The unfolded protein response modulates the autophagy‐mediated egg production in the mosquito Aedes aegypti

Mosquitoes must feed on vertebrate blood for egg development. As a consequence, some mosquito species are vectors for pathogens that cause devastating diseases in humans. Hence, understanding the mechanisms that control egg developmental cycles is important for developing novel approaches for the control of mosquito‐borne diseases. The unfolded protein response (UPR) is a cellular stress response related to endoplasmic reticulum (ER) stress. The UPR is activated in response to an accumulation of unfolded or misfolded proteins in the ER. Massive proteins have been shown to be produced during egg development, and it is obvious that unfolded or misfolded proteins may arise during vitellogenesis. It has been shown that autophagy in the mosquito fat body plays a central role in the progression of gonadotrophic cycles in the mosquito Aedes aegypti. However, the molecular mechanisms underlying the induction of UPR and the correlation between UPR and autophagy remain unclear. Here, we demonstrate that autophagy is activated during vitellogenesis and that the activation of autophagy is correlated with the UPR. We also show that the expressions of UPR and autophagy can be induced in an in vitro fat body culture system through an amino acid treatment. In addition, the expressions of UPR, autophagy‐specific markers and vitellogenin were also induced during dithiothreitol treatment. Interestingly, the silencing of UPR‐related genes significantly reduced the expression of autophagy‐specific markers and inhibited mosquito fecundity. Taken together, we conclude that autophagy‐mediated egg production in the mosquito A. aegypti is regulated by UPR.     

Development of a novel set of Gateway-compatible vectors for live imaging in insect cells

Insect genomics is a growing area of research. To exploit fully the genomic data that are being generated, high‐throughput systems for the functional characterization of insect proteins and their interactomes are required. In this work, a Gateway‐compatible vector set for expression of fluorescent fusion proteins in insect cells was developed. The vector set was designed to express a protein of interest fused to any of four different fluorescent proteins [green fluorescent protein (GFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and mCherry] by either the C‐terminal or the N‐terminal ends. Additionally, a collection of organelle‐specific fluorescent markers was assembled for colocalization with fluorescent recombinant proteins of interest. Moreover, the vector set was proven to be suitable for simultaneously detecting up to three proteins by multiple labelling. The use of the vector set was exemplified by defining the subcellular distribution of Mal de Río Cuarto virus (MRCV) outer coat protein P10 and by analysing the in vivo self‐interaction of the MRCV viroplasm matrix protein P9‐1 in Förster resonance energy transfer (FRET) experiments. In conclusion, we have developed a valuable tool for high‐throughput studies of protein subcellular localization that will aid in the elucidation of the function of newly described insect and virus proteins.     

Identification of an E‐box DNA binding protein,activated protein 4, and its function in regulating the expression of the gene encoding diapause hormone and pheromone biosynthesis‐activating neuropeptide in Helicoverpa armigera

Activated protein 4 (AP‐4), an E‐box DNA‐binding protein, was cloned from the cotton bollworm, Helicoverpa armigera (Har). The expression of Har‐AP‐4 mRNA and the protein that it encodes are significantly higher in nondiapause pupae than in diapause pupae. In vitro‐translated Har‐AP‐4 can bind specifically to the E‐box motif on the promoter of the diapause hormone and pheromone biosynthesis‐activating neuropeptide (DH‐PBAN). Har‐AP‐4, fused with the green fluorescent protein (GFP), is localized to the nucleus, and overexpression of Har‐AP‐4 can significantly activate the promoter of the DH‐PBAN gene that is involved in nondiapause pupal development in H. armigera. These results suggest that Har‐AP‐4, which binds to the promoter of DH‐PBAN, may play a role in regulating pupal development in H. armigera.     

Transgenic characterization of two testis‐specific promoters in the silkworm,Bombyx mori

Sex‐specific regulatory elements are key components for developing insect genetic sexing systems. The current insect genetic sexing system mainly uses a female‐specific modification system whereas little success was reported on male‐specific genetic modification. In the silkworm Bombyx mori, a lepidopteran model insect with economic importance, a transgene‐based, female‐specific lethality system has been established based on sex‐specific alternative splicing factors and a female‐specific promoter BmVgp (vitellogenin promoter) has been identified. However, no male‐specific regulatory elements have yet been identified. Here we report the transgenic identification of two promoters that drive reporter gene expression in a testis‐specific manner in B. mori. Putative promoter sequences from the B. mori Radial spoke head 1 gene (BmR1) and beta‐tubulin 4 gene (Bmβ4) were introduced using piggybac‐based germline transformation. In transgenic silkworms, expression of the reporter gene enhanced green fluorescent protein (EGFP) directed by either BmR1 promoter (BmR1p) or Bmβ4p showed precisely testis‐specific manners from the larval to adult stage. Furthermore, EGFP expression of these two transgenic lines showed different localization in the testis, indicating that BmR1p or Bmβ4p might be used as distinct regulatory elements in directing testis‐specific gene expression. Identification of these testis‐specific promoters not only contributes to a better understanding of testis‐specific gene function in insects, but also has potential applications in sterile insect techniques for pest management.     

The role of G protein‐coupled receptor‐related genes in cytochrome P450‐mediated resistance of the house fly,Musca domestica (Diptera: Muscidae), to imidacloprid

Ninety‐four putative G protein‐coupled receptors (GPCRs) were identified in the Musca domestica genome. They were annotated and compared with their homologues in Drosophila melanogaster. Phylogenetic analyses of the GPCRs from both species revealed that several family members shared a closer relationship based on the domain architecture. The expression profiles of these genes were examined by quantitative real‐time PCR amongst three strains of the house fly, a near‐isogenic line strain with imidacloprid resistance (N‐IRS), the corresponding susceptible strain (CSS) and another strain derived from field populations with imidacloprid resistance (IRS). We found that five GPCR genes were upregulated in the N‐IRS and eight GPCR genes were upregulated in the IRS strains compared to the CSS strain. The transgenic lines of D. melanogaster with the GPCR genes (LOC101899380 in the N‐IRS strain and LOC101895664 in the IRS strain) exhibited significantly increased tolerance to imidacloprid, and higher expression of cytochrome P450 genes. Bioinformatic analysis of LOC101899380 was carried out based on its full‐length nucleic acid sequence and putative amino acid sequence, and it was named Methuselah‐like10 (Mthl10) owing to its homology with D. melanogaster Mthl10. A cell‐base cell counting kit‐8 toxicity assay demonstrated that the expression of the GPCR gene LOC101899380 in Spodoptera frugiperda (Sf9) cells using a baculovirus‐mediated expression system can elevate the cell tolerance to imidacloprid, indirectly supporting the hypothesis that the GPCR gene LOC101899380 plays some role in imidacloprid resistance. These results should be useful for furthering understanding of the regulatory pathway by which house flies develop resistance.     

Dual Variation in SCN5A and CACNB2b Underlies the Development of Cardiac Conduction Disease without Brugada Syndrome

Background: Inherited loss of function mutations in SCN5A have been linked to overlapping syndromes including cardiac conduction disease and Brugada syndrome (BrS). The mechanisms responsible for the development of one without the other are poorly understood. Methods: Direct sequencing was performed in a family with cardiac conduction disease. Wild‐type (WT) and mutant channels were expressed in TSA201 cells for electrophysiological study. Green fluorescent protein (GFP)‐fused WT or mutant genes were used to assess channel trafficking. Results: A novel SCN5A mutation, P1008S, was identified in all family members displaying first‐degree atrioventricular block, but not in unaffected family members nor in 430 reference alleles. Peak P1008S current was 11.77% of WT (P < 0.001). Confocal microscopy showed that WT channels tagged with GFP were localized on the cell surface, whereas GFP‐tagged P1008S channels remained trapped in intracellular organelles. Trafficking could be rescued by incubation at room temperature, but not by incubation with mexiletine (300 μM) at 37°C. We also identified a novel polymorphism (D601E) in CACNB2b that slowed inactivation of L‐type calcium current (I_Ca,L), significantly increased total charge. Using the Luo‐Rudy action potential (AP) model, we show that the reduction in sodium current (I_Na) can cause loss of the right ventricular epicardial AP dome in the absence but not in the presence of the slowed inactivation of I_Ca,L. Slowed conduction was present in both cases. Conclusions: Our results suggest genetic variations leading to a loss‐of‐function in I_Na coupled with a gain of function in I_Ca,L may underlie the development of cardiac conduction disease without BrS. (PACE 2010; 33:274–285)     

Autophagy ATG16L1 rs2241880 impacts the colorectal cancer risk: A case‐control study

BackgroundDespite many efforts to discover the important role of the autophagy process in the pathogenesis of colorectal cancer (CRC), the exact involved molecular mechanism still remains to be elucidated. Recently, a limited number of studies have been employed to discover the impact of autophagy genes’ variants on the development and progression of CRC. Here, we evaluated the association between two single‐nucleotide polymorphisms (SNPs) in the main components of the autophagy genes, ATG16L1 rs2241880, and ATG5 rs1475270, and the CRC risk in an Iranian population.MethodsDuring this investigation, a total of 369 subjects, including 179 CRC patients and 190 non‐cancer controls have been genotyped using Tetra‐primer amplification refractory mutation system‐polymerase chain reaction (TP‐ARMS‐PCR) method.ResultThe results demonstrated that the T allele of the ATG16L1 rs2241880 was significantly associated with the increased risk of CRC in the studied population (OR 1.64, 95% CI: 1.21–2.22, p = 0.0015). Moreover, ATG16L1 rs2241880 TT genotype increased the susceptibility to CRC (OR 3.31, 95% CI: 1.64–6.69, p = 0.0008). Furthermore, a significant association was observed under the recessive and dominant inheritance models (p = 0.0015 and p = 0.017, respectively). No statistically significant differences were found in the ATG5 rs1475270 alleles and genotypes between the cases and controls.ConclusionThe results of the present study may be helpful concerning the risk stratification in CRC patients based on the genotyping approach of autophagy pathways and emphasize the need for further investigations among different populations and ethnicities to refine our conclusions.     

Nuclear factor erythroid‐derived 2–related factor 2 activates glutathione S‐transferase expression in the midgut of Spodoptera litura (Lepidoptera: Noctuidae) in response to phytochemicals and insecticides

Detoxication enzymes play an important role in insect resistance to xenobiotics such as insecticides and phytochemicals. We studied the pathway for activating the expression of glutathione S‐transferases (GSTs) in response to selected xenobiotics. An assay of the promoter activity of GST epsilon 1 (Slgste1) of Spodoptera litura led to the discovery of a cis‐regulating element. An antioxidant response element was activated in response to indole‐3‐carbinol (I3C) and chlorpyrifos (CPF) and was able to bind with the xenobiotic sensor protein nuclear factor erythroid‐derived 2–related factor 2 (SlNrf2). SlNrf2 and Slgste1 were responsive to reactive oxygen species induced by I3C and CPF in a S. litura cell line, as well as in S. litura midguts. SlNrf2 RNA interference (RNAi) reduced the message RNA levels of Slgste1 and the peroxidase activity of GSTs in response to I3C, xanthotoxin, CPF and deltamethrin. SlNrf2 RNAi and inhibitor treatment of GST activity decreased the viability of I3C‐treated cells. These results indicate that SlNrf2 activates the expression of GSTs in response to oxidative stresses caused by exposure to xenobiotics.     

20‐Hydroxyecdysone stimulates nuclear accumulation of BmNep1, a nuclear ribosome biogenesis‐related protein in the silkworm,Bombyx mori

The pathway of communication between endocrine hormones and ribosome biogenesis critical for physiological adaptation is largely unknown. Nucleolar essential protein 1 (Nep1) is an essential gene for ribosome biogenesis and is functionally conserved in many in vertebrate and invertebrate species. In this study, we cloned Bombyx mori Nep1 (BmNep1) due to its high expression in silk glands of silkworms on day 3 of the fifth instar. We found that BmNep1 mRNA and protein levels were upregulated in silk glands during fourth‐instar ecdysis and larval–pupal metamorphosis. By immunoprecipitation with the anti‐BmNep1 antibody and liquid chromatography‐tandem mass spectrometry analyses, it was shown that BmNep1 probably interacts with proteins related to ribosome structure formation. Immunohistochemistry, biochemical fractionation and immunocytochemistry revealed that BmNep1 is localized to the nuclei in Bombyx cells. Using BmN cells originally derived from ovaries, we demonstrated that 20‐hydroxyecdysone (20E) induced BmNep1 expression and stimulated nuclear accumulation of BmNep1. Under physiological conditions, BmNep1 was also upregulated in ovaries during larval–pupal metamorphosis. Overall, our results indicate that the endocrine hormone 20E facilitates nuclear accumulation of BmNep1, which is involved in nuclear ribosome biogenesis in Bombyx.     

Amblyomma americanum tick saliva insulin‐like growth factor binding protein‐related protein 1 binds insulin but not insulin‐like growth factors

Silencing Amblyomma americanum insulin‐like growth factor binding protein‐related protein 1 (AamIGFBP‐rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP‐rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP‐1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP‐rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24–48 h after attachment. Our data suggest that native AamIGFBP‐rP1 is a functional insulin binding protein in that both yeast‐ and insect cell‐expressed rAamIGFBP‐rP1 bound insulin, but not insulin‐like growth factors. When subjected to anti‐blood clotting and platelet aggregation assays, rAamIGFBP‐rP1 did not have any effect. Unlike human IGFBP‐rP1, which is controlled by trypsinization, rAamIGFBP‐rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick‐borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24–48 h of the tick starting to feed makes AamIGFBP‐rP1 an attractive target for antitick vaccine development.     

Mlck1a is expressed in zebrafish thrombocytes and is an essential component of thrombus formation

Summary. Background: We have used the advantages of the zebrafish model system to demonstrate which of the vertebrate Myosin Light Chain Kinase (MLCK) genes is expressed in thrombocytes and important for thrombus formation. Methods and Results: Here we report that Mlck1a is an essential component of thrombus formation. Phylogenetic data revealed four zebrafish orthologous for three human MLCK genes. To investigate expression of the zebrafish mlck genes in thrombocytes we compared GFP‐tagged platelets with other cells by microarray analysis, and showed that mlck1a expression was 4.5‐fold enriched in platelets. Furthermore, mlck1a mRNA and mRNA for the platelet‐specific cd41 co‐localized in thrombi. Expression of other mlck subtypes was lower in GFP‐tagged platelets (mlck1b; 0.77‐fold enriched) and absent in thrombi (mlck1b, ‐2, ‐3). To investigate the role of Mlck1a in thrombus formation, we knocked down mlck1a using two morpholinos. This resulted in impaired morphology changes of platelets adhering on fibrinogen. In a thrombosis model, in which thrombocytes adhere to the vessel wall damaged by laser irradiation, thrombus formation was slowed down in mlck1a‐deficient embryos. Conclusion: We conclude that Mlck1a is the subtype of MLCK that contributes to platelet shape change and thrombus formation.     

The juvenile hormone binding protein of silkworm haemolymph: gene and functional analysis

A cDNA fragment of haemolymph juvenile hormone binding protein (hJHBP) from larvae of Bombyx mori was amplified by RT‐PCR using degenerate primers based on the N‐terminal amino acid sequence of purified hJHBP and a conserved region near the C‐terminus of other lepidopteran hJHBPs. 5′‐ and 3′‐ends were amplified by RACE to yield cDNAs, hJHBP1 and hJHBP2, encoding 225 amino acids with three substitutions. hJHBP‐mRNA levels in the fat body were constant in the 4th instar, but decreased in the 5th. JHBP protein was constant until wandering, then declined. Recombinant hJHBP1 expressed in E. coli migrated on SDS‐PAGE with a M_r of 32 kDa and showed a K_d of 4.5 × 10^?7 M with JH III, both similar to those of native hJHBP.