Fruit Fly Protein Research Articles

Investigating SARS-CoV-2 virus-host interactions and mRNA expression: Insights using three models of D. melanogaster

Responsible for COVID-19, SARS-CoV-2 is a coronavirus in which contagious variants continue to appear. Therefore, some population groups have demonstrated greater susceptibility to contagion and disease progression. For these reasons, several researchers have been studying the SARS-CoV-2/human interactome to understand the pathophysiology of COVID-19 and develop new pharmacological strategies. D. melanogaster is a versatile animal model with approximately 90 % human protein orthology related to SARS-CoV-2/human interactome and is widely used in metabolic studies. In this context, our work assessed the potential interaction between human proteins (ZNF10, NUP88, BCL2L1, UBC9, and RBX1) and their orthologous proteins in D. melanogaster (gl, Nup88, Buffy, ubc9, and Rbx1a) with proteins from SARS-CoV-2 (nsp3, nsp9, E, ORF7a, N, and ORF10) using computational approaches. Our results demonstrated that all the proteins have the potential to interact, and we compared the binding sites between humans and fruit flies. The stability and consistency in the structure of the gl_nsp3 complex, specifically, could be crucial for its specific biological functions. Lastly, to enhance the understanding of the influence of host factors on coronavirus infection, we also analyse the mRNA expression of the five genes (mbo, gl, lwr, Buffy, and Roc1a) responsible for encoding the fruit fly proteins. Briefly, we demonstrated that those genes were differentially regulated according to diets, sex, and age. Two groups showed higher positive gene regulation than others: females in the HSD group and males in the aging group, which could imply a higher virus-host susceptibility. Overall, while preliminary, our work contributes to the understanding of host defense mechanisms and potentially identifies candidate proteins and genes for in vivo viral studies against SARS-CoV-2.

Effect of fruit fly protein bait on the red scale parasitoid, Aphytis lingnanensis (Hymenoptera: Aphelinidae): attraction and toxicity

AbstractProtein bait spray is a tool used widely in tephritid fruit fly pest (Diptera: Tephritidae) control and eradication programs. However, this technique might have a negative effect on non‐target, beneficial insects. Aphytis lingnanensis is a very successful parasitoid of the California red scale, Aonidiella aurantii, a worldwide pest of citrus. Though A. lingnanensis is widely used in augmentative release programs, the impact of protein bait‐insecticide mixtures on the parasitoid is not documented. In this study, we investigated the attraction of A. lingnanensis to Pinnacle® protein, a fruit fly protein bait commonly used in Australia. Following this, the contact toxicity of field weathered residues of protein bait mixed with the insecticides spinosad, fipronil, malathion and chlorpyrifos to A. lingnanensis were evaluated. Finally, the non‐lethal impact of these residues on the capacity of A. lingnanensis to parasitize its host was investigated. Food‐deprived parasitoids were not attracted to protein but were significantly attracted to honey. The lack of attraction of protein, however, increased when sugar was added to it. Relative to a control of only protein, protein bait‐insecticide mixtures applied on citrus leaves and weathered outdoors for 2 h, 3 days, 7 days and 12 days caused high mortality of A. lingnanensis. The residual toxicity of the four weathering periods, for all the protein bait‐insecticide mixtures, produced high mortality rates varying between 90% and 100%. In addition, the capacity of A. lingnanensis to parasitize its host after exposure to weathered residues containing an insecticide was severely affected. The results suggest that A. lingnanensis is highly susceptible to the insecticides evaluated here when used in fruit fly protein baits. Therefore, the use of these insecticides in protein bait sprays, as part of an area wide fruit fly management, should be carefully considered in areas where augmentative release and conservation of A. lingnanensis are also applied.

Comparative transcriptome profiling of heat stress response of the mangrove crab Scylla serrata across sites of varying climate profiles

BackgroundThe fishery and aquaculture of the widely distributed mangrove crab Scylla serrata is a steadily growing, high-value, global industry. Climate change poses a risk to this industry as temperature elevations are expected to threaten the mangrove crab habitat and the supply of mangrove crab juveniles from the wild. It is therefore important to understand the genomic and molecular basis of how mangrove crab populations from sites with different climate profiles respond to heat stress. Towards this, we performed RNA-seq on the gill tissue of S. serrata individuals sampled from 3 sites (Cagayan, Bicol, and Bataan) in the Philippines, under normal and heat-stressed conditions. To compare the transcriptome expression profiles, we designed a 2-factor generalized linear model containing interaction terms, which allowed us to simultaneously analyze within-site response to heat-stress and across-site differences in the response.ResultsWe present the first ever transcriptome assembly of S. serrata obtained from a data set containing 66 Gbases of cleaned RNA-seq reads. With lowly-expressed and short contigs excluded, the assembly contains roughly 17,000 genes with an N50 length of 2,366 bp. Our assembly contains many almost full-length transcripts – 5229 shrimp and 3049 fruit fly proteins have alignments that cover >80% of their sequence lengths to a contig. Differential expression analysis found population-specific differences in heat-stress response. Within-site analysis of heat-stress response showed 177, 755, and 221 differentially expressed (DE) genes in the Cagayan, Bataan, and Bicol group, respectively. Across-site analysis showed that between Cagayan and Bataan, there were 389 genes associated with 48 signaling and stress-response pathways, for which there was an effect of site in the response to heat; and between Cagayan and Bicol, there were 101 such genes affecting 8 pathways.ConclusionIn light of previous work on climate profiling and on population genetics of marine species in the Philippines, our findings suggest that the variation in thermal response among populations might be derived from acclimatory plasticity due to pre-exposure to extreme temperature variations or from population structure shaped by connectivity which leads to adaptive genetic differences among populations.

EVALUATION OF PROTEIN BAIT LACED WITH VARIOUS INSECTICIDES ON THE QUEENSLAND FRUIT FLY (DIPTERA: TEPHRITIDAE): ATTRACTION, FEEDING, MORTALITY AND BAIT PERSISTENCE

The use of malathion in fruit fly protein bait sprays has raised serious concerns due to its adverse effects on non-target organisms. This has necessitated the evaluation of novel reduced-risk compounds. This study evaluated the effects of spinosad, fipronil, malathion and chlorpyrifos mixed with fruit fly protein bait (Mauri Pinnacle protein®) on attraction, feeding and mortality of the Queensland fruit fly, Bactrocera tryoni (Froggatt). The effects of outdoor weathering of these mixtures on fly mortality were also determined. In field-cage experiment, protein-starved flies showed the same level of attraction to baits containing spinosad, fipronil, malathion, chlorpyrifos and protein alone used as control. Female protein-starved flies were deterred from feeding on baits containing malathion and chlorpyrifos compared to baits containing spinosad, fipronil and protein alone. Baits containing malathion and chlorpyrifos caused higher fly mortality and rapid fly knock down than spinosad and fipronil. However, spinosad acted slowly and caused an increase in fly mortality over time, causing up to 90% fly mortality after 72-h. Baits containing malathion and chlorpyrifos, applied on citrus leaves and weathered outdoors, had longer residual effectiveness in killing flies than spinosad and fipronil. Residual effectiveness of the spinosad bait mixture waned significantly after 3 days of outdoor weathering. Results suggest that spinosad and fipronil can be potential alternatives for malathion in protein bait sprays.

Drosophila SNAP-29 Is an Essential SNARE That Binds Multiple Proteins Involved in Membrane Traffic

Each membrane fusion event along the secretory and endocytic pathways requires a specific set of SNAREs to assemble into a 4-helical coiled-coil, the so-called trans-SNARE complex. Although most SNAREs contribute one helix to the trans-SNARE complex, members of the SNAP-25 family contribute two helixes. We report the characterization of the Drosophila homologue of SNAP-29 (dSNAP-29), which is expressed throughout development. Unlike the other SNAP-25 like proteins in fruit fly (i.e., dSNAP-25 and dSNAP-24), which form SDS-resistant SNARE complexes with their cognate SNAREs, dSNAP-29 does not participate in any SDS-resistant complexes, despite its interaction with dsyntaxin1 and dsyntaxin16 in vitro. Immunofluorescence studies indicated that dSNAP-29 is distributed in various tissues, locating in small intracellular puncta and on the plasma membrane, where it associates with EH domain-containing proteins implicated in the endocytic pathway. Overexpression and RNAi studies suggested that dSNAP-29 mediates an essential process in Drosophila development.

Toxicity measurement of spinosad and phloxine B on oriental fruit fly <I>Bactrocera dorsalis</I>

In order to choose the best insecticide for fruit fly protein hydrolysate baits in field,laboratory tests were taken to assess the stomach toxicity of spinosad and phloxine B with torula yeast protein hydrolysate on oriental fruit flies,Bactrocera dorsalis (Hendel) female and male adults.Median lethal concentration values of spinosad and phloxine B on B.dorsalis male and female adults at 5 h were 12.757 0 mg/kg,13.953 5 mg/kg;779.937 8 mg/kg,826.625 9 mg/kg respectively.Median lethal time of spinosad was 4.190 4 h(females) and 4.973 0 h(males) at 16 mg/kg and 100 mg/kg;The median lethal time of spinosad was 2.487 7 h(females) and 2.675 5 h(males).For phloxine B,at 630 mg/kg the median lethal time was 5.350 7 h(females) and 4.946 2 h(males),and at 1,000 mg/kg the median lethal time was 4.765 0 h(females) and 4.571 5 h(males).

Evolutionary pattern of protein architecture in mammal and fruit fly genomes

Mutations, which can alter amino acid constitution, contribute greatly to protein evolution. However, little is reported of their pattern during protein structural evolution. We investigated the distribution of non-synonymous single nucleotide polymorphisms (nsSNPs) and insertions/deletions (indels) along mammal and fruit fly proteins. We found the nsSNPs (and d N) and indels increased in protein boundary regions, and this pattern is inversely correlated with the distribution of protein domain density. Additionally, synonymous substitutions (and d S) are reduced in 5′ and 3′ regions, indicating more variable protein boundaries, compared with central interior. All evidence suggests that the inner part of coding sequences (CDSs) is comparatively conserved, whereas the 5′ and 3′ regions, with higher evolution rates, are more variable. We assumed that due to greater frequencies of nsSNPs and indels in adaptive regions of CDSs it could be easier to ultimately alter, gain, or lose amino acids, thus becoming the front line of protein evolution.

Identification of the Drosophila melanogaster Mitochondrial Citrate Carrier: Bacterial Expression, Reconstitution, Functional Characterization and Developmental Distribution

The mitochondrial carriers are a family of transport proteins that shuttle metabolites, nucleotides and cofactors across the inner mitochondrial membrane. The genome of Drosophila melanogaster encodes at least 46 members of this family. Only four of them have been characterized: the two isoforms of the ADP/ATP translocase, the brain uncoupling protein and the carnitine/acylcarnitine carriers. The transport functions of the remainders cannot be assessed with certainty. One of them, the product of the gene CG6782, shows a fairly close sequence homology to the known sequence of the rat mitochondrial citrate carrier. In this article the fruit fly protein coding by the CG6782 gene has been functionally characterized by over-expression in Escherichia coli and reconstitution into liposomes. It shows to have similar transport properties of the eukaryotic mitochondrial citrate carriers previously biochemically characterized. This indicates that in addition to the protein sequence conservation, insect and mammalian citrate carriers are also significantly related at the functional level suggesting that Drosophila may be used as model organism for the study of mitochondrial solute transporter. The DmCIC expression pattern throughout development was also investigated; the transcripts were detected at equal levels in all stages analysed.

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.

HIERARCHICAL AND TOPOLOGICAL STUDY OF THE PROTEIN–PROTEIN INTERACTION NETWORKS

We employ the random graph theory approach to analyze the protein–protein interaction database DIP. Several global topological parameters are used to characterize the protein–protein interaction networks (PINs) for seven organisms. We find that the seven PINs are well approximated by the scale-free networks, that is, the node degree cumulative distribution Pcum(k) scales with the node degree k (Pcum(k) ~ k-α). We also find that the logarithm of the average clustering coefficient Cave(k) scales with k (Cave(k) ~ k-β), for E. coli and S. cerevisiae. In particular, we determine that the E. coli and the S. cerevisiae PINs are better represented by the stochastic and deterministic hierarchical network models, respectively. The current fruit fly protein–protein interaction dataset does not have convincing evidence in favor of the hierarchical network model. These findings lead us to conclude that, in contrast to scale-free structure, hierarchical structure model applies for certain species' PINs only.We also demonstrate that PINs are robust when subject to random perturbation where up to 50% of the nodes are rewired. Average node degree correlation study supports the fact that nodes of low connectivity are correlated, whereas nodes of high connectivity are not directly linked.

The Evolutionary Relationship of the Domain Architectures in the RhoGEF-containing Proteins

Domain insertions and deletions lead to variations in the domain architectures of the proteins from their common ancestor. In this work, we investigated four groups of the RhoGEF-containing proteins from different organisms with domain architectures RhoGEF-PH-SH3, SH3-RhoGEF-PH, RhoGEF-PH, and SH3-RhoGEF defined in the Pfam database. The phylogenetic trees were constructed using each individual domain and/or the combinations of all the domains. The phylogenetic analysis suggests that RhoGEF-PH-SH3 and SH3-RhoGEF-PH might have evolved from RhoGEF-PH through the insertion of SH3 independently, while SH3-RhoGEF of proteins in fruit fly might have evolved from SH3-RhoGEF-PH by the degeneration of PH domain.

The ‘unique key’ feature of the Iap-binding motifs in RHG proteins

Reaper, Hid, Grim, and Sickle are a group of fruit fly proteins that play pivotal roles in regulating apoptosis during development and in response to cytotoxic stimuli. A rich array of structure/function analysis indicated that two short motifs shared among these proteins contribute to their proapoptotic activity. So far, only in Drosophila melanogaster have all four proteins been identified. Taking advantage of genome sequence projects, we have identified orthologs of these proteins in several divergent Drosophila species. Comparison of the two motifs within and among the ortholog groups revealed a striking distinctiveness of the Iapbinding motif, which is characteristic of each group and is strictly conserved. This finding suggests that these proteins function as ‘unique’ rather than ‘general’ keys to unlock the inhibition of Iap on caspase activation. The GH3 motif, on the other hand, shows differential conservation. Together, these findings support the hypothesis that these proteins are not simply redundant, but each has a distinct role in regulating cell death.

Physiological roles of glycogen synthase kinase-3: potential as a therapeutic target for diabetes and other disorders.

Glycogen synthase kinase-3 (GSK-3) has perplexed signal transduction researchers since its detection in skeletal muscle 25 years ago. The enzyme confounds most of the rules normally associated with protein kinases in that it exhibits significant activity, even in resting, unstimulated cells. However, the protein is highly regulated and potently inactivated in response to signals such as insulin and polypeptide growth factors. The enzyme also displays a distinct and unusual preference for substrates that have been previously phosphorylated by other protein kinases which provides obvious opportunities for cross-talk. Its substrates are diverse and are predominantly regulatory molecules. The molecular cloning of the kinase revealed it to be encoded by two related but distinct genes. Moreover, the mammalian proteins showed remarkable similarity to a fruitfly protein isolated on the basis of its role in cell fate determination. From these humble beginnings, study of the enzyme has accrued further surprises such as its inhibition by lithium, its regulation by serine and tyrosine phosphorylation and its implication in several human disorders including Alzheimers disease, bipolar disorder, cancer and diabetes. Most recently, small molecule inhibitors of GSK-3 have been developed and assessed for therapeutic potential in several of models of pathophysiology. The question is whether modulation of such an "involved" enzyme could lead to selective restoration of defects without multiple unwanted side effects. This review summarizes current knowledge of GSK-3 with respect to its known functions, together with an assessment of its real-life potential as a drug target for chronic conditions such as type 2 diabetes.

Cloning and characterization of dRFX, the Drosophila member of the RFX family of transcription factors.

The RFX family of transcription factors is characterized by a unique DNA binding domain. Five genes have been isolated in mammals, one gene in Caenorhabditis elegans and in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae. Whereas the roles of the RFX genes are beginning to be understood in yeasts, no clear function has been reported in multicellular organisms, except for RFX5, the most divergent member of the family. To study the physiological role of RFX transcription factors using an alternative multicellular model, we report the isolation and characterization of the Drosophila RFX gene (dRFX). The fruit fly protein shares highly conserved domains with the mammalian factors RFX1 to 3 and is more closely related to this subgroup. It binds DNA with the same target specificity as mammalian factors RFX1 to 3. dRFX is located on chromosome III and we characterized the entire locus. dRFX expression was analyzed during embryogenesis. dRFX mRNAs are detected only in the peripheral nervous system and in the brain of the embryo.

Cloning and characterization of the white gene from Anopheles gambiae.

A 14 kb region of genomic DNA containing the X-linked Anopheles gambiae eye colour gene, white, was cloned and sequenced. Genomic clones containing distinct white+ alleles were polymorphic for the insertion of a small transposable element in intron 3, and differed at 1% of nucleotide positions compared. Sequence was also determined from a rare 2914 bp cDNA. Comparison of cDNA and genomic sequences established an intron-exon structure distinct from Drosophila white. Despite a common trend in Anopheles and Drosophila of weak codon bias given low levels of gene expression, codon usage by Anopheles gambiae white was strongly biased. Overall amino acid identity between the predicted mosquito and fruitfly proteins was 64%, but dropped to 14% at the amino terminus. To correlate phenotypically white-eyed strains of A. gambiae with structural lesions in white, five available strains were analysed by PCR and Southern blotting. Although these strains carried allelic mutations, independently generated by gamma radiation (three strains) or spontaneous events (two strains), no white lesions were detected. Significantly, another non-allelic X-linked mutation, causing an identical white-eyed phenotype, has been correlated with a structural defect in the cloned white gene (Benedict et al., 1995). Taken together, these observations suggest that the white-eyed mutants analysed in the present study carry mutations in a second eye colour gene and are most likely white+.

Sequences of two cDNAs encoding silkworm homologues of Drosophila melanogaster squid gene

The squid (sqd) gene of Drosophila melanogaster encodes a few isoforms of a heterogeneous nuclear (hn) RNA-binding protein. We isolated two types of cDNAs coding for homologues of the Sqd protein from the silkworm Bombyx mori. The two predicted amino acid (aa) sequences are identical up to aa 280 and then diverge. The silkworm and fruit fly proteins share 80% homology in the RNA-binding motif region. These cDNAs detect 2.0-, 1.8- and 1-kb mRNAs in the middle and posterior silk glands.

Modulation of the glycogen synthase kinase-3 family by tyrosine phosphorylation.

Glycogen synthase kinase-3 (GSK-3) is a protein serine kinase implicated in the cellular response to insulin. The enzyme is the mammalian homologue of the zeste-white3 (shaggy) homeotic gene of Drosophila melanogaster and has been implicated in the regulation of the c-Jun/AP-1 transcription factor. In mammals this protein serine kinase is encoded by two related genes termed GSK-3 alpha and beta. Here, we demonstrate that these two proteins and the fruit fly protein are phosphorylated on tyrosine in vivo. Moreover, GSK-3 beta activity and function are shown to be dependent on tyrosine phosphorylation. The modified tyrosine residue is conserved in all members of the GSK-3 family and is equivalent to that required for activity by mitogen-activated protein (MAP) kinases. However, unlike MAP kinases, GSK-3 is highly phosphorylated on tyrosine and thus active in resting cells.

Effect of ligands on Drosophila phosphorylase a as monitored by its enzymic inactivation

1. The dephosphorylation of Drosophila phosphorylase a with the catalytic subunit of fruit-fly protein phosphatase-1 was inhibitied by AMP, IMP, ADP, ATP, glucose-6-P, glucose-1-P and UDPG. Glucose, caffeine and glycogen did not influence the reaction. 2. The inhibitory effect of AMP was reduced by glucose and caffeine. 3. The above ligands acted through the modification of phosphorylase a conformation. This conclusion was drawn from the ligands' effect on the dephosporylation of phosphohistone by Drosophila phosphatase-1 and on the tryptic digestion of fruit-fly phosphorylase a.