Identification of a Misfolded Region in Superoxide Dismutase 1 That Is Exposed in Amyotrophic Lateral Sclerosis

Rotunno, Melissa; Auclair, Jared R.; Maniatis, Stephanie; Shaffer, Scott A.; Agar, Jeffrey N.; Bosco, Daryl A.

doi:10.1074/jbc.m114.581801

Cited by 30 publications

(34 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15)(16)(17)(18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).…”

supporting

confidence: 81%

“…Instead, residues comprising the SOD1 dimer interface are spread into discrete regions on each monomer, with a significant fraction solvent-exposed in the SOD1 trimer (Movie S2). Additionally, the recently discovered epitope of the C4F6 Ab (18), which preferentially binds the SOD1 trimer (Fig. S4A) as well as several other disease-linked SOD1 species (15,16) over the native dimer or monomer, is completely exposed on the surface of the trimer structure ( Fig.…”

Section: Sod1 Trimer Features Nonnative Secondary Tertiary and Quatmentioning

confidence: 99%

See 1 more Smart Citation

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Proctor

Fee

Tao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

109

146

View full text Add to dashboard Cite

Since the linking of mutations in the Cu,Zn superoxide dismutase gene (sod1) to amyotrophic lateral sclerosis (ALS) in 1993, researchers have sought the connection between SOD1 and motor neuron death. Disease-linked mutations tend to destabilize the native dimeric structure of SOD1, and plaques containing misfolded and aggregated SOD1 have been found in the motor neurons of patients with ALS. Despite advances in understanding of ALS disease progression and SOD1 folding and stability, cytotoxic species and mechanisms remain unknown, greatly impeding the search for and design of therapeutic interventions. Here, we definitively link cytotoxicity associated with SOD1 aggregation in ALS to a nonnative trimeric SOD1 species. We develop methodology for the incorporation of low-resolution experimental data into simulations toward the structural modeling of metastable, multidomain aggregation intermediates. We apply this methodology to derive the structure of a SOD1 trimer, which we validate in vitro and in hybridized motor neurons. We show that SOD1 mutants designed to promote trimerization increase cell death. Further, we demonstrate that the cytotoxicity of the designed mutants correlates with trimer stability, providing a direct link between the presence of misfolded oligomers and neuron death. Identification of cytotoxic species is the first and critical step in elucidating the molecular etiology of ALS, and the ability to manipulate formation of these species will provide an avenue for the development of future therapeutic strategies.neurodegeneration | protein aggregation | protein misfolding | ALS | structural modeling P rotein misfolding and aggregation are linked to cell death and disease progression in neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS). In these diseases and others, the formation of amyloid plaques, often observed post mortem, has long been thought to play a role in neurodegeneration, but toxicity has never been confirmed (1-3). Recent research has shown that small, soluble oligomers, rather than insoluble amyloids, are likely to be the cytotoxic species causing neurodegeneration (4-14). These small, soluble oligomers undergo aberrant interactions with cell machinery and activate cell death pathways, but their exact stoichiometry is not known and their properties have yet to be characterized. Recently, metastable soluble Cu,Zn superoxide dismutase (SOD1) oligomers have been identified that contain an epitope associated with disease-linked species of SOD1, mutants of which are implicated in a subset of ALS (15-18). Size exclusion chromatography (SEC) of these oligomers revealed a size range of two to four monomers, consistent with previous findings of potentially cytotoxic SOD1 oligomers (19)(20)(21).Knowledge of the structures of these species would not only allow for definitive testing of their toxicity but could potentially lead to an understanding of disease mechanism and therapeutic strategies against diseases for which no ...

show abstract

supporting

confidence: 81%

Section: Sod1 Trimer Features Nonnative Secondary Tertiary and Quatmentioning

confidence: 99%

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Proctor

Fee

Tao

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

109

146

View full text Add to dashboard Cite

show abstract

“…Furthermore, we find strong correlations within the zinc-binding loop (residues 49–60) only in A4V–SOD1, whereas in WT-, T2D-, and T2D/A4V–SOD1 these correlated motions are suppressed (Figure S6). The zinc-binding loop has been suggested to allosterically regulate the conformation of the C4F6 epitope (Bosco et al, 2010), which is directly linked to neural-toxicity of SOD1 in a primary microglia activation assay (Furukawa et al, 2015; Rotunno et al, 2014). We propose that in A4V–SOD1 the cross-correlated motions promote the exposure of the disease-related epitope, and that the T2D/A4V double mutant prevents this effect.…”

Section: Resultsmentioning

confidence: 99%

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

et al. 2016

View full text Add to dashboard Cite

SUMMARY The majority of amyotrophic lateral sclerosis (ALS)-related mutations in the enzyme Cu, Zn superoxide dismutase (SOD1), as well as a post-translational modification, glutathionylation, destabilize the protein and lead to a misfolded oligomer that is toxic to motor neurons. The biophysical role of another physiological SOD1 modification, T2-phosphorylation, has remained a mystery. Here, we find that a phosphomimetic mutation, T2D, thermodynamically stabilizes SOD1 even in the context of a strongly SOD1-destabilizing mutation, A4V, one of the most prevalent and aggressive ALS-associated mutations in North America. This stabilization protects against formation of toxic SOD oligomers and positively impacts motor neuron survival in cellular assays. We solve the crystal structure of T2D-SOD1 and explain its stabilization effect using DMD simulations. These findings imply that T2-phosphorylation may be a plausible innate cellular protection response against SOD1-induced cytotoxicity, and stabilizing the SOD1 native conformation might offer us viable pharmaceutical strategies against currently incurable ALS.

show abstract

“…S4), and the fact that similar m values were determined for all PFN1 variants by the urea denaturation analysis suggested these proteins adopt similar tertiary structures as well (Table 1) (18). To probe further for potential structural differences between PFN1 WT and ALS-linked variants, these proteins were subjected to native gel electrophoresis, a biochemical technique capable of detecting conformational differences between misfolded variants and their WT counterparts (19). PFN1 WT and E117G migrated predominately as single, distinct bands with similar mobility, whereas multiple bands of slower mobility were observed for PFN1 variants C71G, M114T, and G118V (Fig.…”

Section: Als-linked Mutations Induce a Misfolded Conformation Within mentioning

confidence: 99%

Structural basis for mutation-induced destabilization of profilin 1 in ALS

Boopathy

Silvas

Tischbein

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

110

View full text Add to dashboard Cite

Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutationinduced destabilization can account for the high propensity of ALSlinked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.amyotrophic lateral sclerosis | profilin 1 | protein stability | X-ray crystallography | protein misfolding M utations in the profilin 1 gene (PFN1) were recently associated with both familial and sporadic forms of amyotrophic lateral sclerosis (ALS) (1, 2), an incurable and fatal neurodegenerative disease that primarily targets motor neurons (3). The etiology of sporadic ALS is poorly understood, whereas familial ALS is caused by inheritable genetic defects in defined genes such as PFN1 (3). PFN1 is a 15-kDa protein that is best known for its role in actin dynamics in the context of endocytosis, membrane trafficking, cell motility, and neuronal growth and differentiation (4). In addition to binding monomeric or G-actin, PFN1 also binds to a host of different proteins through their poly-L-proline motifs and to lipids such as phosphatidylinositol 4,5-bisphosphate (4, 5). However, little is known about the mechanism(s) associated with PFN1-mediated ALS pathogenesis. The observation that most ALS-linked PFN1 variants are highly prone to aggregation in mammalian cultured cells suggests that diseasecausing mutations induce an altered, or misfolded, conformation within PFN1 (2). Protein misfolding is a hallmark feature of most neurodegenerative diseases, including ALS (3), and can contribute to disease through both gain-of-toxic-function and loss-of-normalfunction mechanisms (6). Although mutations in PFN1 cause ALS through a dominant inheritance mode (2), there is some evidence supporting a loss-of-function mechanism for mutant PFN1. For example, ALS-linked mutations were shown to abrogate the binding of PFN1 to actin (2) and to impair the incorporation of PFN1 into cytoplasmic stress granules during arsenite-induced stress (7) in cultured cells. Moreover, ectopic expression of these variants in murine motor neurons led to a reduction in both axon outgrowth and growth cone size, consistent with a loss of function through a dominant-negative mechanism (2).Although ALS-linked mutations were shown to induce PFN1 aggregation, the ef...

show abstract

Identification of a Misfolded Region in Superoxide Dismutase 1 That Is Exposed in Amyotrophic Lateral Sclerosis

Cited by 30 publications

References 53 publications

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

Nonnative SOD1 trimer is toxic to motor neurons in a model of amyotrophic lateral sclerosis

A Phosphomimetic Mutation Stabilizes SOD1 and Rescues Cell Viability in the Context of an ALS-Associated Mutation

Structural basis for mutation-induced destabilization of profilin 1 in ALS

Contact Info

Product

Resources

About