Vladimir I. Klichko

The peroxiredoxin gene family in Drosophila melanogaster.

Five peroxiredoxin genes have been identified in Drosophila melanogaster on the basis of a genome-wide search. Three of the genes (DPx-4156, DPx-4783, and DPx-5037) fall into the 2-Cys subgroup, while the other two (DPx-2540 and DPx-6005) belong to the 1-Cys subgroup. Using cDNAs, all five were expressed in E. coli and the purified recombinant proteins were shown to reduce H(2)O(2) in the presence of dithiothreitol. The three 2-Cys Prx were also shown to be active in the thioredoxin system and were, consequently, classified as thioredoxin peroxidases. Antisera raised against the DPx-4783 recombinant protein crossreacted with all family members and recognized protein species of the predicted sizes (22-27 kD). All five family members, when individually overexpressed in Drosophila S2 cells, conferred some resistance to H(2)O(2) treatment, as measured by cell viability. Functional diversification of the Drosophila peroxiredoxin family members was suggested by two lines of evidence: (i) the patterns of mRNA accumulation varied for the different genes during development and (ii) recombinant proteins fused to an epitope tag and overexpressed in Drosophila cells, differed in subcellular localizations--three proteins occurred in the cytosol, one was localized to the mitochondria, and one was found to be secreted.

Peroxiredoxin 5 confers protection against oxidative stress and apoptosis and also promotes longevity in Drosophila

Peroxiredoxin 5 is a distinct isoform of the peroxiredoxin gene family. The antioxidative and anti-apoptotic functions of peroxiredoxin 5 have been extensively demonstrated in cell culture experiments. In the present paper, we provide the first functional analysis of peroxiredoxin 5 in a multicellular organism, Drosophila melanogaster. Similar to its mammalian, yeast or human counterparts, dPrx5 (Drosophila peroxiredoxin 5) is expressed in several cellular compartments, including the cytosol, nucleus and the mitochondrion. Global overexpression of dPrx5 in flies increased resistance to oxidative stress and extended their life span by up to 30% under normal conditions. The dprx5(-/-) null flies were comparatively more susceptible to oxidative stress, had higher incidence of apoptosis, and a shortened life span. TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling) analysis revealed that the dprx5(-/-) null mutant had discernible tissue-specific apoptotic patterns, similar to those observed in control flies exposed to paraquat. In addition, apoptosis was particularly notable in oenocytes. During development the dPrx5 levels co-varied with ecdysone pulses, suggesting inter-relationship between ecdystreroids and dPrx5 expression. The importance of dPrx5 for development was further underscored by the embryonic lethal phenotype of progeny derived from the dprx5(-/-) null mutant. Results from the present study suggest that the antioxidant and anti-apoptotic activities of dPrx5 play a critical role in development and aging of the fly.

Overexpression of Glucose-6-phosphate Dehydrogenase Extends the Life Span of Drosophila melanogaster

The redox state of tissues tends to become progressively more prooxidizing during the aging process. The hypothesis tested in this study was that enhancement of reductive capacity by overexpression of glucose-6-phosphate dehydrogenase (G6PD), a key enzyme for NADPH biosynthesis, could protect against oxidative stress and extend the life span of transgenic Drosophila melanogaster. Overexpression of G6PD was achieved by combining a UAS-G6PD responder transgene at one of four independent loci with either a broad expression (armadillo-GAL4, Tubulin-GAL4, C23-GAL4, and da-GAL4) or a neuronal driver (D42-GAL4 and Appl-GAL4). The mean life spans of G6PD overexpressor flies were extended, in comparison with driver and responder controls, as follows: armadillo-GAL4 (up to 38%), Tubulin-GAL4 (up to 29%), C23-GAL4 (up to 27%), da-GAL4 (up to 24%), D42-GAL4 (up to 18%), and Appl-GAL4 (up to 16%). The G6PD enzymatic activity was increased, as were the levels of NADPH, NADH, and the GSH/GSSG ratio. Resistance to experimental oxidative stress was enhanced. Furthermore, metabolic rates and fertility were essentially the same in G6PD overexpressors and control flies. Collectively, the results demonstrate that enhancement of the NADPH biosynthetic capability can extend the life span of a relatively long-lived strain of flies, which supports the oxidative stress hypothesis of aging.

Anaerobic induction of Bacillus anthracis hemolytic activity.

A number of genes in Bacillus anthracis encode for proteins homologous to the membrane-damaging factors known as pathogenic determinants in different bacteria. B. anthracis, however, has been traditionally considered non-hemolytic, and the recently identified hemolytic genes have been suggested to be transcriptionally silent. We found that the hemolytic genes of B. anthracis, collectively designated as anthralysins (Anls), could be induced in strict anaerobic conditions. We also demonstrate that Anl genes are expressed at the early stages of infection within macrophages by vegetating bacilli after spore germination. Cooperative and synergistic enhancement of the pore-forming and phospholipase C (PLC) activities of the Anls was found in hemolytic tests on human, but not sheep, red blood cells (RBC). These findings imply Anls as B. anthracis pathogenic determinants and highlight oxygen limitation as environmental factor controlling their expression at both early and late stages of infection.

Circadian Regulation of Glutathione Levels and Biosynthesis in Drosophila melanogaster

Circadian clocks generate daily rhythms in neuronal, physiological, and metabolic functions. Previous studies in mammals reported daily fluctuations in levels of the major endogenous antioxidant, glutathione (GSH), but the molecular mechanisms that govern such fluctuations remained unknown. To address this question, we used the model species Drosophila, which has a rich arsenal of genetic tools. Previously, we showed that loss of the circadian clock increased oxidative damage and caused neurodegenerative changes in the brain, while enhanced GSH production in neuronal tissue conferred beneficial effects on fly survivorship under normal and stress conditions. In the current study we report that the GSH concentrations in fly heads fluctuate in a circadian clock-dependent manner. We further demonstrate a rhythm in activity of glutamate cysteine ligase (GCL), the rate-limiting enzyme in glutathione biosynthesis. Significant rhythms were also observed for mRNA levels of genes encoding the catalytic (Gclc) and modulatory (Gclm) subunits comprising the GCL holoenzyme. Furthermore, we found that the expression of a glutathione S-transferase, GstD1, which utilizes GSH in cellular detoxification, significantly fluctuated during the circadian day. To directly address the role of the clock in regulating GSH-related rhythms, the expression levels of the GCL subunits and GstD1, as well as GCL activity and GSH production were evaluated in flies with a null mutation in the clock genes cycle and period. The rhythms observed in control flies were not evident in the clock mutants, thus linking glutathione production and utilization to the circadian system. Together, these data suggest that the circadian system modulates pathways involved in production and utilization of glutathione.

Peroxiredoxin 5 modulates immune response in Drosophila.

BACKGROUND Peroxiredoxins are redox-sensing enzymes with multiple cellular functions. Previously, we reported on the potent antioxidant function of Drosophila peroxiredoxin 5 (dPrx5). Studies with mammalian and human cells suggest that peroxiredoxins can modulate immune-related signaling. METHODS Survivorship studies and bacteriological analysis were used to determine resistance of flies to fungal and bacterial infections. RT-PCR and immunoblot analyses determined expression of dPrx5 and immunity factors in response to bacterial challenge. Double mutants for dprx5 gene and genes comprising the Imd/Relish and dTak1/Basket branches of the immune signaling pathways were used in epistatic analysis. RESULTS The dprx5 mutant flies were more resistant to bacterial infection than controls, while flies overexpressing dPrx5 were more susceptible. The enhanced resistance to bacteria was accompanied by rapid induction of the Imd-dependent antimicrobial peptides, phosphorylation of the JNK kinase Basket and altered transcriptional profiling of the transient response genes, puckered, ets21C and relish, while the opposite effects were observed in flies over-expressing dPrx5. Epistatic analysis of double mutants, using attacin D and Puckered as read outs of activation of the Imd and JNK pathways, implicated dPrx5 function in the control of the dTak1-JNK arm of immune signaling. CONCLUSIONS Differential effects on fly survivorship suggested a trade-off between the antioxidant and immune functions of dPrx5. Molecular and epistatic analyses identified dPrx5 as a negative regulator in the dTak1-JNK arm of immune signaling. GENERAL SIGNIFICANCE Our findings suggest that peroxiredoxins play an important modulatory role in the Drosophila immune response.

Catalase expression in Drosophila melanogaster is responsive to ecdysone and exhibits both transcriptional and post-transcriptional regulation.

In the present study, we have examined catalase protein and mRNA levels and the factors that may regulate catalase expression in Drosophila melanogaster during development. Both mRNA and protein changes are in general accord with variations in ecdysteroid titer during development. Differences in mRNA and protein accumulation profiles, particularly in embryos and young adults, suggest that catalase may be regulated at both transcriptional and post-transcriptional levels. It was possible to induce catalase expression by administering exogenous 20-hydroxyecdysone (Ec) in culture at certain stages of development (usually at time points corresponding to previously observed hormone and catalase peaks). Experiments with exogenous administration of Ec, cycloheximide, and actinomycin D suggest a complex interplay of factors affecting catalase expression. In cultured third instar larvae, superinduction of catalase occurred in the presence of both Ec and cycloheximide. If ecdysteroid production was suppressed prior to antibiotic treatment by temperature upshift of the conditional mutant dre4(e55), superinduction occurred mostly at the protein level. In cultured adult abdomens, we observed induction by Ec and superinduction in the presence of hormone and translation or transcription inhibitors. Unlike what was observed in larvae, superinduction of catalase protein was dramatically more pronounced in control flies.

CuZn-SOD promoter-driven expression in the Drosophila central nervous system

The aim of this study was to ascertain the status of CuZn superoxide dismutase (CuZn-SOD) expression in the central nervous system of Drosophila melanogaster. Immunoblot analysis of dissected tissue extracts revealed low levels of the CuZn-SOD protein in adult brains relative to other adult and larval tissues. To explore further this observation, three different reporter constructs containing different elements of the CuZn-SOD promoter domain were used for the generation of transgenic flies. A high level of reporter gene expression occurred during the second wave of neurogenesis (third instar and early pupal stages) in scattered, proliferating neuroblasts (NBs) and in proliferation centers of the optic lobe. In mature, postmitotic neurons, this expression was lower relative to other tissues. In adult flies, at all ages examined, there was little if any detectable reporter gene expression in cells of the central nervous system. These results suggest that one of the key components of the antioxidant defenses, CuZn-SOD, is quite low in postmitotic neural tissue, rendering it particularly susceptible to oxidative damage during aging.

MITOCHONDRIAL PEROXIREDOXINS ARE CRITICAL FOR THE MAINTENANCE OF REDOX STATE AND THE SURVIVAL OF ADULT DROSOPHILA

Drosophila mitochondria contain two peroxidases, peroxiredoxin 3 (dPrx3) and peroxiredoxin 5 (dPrx5), which together constitute the sole known intramitochondrial mechanism for the catalytic removal of hydrogen and organic peroxides. dPrx3 exists exclusively within mitochondria, whereas dPrx5 is also present in some other intracellular compartments. Levels of these two peroxiredoxins were genetically manipulated, singly and together, in D. melanogaster, for the purpose of understanding their respective functions. Underexpression of dPrx3 by 90-95% had no discernable effect on life span under normal or oxidative stress conditions; the dPrx5 null flies were previously reported to exhibit a 10% shortening of mean life span and an increase in sensitivity to oxidative stress. Flies underexpressing both dPrx3 and dPrx5 showed an 80% decrease in life span, a severe disruption in thiol homeostasis, and a massive induction of apoptosis in the muscle and digestive system tissues. The early mortality in flies underexpressing both peroxiredoxins was partially offset by overexpression of thioredoxin reductase but not mitochondrion-targeted catalase. These results suggest that mitochondrial peroxiredoxins confer specific protection for thioredoxin/glutathione systems, play a critical role in the maintenance of global thiol homeostasis, and prevent the age-associated apoptosis and premature death.

The effect of peroxiredoxin 4 on fly physiology is a complex interplay of antioxidant and signaling functions

Peroxiredoxin 4 (Prx4) has been implicated in a wide variety of biological processes, including development, progression of cancer, inflammation, and antioxidant function. The purpose of this study was to provide further insight into its multiple roles at the whole‐animal level, using Drosophila. Reduced expression of dPrx4 (up to 90%) resulted in greater sensitivity to oxidative stress, an elevated H2O2 flux, and increases in lipid peroxidation, but no effect on longevity. Overexpression at low levels (<2‐fold) gave reduced levels of oxidative damage and tended to show an increase in longevity. Flies expressing dPrx4 globally at high levels (>5‐fold) had a dramatically reduced life span (by 20–80%) and increased apoptosis. Analysis of these overexpressors revealed an aberrant redistribution of the dPrx4 protein from the endoplasmic reticulum (ER) to cytosol and hemolymph. In addition to the known proapoptotic effects of the cytosolic form of dPrx4, dPrx4 overexpression triggered an NF‐κB‐mediated proinflammatory response, similar to that observed in cells under ER stress or when microbially challenged. Finally, we provide the first evidence that dPrx4, on secretion into the hemolymph, elicits a JAK/STAT‐mediated response. The effects on fly survival and homeostasis appear to represent a combination of differential effects dictated in large part by dPrx4 subcellular and tissue‐specific localization.—Radyuk, S. N., Klichko, V. I., Michalak, K., Orr, W. C. The effect of peroxiredoxin 4 on fly physiology is a complex interplay of antioxidant and signaling functions. FASEB J. 27, 1426–1438 (2013). www.fasebj.org