Pathogenic superoxide dismutase structure, folding, aggregation and turnover

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Significant advances have been made during the past two years toward an understanding of the molecular basis for how mutations in human cytosolic copper-zinc superoxide dismutase (SOD1) cause the inherited form of amyotrophic lateral sclerosis (ALS). Biophysical studies suggest that the pathogenic mutations destabilize loop or β-barrel structural elements of the protein. With few exceptions, the loss of metal ions and reduction of the intrasubunit disulfide bond enhance this destabilization. In mouse models of the disease, the formation of visible aggregates containing mutant SOD1 occurs relatively late in the lifespan, hinting that the quality control and protein turnover systems of motor neurons eventually become overwhelmed or compromised. Studies probing SOD1 turnover have suggested the possibility that proteolytic breakdown products may play a role in pathogenesis.

Introduction

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the progressive destruction of motor neurons. Approximately 10% of cases are inherited and are termed ‘familial’ (fALS). A subset of these cases are linked to dominant mutations in the cytosolic antioxidant enzyme copper-zinc superoxide dismutase (SOD1) [1, 2]. Mice expressing human fALS SOD1 polypeptides in addition to their endogenous SOD1 develop paralytic symptoms, whereas SOD1 knockout mice do not [3, 4]. SOD1-linked ALS is thus not a loss-of-function disorder, as the expression of the mutant SOD1 polypeptides is sufficient to cause motor neuron death through an as-yet unidentified molecular mechanism. The mouse models have provided a clue, however, in that aggregates containing pathogenic SOD1 become manifest in the motor neurons of animals exhibiting paralytic symptoms [3]. It is now generally accepted that pathogenic SOD1 aggregation is linked to toxicity. This review focuses on the advances since 2003 that have shaped our understanding of the molecular and biophysical properties of the fALS SOD1 mutants in the context of protein structure, misfolding, aggregation and turnover. Studies aimed at developing therapies or prolonging the lifespan of transgenic animals are not covered. Two excellent recent comprehensive reviews of the link between SOD1 and ALS can be found in [5, 6].

Section snippets

Spatial distribution and classification of pathogenic SOD1 mutations

Human SOD1 is a 32 kDa homodimeric protein found in the cytosol, nucleus and intermembrane space of mitochondria [5]. Each monomer folds as an eight-stranded Greek key β-barrel, binds one copper and one zinc ion, and contains an intrasubunit disulfide bond. Approximately 110 fALS mutations have now been identified. The majority of lesions give rise to point mutations, but a few result in truncations or amino acid insertions/deletions. The fALS SOD1 proteins are divided into two groups on the

Structures of pathogenic SOD1 mutants

Three-dimensional structures have been published for the metal-replete WTL mutants A4V [8•, 9], G37R [10], H43R [11], G93A [12] and I113T [8•]. They are all similar to the wild-type protein except for perturbations at or near the site of mutation. The A4V and I113T structures reveal a slight reorientation of the monomers relative to the wild-type enzyme [8•]. Solution small-angle X-ray scattering studies suggest that this reorientation may be more pronounced than that observed in the crystal,

SOD1 folding and quaternary structure

Human SOD1 is a remarkably stable dimeric molecule that remains active under a broad range of harsh denaturing conditions. The intrasubunit disulfide bond formed between Cys57 and Cys146 in each monomer is a rare feature for a protein found in the reducing environment of the cytosol. Although the stabilizing effect of metal ions has long been known, a recent series of studies on the wild-type enzyme have shed light on the structural and functional role of this unusual disulfide [20•, 21•, 22•].

Pathogenic SOD1 aggregation

Studies of pathogenic SOD1 aggregation in vitro are now beginning to appear. The mutants A4V and H43R can form filamentous aggregates that bind Congo red [11]. Khare and colleagues proposed that SOD1 aggregation is a multistep reaction consisting of dimer dissociation, metal loss from the monomers, followed by oligomerization. They also calculated rate and equilibrium constants for the dissociation event [31]. In both studies mentioned above, the experimental conditions were non-native (pH

Pathogenic SOD1 and molecular chaperones

The propensity of pathogenic SOD1 to aggregate in vivo is linked to its concentration, the ability of molecular chaperones to prevent or reverse misfolding, and the rate of turnover by the protein degradation machinery. In presymptomatic G85R and G93A mice, a reduction in total chaperone activity was observed beginning at 60 days of age [42]. However, the small chaperones Hsp25 (Hsp27 in humans) and αβ-crystallin were shown to be upregulated in astrocytes (but not motor neurons) of symptomatic

Pathogenic SOD1 and the proteasome

Studies focused on degradation of pathogenic SOD1 proteins by the proteasome are in their infancy. Two groups demonstrated that the co-chaperone CHIP (C-terminus of Hsc70-interacting protein) associates with mutant SOD1 and promotes its degradation by the ubiquitin-proteasome system [46, 47]. The ubiquitin ligase NEDL1 was shown to bind and ubiquitinate fALS mutants but not wild-type SOD1 [48]. Proteasome inhibition resulted in aggregation in spinal cord slices of G93A mice and subsequent

Conclusions

Figure 4 reflects a summary of current thinking on pathogenic SOD1 aggregation, turnover and toxicity. With few exceptions, the fALS mutations destabilize the structure through metal ion loss or perturbations of the protein fold, and these destabilized SOD1 proteins either aggregate or are turned over rapidly when compared with the wild-type enzyme. It remains unclear exactly how the soluble oligomers, the insoluble aggregates, or the proteolytic breakdown products of pathogenic SOD1 are toxic,

Update

Recently, protein components of secretory large dense-core vesicles in neurons, interneurons and endocrine cells, were found to interact with pathogenic SOD1 proteins, but not with the wild-type enzyme [56••]. These proteins, termed chromogranins, are moved though the trans-Golgi network, translocating to the cell periphery during the maturation process. Data are presented that suggest that chromogranins may act as chaperone-like proteins to facilitate secretion of misfolded SOD1 mutants that

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

I thank members of the Borchelt, Hart, Hayward, Hasnain, Levine and Valentine laboratories for their many helpful discussions and insights, and Stephen Holloway and Alex Taylor for help with the figures. I also thank David Eisenberg, Jon Robertus and Joan Valentine for their guidance and friendship over the years. Work on SOD1 in my laboratory has been supported at various times by the NIH/NINDS, the ALS Association, and the Robert A Welch Foundation. Support for the X-ray Crystallography Core

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