Dataset Information

Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS).

ABSTRACT: Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and whether they behave similarly to their untagged SOD1 counterparts.Here we compare the physicochemical properties of SOD1 and the effects of GFP-tagging on its intracellular behaviour. Immunostaining demonstrated that SOD1 alone and GFP-SOD1 have an indistinguishable intracellular distribution in PC12 cells. Cultured primary motor neurons expressing GFP or GFP-SOD1 showed identical patterns of cytoplasmic expression and of movement within the axon. However, GFP tagging of SOD1 was found to alter some of the intrinsic properties of SOD1, including stability and specific activity. Evaluation of wildtype and mutant SOD1, tagged at either the N- or C-terminus with GFP, in PC12 cells demonstrated that some chimaeric proteins were degraded to the individual proteins, SOD1 and GFP.Our findings indicate that most, but not all, properties of SOD1 remain the same with a GFP tag.

SUBMITTER: Stevens JC

PROVIDER: S-EPMC2833207 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS).

Stevens James C JC Chia Ruth R Hendriks William T WT Bros-Facer Virginie V van Minnen Jan J Martin Joanne E JE Jackson Graham S GS Greensmith Linda L Schiavo Giampietro G Fisher Elizabeth M C EM

PloS one 20100308 3

<h4>Background</h4>Since the discovery that mutations in the enzyme SOD1 are causative in human amyotrophic lateral sclerosis (ALS), many strategies have been employed to elucidate the toxic properties of this ubiquitously expressed mutant protein, including the generation of GFP-SOD1 chimaeric proteins for studies in protein localization by direct visualization using fluorescence microscopy. However, little is known about the biochemical and physical properties of these chimaeric proteins, and ...[more]

PMID: 20221404

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Project description:Amyotrophic Lateral Sclerosis (ALS) is the most frequent motor neuron disorder, with a significant social and economic burden. ALS remains incurable, and the only drugs approved for its treatments confers a survival benefit of a few months for the patients. Missense mutations in superoxide dismutase 1 (SOD1), a major cytoplasmic antioxidant enzyme, has been associated with ALS development, accounting for 23% of its familial cases and 7% of all sporadic cases. This work aims to characterize in silico the structural and functional effects of SOD1 protein variants. Missense mutations in SOD1 were compiled from the literature and databases. Twelve algorithms were used to predict the functional and stability effects of these mutations. ConSurf was used to estimate the evolutionary conservation of SOD1 amino-acids. GROMACS was used to perform molecular dynamics (MD) simulations of SOD1 wild-type and variants A4V, D90A, H46R, and I113T, which account for approximately half of all ALS-SOD1 cases in the United States, Europe, Japan, and United Kingdom, respectively. 233 missense mutations in SOD1 protein were compiled from the databases and literature consulted. The predictive analyses pointed to an elevated rate of deleterious and destabilizing predictions for the analyzed variants, indicating their harmful effects. The ConSurf analysis suggested that mutations in SOD1 mainly affect conserved and possibly functionally essential amino acids. The MD analyses pointed to flexibility and essential dynamics alterations at the electrostatic and metal-binding loops of variants A4V, D90A, H46R, and I113T that could lead to aberrant interactions triggering toxic protein aggregation. These alterations may have harmful implications for SOD1 and explain their association with ALS. Understanding the effects of SOD1 mutations on protein structure and function facilitates the design of further experiments and provides relevant information on the molecular mechanism of pathology, which may contribute to improvements in existing treatments for ALS.

Dataset Information

Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS).

Publications

Modification of superoxide dismutase 1 (SOD1) properties by a GFP tag--implications for research into amyotrophic lateral sclerosis (ALS).

Similar Datasets

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