potential even in the context of other aggregate forming SOD1 mutations. These results demonstrate that various mutations have different effects on SOD1 aggregation potential and that the H80G mutation appears to uniquely act as a dominant inhibitor of SOD1 aggregation.     

Protein aggregation and mitigation strategy in low pH viral inactivation for monoclonal antibody purification

ABSTRACT

Significant amounts of soluble product aggregates were observed during low-pH viral inactivation (VI) scale-up for an IgG4 monoclonal antibody (mAb IgG4-N1), while small-scale experiments in the same condition showed negligible aggregation. Poor mixing and product exposure to low pH were identified as the root cause. To gain a mechanistic understanding of the problem, protein aggregation properties were studied by varying critical parameters including pH, hold time and protein concentration. Comprehensive biophysical characterization of product monomers and aggregates was performed using fluorescence-size-exclusion chromatography, differential scanning fluorimetry, fluorescence spectroscopy, and dynamic light scattering. Results showed IgG4-N1 partially unfolds at about pH 3.3 where the product molecules still exist largely as monomers owing to strong inter-molecular repulsions and favorable colloidal stability. In the subsequent neutralization step, however, the conformationally changed monomers are prone to aggregation due to weaker inter-molecular repulsions following the pH transition from 3.3 to 5.5. Surface charge calculations using homology modeling suggested that intra-molecular repulsions, especially between CH2 domains, may contribute to the IgG4-N1 unfolding at ≤ pH 3.3. Computational fluid dynamics (CFD) modeling was employed to simulate the conditions of pH titration to reduce the risk of aggregate formation. The low-pH zones during acid addition were characterized using CFD modeling and correlated to the condition causing severe product aggregation. The CFD tool integrated with the mAb solution properties was used to optimize the VI operating parameters for successful scale-up demonstration. Our research revealed the governing aggregation mechanism for IgG4-N1 under acidic conditions by linking its molecular properties and various process-related parameters to macroscopic aggregation phenomena. This study also provides useful insights into the cause and mitigation of low-pH-induced IgG4 aggregation in downstream VI operation.     

Biophysical effect of amino acids on the prevention of protein aggregation

Each protein folds into a unique and native structure spontaneously. However, during the unfolding or refolding process, a protein often tends to form aggregates. To establish a method to prevent undesirable protein aggregation and to increase the stability of native protein structures under deterioration conditions, two types of aggregation conditions, thermal unfolding-induced aggregation and dilution-induced aggregation from denatured state, were studied in the presence of additional amino acids and ions using lysozyme as a model protein. Among 15 amino acids tested, arginine exhibited the best results in preventing the formation of aggregates in both cases. Further biophysical studies revealed that arginine did not change the thermal denaturation temperature (T(m)) of the lysozyme. The preventive effect of arginine on aggregation was not dependent on the size or isoelectric point of eight kinds of proteins tested.     

Toxic SOD1 trimers are off-pathway in the formation of amyloid-like fibrils in ALS

Accumulation of insoluble amyloid fibrils is widely studied as a critical factor in the pathology of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease. Misfolded Cu, Zn superoxide dismutase (SOD1) was the first protein linked to ALS, and non-native SOD1 trimeric oligomers were recently linked to cytotoxicity, while larger oligomers were protective to cells. The balance between trimers and larger aggregates in the process of SOD1 aggregation is, thus, a critical determinant of potential therapeutic approaches to treat ALS. However, it is unknown whether these trimeric oligomers are a necessary intermediate for larger aggregate formation or a distinct off-pathway species competing with fibril formation. Depending on the on- or off-pathway scenario of trimer formation, we expect drastically different therapeutic approaches. Here, we show that the toxic SOD1 trimer is an off-pathway intermediate competing with protective fibril formation. We design mutant SOD1 constructs that remain in a trimeric state (super-stable trimers) and show that stabilizing the trimeric SOD1 prevents formation of fibrils in vitro and in a motor neuron-like cell model (NSC-34). Using size exclusion chromatography, we track the aggregation kinetics of purified SOD1 and show direct competition of trimeric SOD1 with larger oligomer and fibril formation. Finally, we show the trimer is structurally independent of both larger soluble oligomers and insoluble fibrils using circular dichroism spectroscopy and limited proteolysis.     

Molecular dynamics simulations of PEI mediated DNA aggregation

Understanding the molecular mechanism of polycation induced DNA aggregation and condensation is important for optimal design of gene delivery carriers. In this work, we performed a series of all-atom molecular dynamics (MD) simulations to investigate polyethylenimine (PEI) mediated DNA aggregation. We found that PEIs condense DNA through two mechanisms: polyion bridging and electrostatic screening of the DNA charges. At PEI/DNA charge ratio >1, PEIs can completely neutralize DNAs at a short distance (～12 ? from the C1' atoms), and this distance is found to be insensitive to the exact value of the charge ratio. When excess PEIs are added to a formed DNA-PEI aggregate, they are found to bind to the aggregate and increase its cationic charge. The added PEIs can also replace the PEIs previously bound to the aggregate. The excess PEIs, however, do not change the spacing of the DNAs in the aggregates. Our simulation results shed light on the mechanisms of PEI, and more generally polycation, mediated DNA aggregation and condensation.     

Unsaturated fatty acids induce cytotoxic aggregate formation of amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutants

Formation of misfolded protein aggregates is a remarkable hallmark of various neurodegenerative diseases including Alzheimer disease, Parkinson disease, Huntington disease, prion encephalopathies, and amyotrophic lateral sclerosis (ALS). Superoxide dismutase 1 (SOD1) immunoreactive inclusions have been found in the spinal cord of ALS animal models and patients, implicating the close involvement of SOD1 aggregates in ALS pathogenesis. Here we examined the molecular mechanism of aggregate formation of ALS-related SOD1 mutants in vitro. We found that long-chain unsaturated fatty acids (FAs) promoted aggregate formation of SOD1 mutants in both dose- and time-dependent manners. Metal-deficient SOD1s, wild-type, and mutants were highly oligomerized compared with holo-SOD1s by incubation in the presence of unsaturated FAs. Oligomerization of SOD1 is closely associated with its structural instability. Heat-treated holo-SOD1 mutants were readily oligomerized by the addition of unsaturated FAs, whereas wild-type SOD1 was not. The monounsaturated FA, oleic acid, directly bound to SOD1 and was characterized by a solid-phase FA binding assay using oleate-Sepharose. The FA binding characteristics were closely correlated with the oligomerization propensity of SOD1 proteins, which indicates that FA binding may change SOD1 conformation in a way that favors the formation of aggregates. High molecular mass aggregates of SOD1 induced by FAs have a granular morphology and show significant cytotoxicity. These findings suggest that SOD1 mutants gain FA binding abilities based on their structural instability and form cytotoxic granular aggregates.     

The small heat shock proteins αB-crystallin and Hsp27 suppress SOD1 aggregation in vitro

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease. The mechanism that underlies amyotrophic lateral sclerosis (ALS) pathology remains unclear, but protein inclusions are associated with all forms of the disease. Apart from pathogenic proteins, such as TDP-43 and SOD1, other proteins are associated with ALS inclusions including small heat shock proteins. However, whether small heat shock proteins have a direct effect on SOD1 aggregation remains unknown. In this study, we have examined the ability of small heat shock proteins αB-crystallin and Hsp27 to inhibit the aggregation of SOD1 in vitro. We show that these chaperone proteins suppress the increase in thioflavin T fluorescence associated with SOD1 aggregation, primarily through inhibiting aggregate growth, not the lag phase in which nuclei are formed. αB-crystallin forms high molecular mass complexes with SOD1 and binds directly to SOD1 aggregates. Our data are consistent with an overload of proteostasis systems being associated with pathology in ALS.