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Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers.

ABSTRACT: Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologically prevalent post-translational oxidative modification, on the stabilities of wild type SOD1 and two phenotypically diverse FALS mutants, A4V and I112T. Glutathionylation results in profound destabilization of SOD1(WT) dimers, increasing the equilibrium dissociation constant K(d) to ~10-20 ?M, comparable to that of the aggressive A4V mutant. SOD1(A4V) is further destabilized by glutathionylation, experiencing an ~30-fold increase in K(d). Dissociation kinetics of glutathionylated SOD1(WT) and SOD1(A4V) are unchanged, as measured by surface plasmon resonance, indicating that glutathionylation destabilizes these variants by decreasing association rate. In contrast, SOD1(I112T) has a modestly increased dissociation rate but no change in K(d) when glutathionylated. Using computational structural modeling, we show that the distinct effects of glutathionylation on different SOD1 variants correspond to changes in composition of the dimer interface. Our experimental and computational results show that Cys-111 glutathionylation induces structural rearrangements that modulate stability of both wild type and FALS mutant SOD1. The distinct sensitivities of SOD1 variants to glutathionylation, a modification that acts in part as a coping mechanism for oxidative stress, suggest a novel mode by which redox regulation and aggregation propensity interact in ALS.

SUBMITTER: Redler RL

PROVIDER: S-EPMC3281512 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers.

Redler Rachel L RL Wilcox Kyle C KC Proctor Elizabeth A EA Fee Lanette L Caplow Michael M Dokholyan Nikolay V NV

Biochemistry 20110718 32

Mutation of the ubiquitous cytosolic enzyme Cu/Zn superoxide dismutase (SOD1) is hypothesized to cause familial amyotrophic lateral sclerosis (FALS) through structural destabilization leading to misfolding and aggregation. Considering the late onset of symptoms as well as the phenotypic variability among patients with identical SOD1 mutations, it is clear that nongenetic factor(s) impact ALS etiology and disease progression. Here we examine the effect of Cys-111 glutathionylation, a physiologica ...[more]

PMID: 21739997

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Project description:95% of patients with ductal pancreatic cancer carry 12th codon activating mutations in their KRAS2 oncogenes. Early whole body imaging of mutant KRAS2 mRNA activation in pancreatic cancer would contribute to disease management. Scintigraphic hybridization probes to visualize gene activity in vivo constitute a new paradigm in molecular imaging. We have previously imaged mutant KRAS2 mRNA activation in pancreatic cancer xenografts by positron emission tomography (PET) based on a single radiometal, (64)Cu, chelated to a 1,4,7,10-tetra(carboxymethylaza)cyclododecane (DOTA) chelator, connected via a flexible, hydrophilic spacer, aminoethoxyethoxyacetate (AEEA), to the N-terminus of a mutant KRAS2 peptide nucleic acid (PNA) hybridization probe. A peptide analogue of insulin-like growth factor 1 (IGF1), connected to a C-terminal AEEA, enabled receptor-mediated endocytosis. We hypothesized that a polydiamidopropanoyl (PDAP) dendrimer (generation m), with increasing numbers (n) of DOTA chelators, extended via an N-terminal AEEA from a mutant KRAS2 PNA with a C-terminal AEEA and IGF1 analogue could enable more intense external imaging of pancreatic cancer xenografts that overexpress IGF1 receptor and mutant KRAS2 mRNA. ([(111)In]DOTA-AEEA)(n)-PDAP(m)-AEEA(2)-KRAS2 PNA-AEEA-IGF1 analogues were prepared and administered intravenously into immunocompromised mice bearing human AsPC1 (G12D) pancreatic cancer xenografts. CAPAN2 (G12 V) pancreatic cancer xenografts served as a cellular KRAS2 mismatch control. Scintigraphic tumor/muscle image intensity ratios for complementary [(111)In](n)-PDAP(m)-KRAS2 G12D probes increased from 3.1 +/- 0.2 at n = 2, m = 1, to 4.1 +/- 0.3 at n = 8, m = 3, to 6.2 +/- 0.4 at n = 16, m = 4, in AsPC1 (G12D) xenografts. Single mismatch [(111)In](n)-PDAP(m)-KRAS2 G12 V control probes showed lower tumor/muscle ratios (3.0 +/- 0.6 at n = 2, m = 1, 2.6 +/- 0.9 at n = 8, m = 3, and 3.7 +/- 0.3 at n = 16, m = 4). The mismatch results were comparable to the PNA-free [(111)In]DOTA control results. Simultaneous administration of nonradioactive Gd(n)-KRAS2 G12 V probes (n = 2 or 8) increased accumulation of [(111)In](8)KRAS2 G12 V probes 3-6-fold in pancreatic cancer CAPAN2 xenografts and other tissues, except for a 2-fold decrease in the kidneys. As a result, tissue distribution tumor/muscle ratios of (111)In uptake increased from 3.1 +/- 0.5 to 6.5 +/- 1.0, and the kidney/tumor ratio of (111)In uptake decreased by more than 5-fold from 174.8 +/- 17.5 to 30.8 +/- 3.1. Thus, PDAP dendrimers with up to 16 DOTA chelators attached to PNA-IGF1 analogs, as well as simultaneous administration of the elevated dose of nonradioactive Gd(n)-KRAS2 G12 V probes, enhanced tumor uptake of [(111)In](n)KRAS2 PNA probes. These results also imply that Gd(III) dendrimeric hybridization probes might be suitable for magnetic resonance imaging of gene expression in tumors, because the higher generations of the dendrimers, including the NMR contrast Gd(n)-KRAS2 G12 V probes, improved tumor accumulation of the probes and specificity of tumor imaging.

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Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers.

Publications

Glutathionylation at Cys-111 induces dissociation of wild type and FALS mutant SOD1 dimers.

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