Immature ALS-associated mutant superoxide dismutases form variable aggregate structures through distinct oligomerization processes.

Protein misfolding and aggregation are hallmarks of many diseases, including amyotrophic lateral sclerosis (ALS). In familial ALS, aberrant self-association of mutant Cu,Zn-superoxide dismutase (SOD1) is implicated as a key contributor to disease. Mutations have the largest impacts on the stability of the most immature form of SOD1, the unmetallated, disulfide-reduced monomer (apoSH SOD1). Here we demonstrate that, despite the marginal stability of apoSH SOD1, aggregation is little correlated with the degree of protein unfolding, and multiple modes of aggregation occur, depending on the mutation and solution conditions. Light scattering and atomic force microscopy reveal two distinct mutant SOD1 behaviours: high aggregator mutants form abundant small assemblies, while low aggregator mutants form fewer, more fibre-like aggregates. Attenuated total reflectance-Fourier transform infrared spectroscopy and Thioflavin T binding show the aggregates maintain native-like anti-parallel beta structure. These results provide new evidence that ALS-associated mutations promote the aggregation of apoSH SOD1 through multiple pathways, with broad implications for understanding mechanisms of protein self-association in disease and biotechnology.

Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that, in some cases, has been linked with mutations to the antioxidant metalloenzyme superoxide dismutase (SOD1). Although the mature form of this enzyme is highly stable and resistant to aggregation, the most immature form, lacking metal and a stabilizing intrasubunit disulfide bond, apoSOD12SH, is dynamic and hypothesized to be a major cause of toxicity in vivo. Previous solution NMR studies of wild-type apoSOD12SH have shown that the ground state interconverts with a series of sparsely populated and transiently formed conformers, some of which have aberrant nonnative structures. Here, we study seven disease mutants of apoSOD12SH and characterize their free energy landscapes as a first step in understanding the initial stages of disease progression and, more generally, to evaluate the plasticity of low-lying protein conformational states. The mutations lead to little change in the structures and dynamics of the ground states of the mutant proteins. By contrast, the numbers of low-lying excited states that are accessible to each of the disease mutants can vary significantly, with additional conformers accessed in some cases. Our study suggests that the diversity of these structures can provide alternate interaction motifs for different mutants, establishing additional pathways for new and often aberrant intra- and intermolecular contacts. Further, it emphasizes the potential importance of conformationally excited states in directing both folding and misfolding processes.

ALS-linked misfolded SOD1 species have divergent impacts on mitochondria.

Approximately 20 % of familial Amyotrophic Lateral Sclerosis (ALS) is caused by mutations in superoxide dismutase (SOD1), which leads to misfolding of the SOD1 protein, resulting in a toxic gain of function. Several conformation-restricted antibodies have been generated that specifically recognize misfolded SOD1 protein, and have been used as therapeutics in pre-clinical models. Misfolded SOD1 selectively associates with spinal cord mitochondria in SOD1 rodent models. Using the SOD1(G93A) rat model, we find that SOD1 conformational specific antibodies AMF7-63 and DSE2-3H1 labeled a fibrillar network concentrated in the anterior horn; while A5C3, B8H10, C4F6 and D3H5 labeled motor neurons as well as puncta in the neuropil. There is a time-dependent accumulation of misfolded SOD1 at the surface of spinal cord mitochondria with AMF7-63-labeled mitochondria having increased volume in contrast to a mitochondrial subset labeled with B8H10. In spinal cord homogenates and isolated mitochondria, AMF7-63, DSE2-3H1 and B8H10 detect misfolded SOD1 aggregates. SOD1 that lacks its metal cofactors has an increased affinity for naïve mitochondria and misfolded SOD1 antibodies B8H10 and DSE2-3H1 readily detect demetalated mutant and wild-type SOD1. Together, these data suggest that multiple non-native species of misfolded SOD1 may exist, some of which are associated with mitochondrial damage. Conformational antibodies are invaluable tools to identify and characterize the variation in misfolded SOD1 species with regards to biochemical characteristics and toxicity. This information is highly relevant to the further development of these reagents as therapeutics.

Concurrent Increases and Decreases in Local Stability and Conformational Heterogeneity in Cu, Zn Superoxide Dismutase Variants Revealed by Temperature-Dependence of Amide Chemical Shifts.

The chemical shifts of backbone amide protons in proteins are sensitive reporters of local structural stability and conformational heterogeneity, which can be determined from their readily measured linear and nonlinear temperature-dependences, respectively. Here we report analyses of amide proton temperature-dependences for native dimeric Cu, Zn superoxide dismutase (holo pWT SOD1) and structurally diverse mutant SOD1s associated with amyotrophic lateral sclerosis (ALS). Holo pWT SOD1 loses structure with temperature first at its periphery and, while having extremely high global stability, nevertheless exhibits extensive conformational heterogeneity, with ∼1 in 5 residues showing evidence for population of low energy alternative states. The holo G93A and E100G ALS mutants have moderately decreased global stability, whereas V148I is slightly stabilized. Comparison of the holo mutants as well as the marginally stable immature monomeric unmetalated and disulfide-reduced (apo(2SH)) pWT with holo pWT shows that changes in the local structural stability of individual amides vary greatly, with average changes corresponding to differences in global protein stability measured by differential scanning calorimetry. Mutants also exhibit altered conformational heterogeneity compared to pWT. Strikingly, substantial increases as well as decreases in local stability and conformational heterogeneity occur, in particular upon maturation and for G93A. Thus, the temperature-dependence of amide shifts for SOD1 variants is a rich source of information on the location and extent of perturbation of structure upon covalent changes and ligand binding. The implications for potential mechanisms of toxic misfolding of SOD1 in disease and for general aspects of protein energetics, including entropy-enthalpy compensation, are discussed.

Combined Isothermal Titration and Differential Scanning Calorimetry Define Three-State Thermodynamics of fALS-Associated Mutant Apo SOD1 Dimers and an Increased Population of Folded Monomer.

Many proteins are naturally homooligomers, homodimers most frequently. The overall stability of oligomeric proteins may be described in terms of the stability of the constituent monomers and the stability of their association; together, these stabilities determine the populations of different monomer and associated species, which generally have different roles in the function or dysfunction of the protein. Here we show how a new combined calorimetry approach, using isothermal titration calorimetry to define monomer association energetics together with differential scanning calorimetry to measure total energetics of oligomer unfolding, can be used to analyze homodimeric unmetalated (apo) superoxide dismutase (SOD1) and determine the effects on the stability of structurally diverse mutations associated with amyotrophic lateral sclerosis (ALS). Despite being located throughout the protein, all mutations studied weaken the dimer interface, while concomitantly either decreasing or increasing the marginal stability of the monomer. Analysis of the populations of dimer, monomer, and unfolded monomer under physiological conditions of temperature, pH, and protein concentration shows that all mutations promote the formation of folded monomers. These findings may help rationalize the key roles proposed for monomer forms of SOD1 in neurotoxic aggregation in ALS, as well as roles for other forms of SOD1. Thus, the results obtained here provide a valuable approach for the quantitative analysis of homooligomeric protein stabilities, which can be used to elucidate the natural and aberrant roles of different forms of these proteins and to improve methods for predicting protein stabilities.

Destabilization of the dimer interface is a common consequence of diverse ALS-associated mutations in metal free SOD1.

Neurotoxic misfolding of Cu, Zn-superoxide dismutase (SOD1) is implicated in causing amyotrophic lateral sclerosis, a devastating and incurable neurodegenerative disease. Disease-linked mutations in SOD1 have been proposed to promote misfolding and aggregation by decreasing protein stability and increasing the proportion of less folded forms of the protein. Here we report direct measurement of the thermodynamic effects of chemically and structurally diverse mutations on the stability of the dimer interface for metal free (apo) SOD1 using isothermal titration calorimetry and size exclusion chromatography. Remarkably, all mutations studied, even ones distant from the dimer interface, decrease interface stability, and increase the population of monomeric SOD1. We interpret the thermodynamic data to mean that substantial structural perturbations accompany dimer dissociation, resulting in the formation of poorly packed and malleable dissociated monomers. These findings provide key information for understanding the mechanisms and energetics underlying normal maturation of SOD1, as well as toxic SOD1 misfolding pathways associated with disease. Furthermore, accurate prediction of protein-protein association remains very difficult, especially when large structural changes are involved in the process, and our findings provide a quantitative set of data for such cases, to improve modelling of protein association.

Thermal fluctuations of immature SOD1 lead to separate folding and misfolding pathways.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease involving cytotoxic conformations of Cu, Zn superoxide dismutase (SOD1). A major challenge in understanding ALS disease pathology has been the identification and atomic-level characterization of these conformers. Here, we use a combination of NMR methods to detect four distinct sparsely populated and transiently formed thermally accessible conformers in equilibrium with the native state of immature SOD1 (apoSOD1(2SH)). Structural models of two of these establish that they possess features present in the mature dimeric protein. In contrast, the other two are non-native oligomers in which the native dimer interface and the electrostatic loop mediate the formation of aberrant intermolecular interactions. Our results show that apoSOD1(2SH) has a rugged free energy landscape that codes for distinct kinetic pathways leading to either maturation or non-native association and provide a starting point for a detailed atomic-level understanding of the mechanisms of SOD1 oligomerization.

Many roads lead to Rome? Multiple modes of Cu,Zn superoxide dismutase destabilization, misfolding and aggregation in amyotrophic lateral sclerosis.

ALS (amyotrophic lateral sclerosis) is a fatal neurodegenerative syndrome characterized by progressive paralysis and motor neuron death. Although the pathological mechanisms that cause ALS remain unclear, accumulating evidence supports that ALS is a protein misfolding disorder. Mutations in Cu,Zn-SOD1 (copper/zinc superoxide dismutase 1) are a common cause of familial ALS. They have complex effects on different forms of SOD1, but generally destabilize the protein and enhance various modes of misfolding and aggregation. In addition, there is some evidence that destabilized covalently modified wild-type SOD1 may be involved in disease. Among the multitude of misfolded/aggregated species observed for SOD1, multiple species may impair various cellular components at different disease stages. Newly developed antibodies that recognize different structural features of SOD1 represent a powerful tool for further unravelling the roles of different SOD1 structures in disease. Evidence for similar cellular targets of misfolded/aggregated proteins, loss of cellular proteostasis and cell-cell transmission of aggregates point to common pathological mechanisms between ALS and other misfolding diseases, such as Alzheimer's, Parkinson's and prion diseases, as well as serpinopathies. The recent progress in understanding the molecular basis for these devastating diseases provides numerous avenues for developing urgently needed therapeutics.

Energetics of oligomeric protein folding and association.

In nature, proteins most often exist as complexes, with many of these consisting of identical subunits. Understanding of the energetics governing the folding and misfolding of such homooligomeric proteins is central to understanding their function and misfunction, in disease or biotechnology. Much progress has been made in defining the mechanisms and thermodynamics of homooligomeric protein folding. In this review, we outline models as well as calorimetric and spectroscopic methods for characterizing oligomer folding, and describe extensive results obtained for diverse proteins, ranging from dimers to octamers and higher order aggregates. To our knowledge, this area has not been reviewed comprehensively in years, and the collective progress is impressive. The results provide evolutionary insights into the development of subunit interfaces, mechanisms of oligomer folding, and contributions of oligomerization to protein stability, function and regulation. Thermodynamic analyses have also proven valuable for understanding protein misfolding and aggregation mechanisms, suggesting new therapeutic avenues. Successful recent designs of novel, functional proteins demonstrate increased understanding of oligomer folding. Further rigorous analyses using multiple experimental and computational approaches are still required, however, to achieve consistent and accurate prediction of oligomer folding energetics. Modeling the energetics remains challenging but is a promising avenue for future advances.