Dataset Information

Paraquat Exposure and Sod2 Knockdown have Dissimilar Impacts on the Drosophila melanogaster Carbonylated Protein Proteome

ABSTRACT: Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of superoxide, a progenitor reactive oxygen species, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since reactive carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin-coated beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ mass spectrometry-based proteomics approach. While Paraquat exposure and Sod2 knockdown have similar phenotypes, differences in protein carbonylation were anticipated because Paraquat exposure was expected to increase the concentration of superoxide throughout the cell while Sod2 knockdown was only expected to raise the concentration of superoxide in the mitochondrial matrix. Paraquat exposure resulted in widespread increases in carbonylated protein relative abundance: the median Paraquat-exposed to control carbonylated protein relative abundance ratio was 1.53. For Sod2 knockdown, in contrast, the median carbonylated protein relative abundance ratios were 1.13 versus the RNA interference driver control and 1.05 versus the RNA interference transgene control. However, some proteins did show large increases in carbonylated protein relative abundance on Sod2 knockdown, most notably cytochrome c oxidase subunit Vb, possibly providing some indication of the molecular basis of the Sod2-knockdown phenotype.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Drosophila Melanogaster (fruit Fly)

TISSUE(S): Whole Body

SUBMITTER: David Simpson

LAB HEAD: Scott Gronert

PROVIDER: PXD001528 | Pride | 2014-11-26

REPOSITORIES: Pride

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Publications

Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome.

Narayanasamy Suresh K SK Simpson David C DC Martin Ian I Grotewiel Mike M Gronert Scott S

Proteomics 20140919 21-22

Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disord ...[more]

PMID: 25091824

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Project description:The mitochondrial superoxide dismutase (SOD2) is a major antioxidant protein which detoxifies superoxide anion radicals generated by mitochondrial respiration (Weisiger and Fridovich, J. Biol. Chem. 1973). We designed a model of oxidative stress-induced anemia caused by SOD2-deficiency (Friedman et al. J. Exp. Med. 2001). Our previous work showed that mice reconstituted with SOD2-deficient hematopoietic stem cells develop an anemia with striking similarity to human sideroblastic anemia (SA) (Friedman et al. Blood 2004; Martin et al. Exp Hematol 2005). Our overall goal was to define early events in the pathogenesis of SOD2-deficiency SA and, in particular, to identify genes involved in the response of erythroid progenitors to oxidative stress. We compared gene expression of sorted TER-119+ CD71+ erythroblasts from SOD2-/- ('KO') versus Sod2+/+ ('WT') hematopoietic stem cell recipients using cDNA microarrays. Samples used in this study were re-hybridized with GLYCOv2 oligonucleotide arrays. GLYCOv2 oligonucleotide arrays are custom Affymetrix GeneChip arrays (Affymetrix, Santa Clara, CA) designed for the Functional Glycomics Gateway, collaboration between the Consortium for Functional Glycomics (CFG, http://www.functionalglycomics.org/) and Nature Publishing Group. A complete description of the array can be found at http://www.functionalglycomics.org/static/consortium/resources/resourcecoree.shtml . Hybridization results and quality control details can be found at https://www.functionalglycomics.org/glycomics/publicdata/microarray.jsp , by clicking on the âRaw Dataâ icon link for microarray experiment âJeff Friedman 1: Sod2 KO anemic miceâ. Briefly but importantly, one Sod2-/- sample, which was prepared separately, and whose GAPDH 3'/5' ratio was off range, was legitimately removed from the analysis of the GLYCOv2 and 430 2.0 arrays. Experiment Overall Design: We sorted Sod2-/- and Sod2+/+ size-matched mouse erythroblasts based on the expression of two developmental surface markers, CD71 and TER-119. Total RNA from 4 biological replicates per genotype were extracted and hybridized on 8 Affymetrix Mouse Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA). After quality controls (see scan protocol), analysis was performed with GeneSifter (VizX Labs, Seattle, WA) using 4 Sod2+/+ (control samples, WT) and 3 Sod2-/- (KO) replicates.

Dataset Information

Paraquat Exposure and Sod2 Knockdown have Dissimilar Impacts on the Drosophila melanogaster Carbonylated Protein Proteome

Publications

Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome.

Similar Datasets

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