hCOA3 Stabilizes Cytochrome c Oxidase 1 (COX1) and Promotes Cytochrome c Oxidase Assembly in Human Mitochondria

Cytochrome c oxidase (COX) or complex IV of the mitochondrial respiratory chain plays a fundamental role in energy production of aerobic cells. In humans, COX deficiency is the most frequent cause of mitochondrial encephalomyopathies. Human COX is composed of 13 subunits of dual genetic origin, whose assembly requires an increasing number of nuclear-encoded accessory proteins known as assembly factors. Here, we have identified and characterized human CCDC56, an 11.7-kDa mitochondrial transmembrane protein, as a new factor essential for COX biogenesis. CCDC56 shares sequence similarity with the yeast COX assembly factor Coa3 and was termed hCOA3. hCOA3-silenced cells display a severe COX functional alteration owing to a decreased stability of newly synthesized COX1 and an impairment in the holoenzyme assembly process. We show that hCOA3 physically interacts with both the mitochondrial translation machinery and COX structural subunits. We conclude that hCOA3 stabilizes COX1 co-translationally and promotes its assembly with COX partner subunits. Finally, our results identify hCOA3 as a new candidate when screening for genes responsible for mitochondrial diseases associated with COX deficiency.     

Conservation of oxidative protein stabilization in an insect homologue of parkinsonism-associated protein DJ-1

DJ-1 is a conserved, disease-associated protein that protects against oxidative stress and mitochondrial damage in multiple organisms. Human DJ-1 contains a functionally essential cysteine residue (Cys106) whose oxidation is important for regulating protein function by an unknown mechanism. This residue is well-conserved in other DJ-1 homologues, including two (DJ-1α and DJ-1β) in Drosophila melanogaster. Because D. melanogaster is a powerful model system for studying DJ-1 function, we have determined the crystal structure and impact of cysteine oxidation on Drosophila DJ-1β. The structure of D. melanogaster DJ-1β is similar to that of human DJ-1, although two important residues in the human protein, Met26 and His126, are not conserved in DJ-1β. His126 in human DJ-1 is substituted with a tyrosine in DJ-1β, and this residue is not able to compose a putative catalytic dyad with Cys106 that was proposed to be important in the human protein. The reactive cysteine in DJ-1 is oxidized readily to the cysteine-sulfinic acid in both flies and humans, and this may regulate the cytoprotective function of the protein. We show that the oxidation of this conserved cysteine residue to its sulfinate form (Cys-SO(2)(-)) results in considerable thermal stabilization of both Drosophila DJ-1β and human DJ-1. Therefore, protein stabilization is one potential mechanism by which cysteine oxidation may regulate DJ-1 function in vivo. More generally, most close DJ-1 homologues are likely stabilized by cysteine-sulfinic acid formation but destabilized by further oxidation, suggesting that they are biphasically regulated by oxidative modification.     

The effects of dietary coenzyme Q on Drosophila life span

Reactive oxygen species (ROS), generated as by-products of aerobic metabolism, cause damage to proteins and cellular membranes, and are thus thought to influence senescence. Caenorhabditis elegans fed on diets lacking in ubiquinone coenzyme Q (CoQ), a coenzyme in the oxidative phosphorylation pathway, show increased longevity, possibly because of reduced ROS generation. We test the role of dietary CoQ in determining Drosophila melanogaster longevity by measuring survival and cytochrome c-oxidase activity (a proxy for aerobic metabolic performance) in flies fed wild-type yeast, CoQ-less yeast, or respiratory control (RC) yeast replete with CoQ but independently deficient in mitochondrial respiration. We find no evidence that dietary manipulation of CoQ in D. melanogaster increases life span or decreases age-dependent decline in cytochrome c oxidase activity. Instead, we find evidence that flies fed a diet of respiratory-deficient yeast (CoQ-less or RC) tend to have decreased longevity and increased rates of decline in cytochrome c-oxidase activity [corrected]     

Thioredoxin-2 affects lifespan and oxidative stress in Drosophila

Thioredoxins are proteins that have thiol-reducing activity and a characteristic conserved active site (WCGPC). They have several documented functions, e.g. roles in defences against oxidative stress and as electron donors for ribonucleotide-reductase. In Drosophila melanogaster there are three "classical" thioredoxins with the conserved active site: deadhead, ThioredoxinT and Thioredoxin-2. Here, we report the creation of null-mutations in the Thioredoxin-2 (Trx-2) gene. Characterization of two Trx-2 mutants indicated that Trx-2 affects the lifespan of D. melanogaster, and is involved in the organism's oxidative stress protection system. We found that the mutants have a shorter lifespan than wild-type flies, and thioredoxin double mutant flies showed lower tolerance to oxidative stress than wild-type flies, while flies carrying multiple copies of a Trx-2 rescue construct showed higher tolerance. These findings suggest that Trx-2 has modest or redundant functions in Drosophila physiology under unstressed conditions, but could be important during times of environmental stress.     

DMAP-85: A τ-Like Protein from Drosophila melanogaster Larvae

Abstract: Microtubule-associated proteins (MAPs) play major regulatory roles in the organization and integrity of the cytoskeletal network. Our main interest in this study was the identification and the analysis of structural and functional aspects of Drosophila melanogaster MAPs. A novel MAP with a relative molecular mass of 85 kDa from Drosophila larvae was found associated with taxol-polymerized microtubules. In addition, this protein bound to mammalian tubulin in an overlay assay and coassembled with purified bovine brain tubulin in microtubule sedimentation experiments. The estimated stoichiometry of 85-kDa protein versus tubulin in the polymers was 1:5.3 ± 0.2 mol/mol. It was shown that the 85-kDa protein bound specifically to an affinity column of Sepharose-βII-(422–434) tubulin peptide, which contains the sequence of the MAP binding domain on βII-tubulin. Affinity-purified 85-kDa protein enhanced microtubule assembly in a concentration-dependent manner. This effect was significantly decreased by the presence of the βII-(422–434) peptide in the assembly assays, thus confirming the specificity of the 85-kDa protein interaction with the C-terminal domain on tubulin. Furthermore, this protein also exhibited a strong affinity for calmodulin, based on affinity chromatographic assays. Monoclonal and polyclonal anti-τ antibodies, including sequence-specific probes that recognize repeated microtubule-binding motifs on τ, MAP-2, and MAP-4 and specific N-terminal sequences of τ, cross-reacted with the 85-kDa protein from Drosophila larvae. These results suggest that τ and Drosophila 85-kDa protein share common functional and structural epitopes. We have named this protein as DMAP-85 for Drosophila MAP. The finding on a Drosophila protein with functional homology and structural similarities to mammalian τ opens new perspectives to understand the cellular roles of MAPs.     

Analyses of fruit flies that do not express selenoproteins or express the mouse selenoprotein, methionine sulfoxide reductase B1, reveal a role of selenoproteins in stress resistance

Selenoproteins are essential in vertebrates because of their crucial role in cellular redox homeostasis, but some invertebrates that lack selenoproteins have recently been identified. Genetic disruption of selenoprotein biosynthesis had no effect on lifespan and oxidative stress resistance of Drosophila melanogaster. In the current study, fruit flies with knock-out of the selenocysteine-specific elongation factor were metabolically labeled with (75)Se; they did not incorporate selenium into proteins and had the same lifespan on a chemically defined diet with or without selenium supplementation. These flies were, however, more susceptible to starvation than controls, and this effect could be ascribed to the function of selenoprotein K. We further expressed mouse methionine sulfoxide reductase B1 (MsrB1), a selenoenzyme that catalyzes the reduction of oxidized methionine residues and has protein repair function, in the whole body or the nervous system of fruit flies. This exogenous selenoprotein could only be expressed when the Drosophila selenocysteine insertion sequence element was used, whereas the corresponding mouse element did not support selenoprotein synthesis. Ectopic expression of MsrB1 in the nervous system led to an increase in the resistance against oxidative stress and starvation, but did not affect lifespan and reproduction, whereas ubiquitous MsrB1 expression had no effect. Dietary selenium did not influence lifespan of MsrB1-expressing flies. Thus, in contrast to vertebrates, fruit flies preserve only three selenoproteins, which are not essential and play a role only under certain stress conditions, thereby limiting the use of the micronutrient selenium by these organisms.     

Developmental changes in Drosophila melanogaster following exposure to alternating electromagnetic fields

This study investigated the biological effects of alternating electromagnetic fields (EMFs) on developmental stages of Drosophila melanogaster eggs and the first, second and third instar larvae stages. D. melanogaster eggs and larval stages were exposed to a 11 mT 50 Hz field produced by a pair of Helmholtz coils. Each stage was exposed to aEMFs for 2, 4, 6 and 8 h. Features of adult flies such as head, thorax, abdomen and other morphological changes were studied and compared. The frequency of abnormal flies was calculated using statistical methods at P <.05. The results obtained from exposing larvae in different stages of development showed a significant increase in the number of abnormal adult flies, whereas no significant increase was observed in the group arising from eggs exposed to aEMFs. Also, it appeared that duration of exposure correlates with the increase in the number of abnormal flies. There was no significant difference in mortality rate and sex distribution of the abnormal flies between field exposed and the control groups.     

A novel fruitfly protein under developmental control degrades uracil-DNA

Uracil in DNA may arise by cytosine deamination or thymine replacement and is removed during DNA repair. Fruitfly larvae lack two repair enzymes, the major uracil-DNA glycosylase and dUTPase, and may accumulate uracil-DNA. We asked if larval tissues contain proteins that specifically recognize uracil-DNA. We show that the best hit of pull-down on uracil-DNA is the protein product of the Drosophila melanogaster gene CG18410. This protein binds to both uracil-DNA and normal DNA but degrades only uracil-DNA; it is termed Uracil-DNA Degrading Factor (UDE). The protein has detectable homology only to a group of sequences present in genomes of pupating insects. It is under detection level in the embryo, most of the larval stages and in the imago, but is strongly upregulated right before pupation. In Schneider 2 cells, UDE mRNA is upregulated by ecdysone. UDE represents a new class of proteins that process uracil-DNA with potential involvement in metamorphosis.     

Studies on the glutathione S-transferase proteome of adult Drosophila melanogaster: responsiveness to chemical challenge

GSTs from adult Drosophila melanogaster have been partially purified using three different affinity chromatography media and separated by 2-DE. Nine GSTs have been identified by MALDI-TOF MS. In the absence of special treatments, eight GSTs could be positively identified. These were DmGSTs D1 (the dominant Delta isoform which was present in five protein zones of differing pI) and D3 (and possibly also D5); the Epsilon-class GSTs E3, 6, 7 and 9 and a previously uncharacterised, probable member of the class, CG16936. The Sigma-class DmGSTS1 was prominent. DmGSTD2 was detected only after pretreatment of the flies with Phenobarbital (PhB). Treatment with Paraquat (PQ) led to an increase in the total GST activity, as measured with the substrates 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloro-nitrobenzene (DCNB) and an increase in the relative amounts of the D1, D3, E6 and E7 isoforms. PhB treatment led to increases in the relative amounts of the D1, D2, E3, E6, E7 and E9 isoforms detected with a possible depression in the relative amount of GSTS1. CG16936 was unaffected by either pretreatment.     

Sodium nitroprusside toxicity in Drosophila melanogaster: delayed pupation, reduced adult emergence, and induced oxidative/nitrosative stress in eclosed flies

The toxicity of sodium nitroprusside (SNP) was tested on the Drosophila melanogaster model system. Fly larvae were raised on food supplemented with SNP at concentrations of 0.01-1.5 mM. Food supplementation with SNP caused a developmental delay in flies and reduced adult eclosion. Biochemical analyses such as levels of oxidative stress markers and activities of antioxidant and associated enzymes were carried out on 2-day-old flies emerged from control and SNP-fed larvae. Larval exposure to SNP resulted in lower activities of aconitase and catalase in adult flies relative to the control cohort. However, larval treatment with SNP led to higher carbonyl protein content and higher activities of superoxide dismutase, glucose-6-phosphate dehydrogenase, thioredoxin reductase, and glutathione-S-transferase in flies. Among the parameters tested, aconitase activity and developmental end points may be useful early indicators of toxicity caused by SNP. The study also suggests that the toxicity of SNP may arise not just from its direct effects, but also from its decomposition products such as nitric oxide and iron ions.     

Adaptive evolution of relish, a Drosophila NF-kappaB/IkappaB protein

NF-kappaB and IkappaB proteins have central roles in regulation of inflammation and innate immunity in mammals. Homologues of these proteins also play an important role in regulation of the Drosophila immune response. Here we present a molecular population genetic analysis of Relish, a Drosophila NF-kappaB/IkappaB protein, in Drosophila simulans and D. melanogaster. We find strong evidence for adaptive protein evolution in D. simulans, but not in D. melanogaster. The adaptive evolution appears to be restricted to the IkappaB domain. A possible explanation for these results is that Relish is a site of evolutionary conflict between flies and their microbial pathogens.     

Analysis of hunger-driven gene expression in the Drosophila melanogaster larval central nervous system

A transposon-inserted mutant of Drosophila melanogaster was recently identified, and the larvae show no food preference (Ryuda and Hayakawa, 2005). To reveal the genetic mechanism underlying the preference change in this mutant, a large-scale oligo-DNA microarray screening was carried out to identify genes whose expression is different in control and mutant strains. We focused especially on hunger-driven changes in gene expression in the larval central nervous system (CNS) of both strains, because the state of food depletion should promote a feeding response due to changed expression of certain genes in the CNS. We identified 22 genes whose expression changed after starvation in either or both of the two strains. Quantitative RT-PCR analyses confirmed the expression changes in four genes, CG6271, CG6277, CG7953, and new glue 3 (ng3, encoding a putative structural molecule). CG6271 and CG6277 encode triacylglycerol lipase, and CG7953 produces a protein homologous to a juvenile hormone (JH) binding protein. The expression of these two groups of genes was enhanced in control strain larvae with a normal food preference but not in GS1189 strain larvae. Given that these genes contribute to mediating hunger-driven changes in food preference and intake in D. melanogaster larvae, the dysfunction of these key genes could cause the defect in food preference observed in GS1189-strain larvae.