Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral  sclerosis show enhanced formation of aggregates
            <i>in vitro</i>

Stathopulos, Peter B.; Rumfeldt, Jessica A.O.; Scholz, Guenter A.; Irani, Roxanna A.; Frey, H.E.; Hallewell, Robert A.; Lepock, James R.; Meiering, Elizabeth M.

doi:10.1073/pnas.1237797100

Cited by 246 publications

(295 citation statements)

References 38 publications

(50 reference statements)

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“…This conclusion emphasizes the observed correlation between the effects of mutations and the onset of diseases (12,17,23,26). It is a reassuring result in the context of studies of this type that the physicochemical principles observed through experiments involving simplified noncellular environments are so relevant to the systems in vivo.…”

Section: Evaluation Of the Stabilities And Aggregation Propensities Omentioning

confidence: 60%

Investigating the Effects of Mutations on Protein Aggregation in the Cell

Calloni

Zoffoli

Stefani

et al. 2005

Journal of Biological Chemistry

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The conversion of peptides and proteins into highly ordered and intractable aggregates is associated with a range of debilitating human diseases and represents a widespread problem in biotechnology. Protein engineering studies carried out in vitro have shown that mutations promote aggregation when they either destabilize the native state of a globular protein or accelerate the conversion of unfolded or partially folded conformations into oligomeric structures. We have extended such studies to investigate protein aggregation in vivo where a number of additional factors able to modify dramatically the aggregation behavior of proteins are present. We have expressed, in Escherichia coli cells, an E. coli protein domain, HypF-N. The results for a range of mutational variants indicate that although mutants with a conformational stability similar to that of the wild-type protein are soluble in the E. coli cytosol, variants with single point mutations predicted to destabilize the protein invariably aggregate after expression. We show, however, that aggregation of destabilized variants can be prevented by incorporating multiple mutations designed to reduce the intrinsic propensity of the polypeptide chain to aggregate; in the cases discussed here, this is achieved by an increase in the net charge of the protein. These results suggest that the principles being established to rationalize aggregation behavior in vitro have general validity for situations in vivo where aggregation has both biotechnological and medical relevance.

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Section: Evaluation Of the Stabilities And Aggregation Propensities Omentioning

confidence: 60%

Investigating the Effects of Mutations on Protein Aggregation in the Cell

Calloni

Zoffoli

Stefani

et al. 2005

Journal of Biological Chemistry

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“…For most experiments herein, we employed a well-established pseudo WT (pWT) construct in order to facilitate measurements of stability and aggregation of reduced apo SOD1s and avoid complications caused by aberrant disulfide bond formation (8,(14)(15)(16)(20)(21)(22). In pWT, the nonconserved free cysteines at residues 6 and 111 are replaced by alanine and serine, respectively, whereas the highly conserved cysteines at residues 57 and 146 are retained (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…S1A). Numerous in vivo and in vitro studies have shown that various immature, destabilized forms of SOD1 are prone to aggregate, and this is often enhanced by disease-associated mutations (8)(9)(10)(11)(12)(13)(14)(15)(16). Recently, attention has focused on aggregation of the most immature form of SOD1, in which the disulfide bond is reduced and no metals are bound (reduced apo).…”

mentioning

confidence: 99%

Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

Vassall¹,

Stubbs²,

Primmer³

et al. 2011

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

100

145

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Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS), where aggregation of Cu/Zn superoxide dismutase (SOD1) is implicated in causing neurodegeneration. Recent studies have suggested that destabilization and aggregation of the most immature form of SOD1, the disulfide-reduced, unmetallated (apo) protein is particularly important in causing ALS. We report herein in depth analyses of the effects of chemically and structurally diverse ALS-associated mutations on the stability and aggregation of reduced apo SOD1. In contrast with previous studies, we find that various reduced apo SOD1 mutants undergo highly reversible thermal denaturation with little aggregation, enabling quantitative thermodynamic stability analyses. In the absence of ALS-associated mutations, reduced apo SOD1 is marginally stable but predominantly folded. Mutations generally result in slight decreases to substantial increases in the fraction of unfolded protein. Calorimetry, ultracentrifugation, and light scattering show that all mutations enhance aggregation propensity, with the effects varying widely, from subtle increases in most cases, to pronounced formation of 40–100 nm soluble aggregates by A4V, a mutation that is associated with particularly short disease duration. Interestingly, although there is a correlation between observed aggregation and stability, there is minimal to no correlation between observed aggregation, predicted aggregation propensity, and disease characteristics. These findings suggest that reduced apo SOD1 does not play a dominant role in modulating disease. Rather, additional and/or multiple forms of SOD1 and additional biophysical and biological factors are needed to account for the toxicity of mutant SOD1 in ALS.

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“…Overexpression of SOD1 mutants in rodents recapitulates ALS clinical and pathological hallmarks through a toxic gain of function (1). Many mutations in SOD1 decrease its stability and increase its unfolding rates and propensity to aggregate (2). High molecular weight complexes of SOD1 are observed in mammalian cells and spinal cords of transgenic mice expressing this mutant protein (3).…”

Section: Neurodegeneration ͉ Protein Misfoldingmentioning

confidence: 99%

Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model

Kikuchi

Almer

Yamashita

et al. 2006

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

282

231

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Mutation in superoxide dismutase-1 (SOD1), which is a cause of ALS, alters the folding patterns of this protein. Accumulation of misfolded mutant SOD1 might activate endoplasmic reticulum (ER) stress pathways. Here we show that transgenic mice expressing ALS-linked SOD1 mutants exhibit molecular alterations indicative of a recruitment of ER's signaling machinery. We demonstrate by biochemical and morphological methods that mutant SOD1 accumulates inside the ER, where it forms insoluble high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein. These alterations are age-and regionspecific, because they develop over the course of the disease and occur in the affected spinal cord but not in the nonaffected cerebellum in transgenic mutant SOD1 mice. Our results suggest a toxic mechanism for mutant SOD1 by which this ubiquitously expressed pathogenic protein could affect motor neuron survival and contribute to the selective motor neuronal degeneration in ALS.neurodegeneration ͉ protein misfolding A LS is the most common adult-onset paralytic disease characterized by a loss of motor neurons in the cerebral cortex, brainstem, and spinal cord. Insights into the neurodegenerative mechanisms followed the discovery that dominant mutations in the gene for superoxide dismutase-1 (SOD1) cause familial ALS. Overexpression of SOD1 mutants in rodents recapitulates ALS clinical and pathological hallmarks through a toxic gain of function (1). Many mutations in SOD1 decrease its stability and increase its unfolding rates and propensity to aggregate (2). High molecular weight complexes of SOD1 are observed in mammalian cells and spinal cords of transgenic mice expressing this mutant protein (3). In these animals, intracellular ubiquitin-positive proteinaceous inclusions are also often seen in spinal cord motor neurons and, in some cases, in neighboring astrocytes (3-5). These findings posit that accumulation of misfolded mutant SOD1 could contribute to the demise of motor neurons.Endoplasmic reticulum (ER) stress signaling, otherwise known as the unfolded protein response (UPR), is triggered by an increased load of misfolded proteins in the organelle (6). Herein, we show that transgenic mice expressing mutant SOD1 exhibit age-and region-specific molecular alterations indicative of a broad recruitment of ER signaling pathways, including caspase-12, a prototypical ER cell death effector (7). We also show that mutant SOD1, and to a lesser extent wild-type SOD1 (SOD1 WT ), do accumulate in the ER. Within this organelle, mutant, but not SOD1 WT , forms high molecular weight species and interacts with the ER chaperone immunoglobulin-binding protein (BiP), which is a key component of the ER misfolded protein recognition machinery (6). The preferential accumulation of mutant SOD1 in the ER in the spinal cord cells and the ensuing stress response may represent novel aspects of motor neuron degeneration in this ALS model.

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Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro

Cited by 246 publications

References 38 publications

Investigating the Effects of Mutations on Protein Aggregation in the Cell

Investigating the Effects of Mutations on Protein Aggregation in the Cell

Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

Spinal cord endoplasmic reticulum stress associated with a microsomal accumulation of mutant superoxide dismutase-1 in an ALS model

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