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Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

ABSTRACT: This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn(2+) stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at lysosomal pH-than values predicted from standard pKa values of amino acids and formal oxidation states of metal ions. At pH 7.4, the G85R, D90A, and G93R substitutions diminished the net negative charge of dimeric SOD1 by up to +2.29 units more than predicted; E100K lowered net charge by less than predicted. The binding of a single Zn(2+) to mutant SOD1 lowered its net charge by an additional +2.33?±?0.01 to +3.18?±?0.02 units, however, each protein regulated net charge when binding a second, third, or fourth Zn(2+) (?Z?

SUBMITTER: Shi Y

PROVIDER: S-EPMC4287002 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

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Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

Shi Yunhua Y Abdolvahabi Alireza A Shaw Bryan F BF

Protein science : a publication of the Protein Society 20140807 10

This article utilized "protein charge ladders"-chemical derivatives of proteins with similar structure, but systematically altered net charge-to quantify how missense mutations that cause amyotrophic lateral sclerosis (ALS) affect the net negative charge (Z) of superoxide dismutase-1 (SOD1) as a function of subcellular pH and Zn(2+) stoichiometry. Capillary electrophoresis revealed that the net charge of ALS-variant SOD1 can be different in sign and in magnitude-by up to 7.4 units per dimer at l ...[more]

PMID: 25052939

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Project description:Over 130 mutations to copper, zinc superoxide dismutase (SOD) are implicated in the selective death of motor neurons found in 25% of patients with familial amyotrophic lateral sclerosis (ALS). Despite their widespread distribution, ALS mutations appear positioned to cause structural and misfolding defects. Such defects decrease SOD's affinity for zinc, and loss of zinc from SOD is sufficient to induce apoptosis in motor neurons in vitro. To examine the importance of the zinc site in the structure and pathogenesis of human SOD, we determined the 2.0-A-resolution crystal structure of a designed zinc-deficient human SOD, in which two zinc-binding ligands have been mutated to hydrogen-bonding serine residues. This structure revealed a 9 degrees twist of the subunits, which opens the SOD dimer interface and represents the largest intersubunit rotational shift observed for a human SOD variant. Furthermore, the electrostatic loop and zinc-binding subloop were partly disordered, the catalytically important Arg143 was rotated away from the active site, and the normally rigid intramolecular Cys57-Cys146 disulfide bridge assumed two conformations. Together, these changes allow small molecules greater access to the catalytic copper, consistent with the observed increased redox activity of zinc-deficient SOD. Moreover, the dimer interface is weakened and the Cys57-Cys146 disulfide is more labile, as demonstrated by the increased aggregation of zinc-deficient SOD in the presence of a thiol reductant. However, equimolar Cu,Zn SOD rapidly forms heterodimers with zinc-deficient SOD (t1/2 approximately 15 min) and prevents aggregation. The stabilization of zinc-deficient SOD as a heterodimer with Cu,Zn SOD may contribute to the dominant inheritance of ALS mutations. These results have general implications for the importance of framework stability on normal metalloenzyme function and specific implications for the role of zinc ion in the fatal neuropathology associated with SOD mutations.

Project description:Mutations in superoxide dismutase 1 (SOD1), which are one cause of familial amyotrophic lateral sclerosis (fALS), induce misfolding and aggregation of the protein. Misfolding can be detected by the binding of antibodies raised against peptide epitopes that are normally buried in the native conformation, shifts in solubility in non-ionic detergents, and the formation of macromolecular inclusions. In the present study, we investigate the relationship between detergent-insoluble and sedimentable forms of mutant SOD1, forms of mutant SOD1 with aberrantly accessible epitopes, and mutant protein in inclusions with the goal of defining the spectrum of misfolded states that mutant SOD1 can adopt.Using combined approaches in cultured cell models, we demonstrate that a substantial fraction of mutant SOD1 adopts a non-native conformation that remains soluble and freely mobile. We also show that mutant SOD1 can produce multimeric assemblies of which some are insoluble in detergent and large enough to sediment by ultracentrifugation and some are large enough to detect visually. Three conformationally restricted antibodies were found to be useful in discriminating mal-folded forms of mutant SOD1. An antibody termed C4F6 displays properties consistent with recognition of soluble, freely mobile, mal-folded mutant SOD1. An antibody termed SEDI, which recognizes C-terminal residues, detects larger inclusion structures as well as soluble misfolded entities. An antibody termed hSOD1, which recognizes aa 24-36, detects an epitope shared by soluble non-natively folded WT and mutant SOD1. This epitope becomes inaccessible in aggregates of mutant SOD1.Our studies demonstrate how different methods of detecting misfolding and aggregation of mutant SOD1 reveal different forms of aberrantly folded protein. Immunological and biochemical methods can be used in combination to detect soluble and insoluble misfolded forms of mutant SOD1. Our findings support the view that mutant SOD1 can adopt multiple misfolded conformations with the potential that different structural variants mediate different aspects of fALS.

Project description:The development of molecular biology has led to the identification of protein-based therapeutics as an intriguing approach for the treatment of a wide range of diseases. To manufacture transcellular protein delivery shuttles, we attempted charge reversal chemistry on native proteins [e.g., superoxide dismutase (SOD): an enzyme capable of scavenging detrimental reactive oxygen species] by the selective conversion of the positively charged amino residues of native SOD to conjugated negatively charged citraconic moieties, eliciting overall negatively charged polyelectrolytes for the subsequent electrostatic self-assembly with cationic metal-organic framework (MOF) derivatives into protein delivery systems. Please note that the charge conversion was reversible, restoring the original amino groups in intracellular acidic endosome compartments (pH 5), which afforded facile charge reversible functions to reclaim the active SOD in the cell interior. In particular, the strategic manufacture of dendritic MOF supramolecular architectures as transcellular shuttles for the aforementioned charge-reversible SOD derivatives is noteworthy. The MOF was surface-functionalized with several polycationic segments, thus contributing to the hyper-charged architecture for the easy accommodation of the negatively charged SOD derivatives. Consequently, the SOD derivatives managed to internalize into cells by hitchhiking via endocytosis of the positively charged MOF. Once they resided in the acidic endosomes, the charge reversal of the SOD derivatives could occur smoothly and result in reduced interactions between the charged-reversed SOD and MOF, leading to the release of active SOD. Simultaneously, the dendritic MOF due to protein release presented a highly positive-charged architecture to provoke endosome membrane disruption, consequently spurring the translocation of SOD to the cytosol for the execution of its enzymatic activities. Herein, the intracellular ROS level of the activated macrophages was validated to be markedly suppressed by our proposed transcellular SOD shuttles, thereby indicating their wide availability to diverse functional proteins for biomedical applications.

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Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

Publications

Protein charge ladders reveal that the net charge of ALS-linked superoxide dismutase can be different in sign and magnitude from predicted values.

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