Expression and characterization of the PEBP homolog genes from Drosophila

The phosphatidylethanolamine binding proteins (PEBPs) family is evolutionarily conserved and involved in different physiological phenomena. PEBPs were found in many species from bacteria to mammals. Despite numerous studies, PEBPs' biological function and mode of action remain elusive. Based on sequence homology, seven PEBP genes were detected in the Drosophila genome. Only one of them, the odorant binding protein (OBP), has been characterized. To date nothing is known concerning the expression pattern and biological roles of the six other PEBP genes. By RT‐PCR and Western blot analysis, we examined expression of the PEBPs in different tissues and embryos. The 6 PEBPs were differentially expressed. Only one, CG10298, is specific of only one tissue: the testis. Additionally, by comparing in wild type and male‐sterile mutants we show that CG10298 is present only during spermatid differentiation. Furthermore, by comparing structural parameters of the six PEBP proteins with those of human PEBP‐1, we have established that PEBP CG10298 is most closely related to human PEBP. © 2009 Wiley Periodicals, Inc.     

Small-scale, semi-automated purification of eukaryotic proteins for structure determination

A simple approach that allows cost-effective automated purification of recombinant proteins in levels sufficient for functional characterization or structural studies is described. Studies with four human stem cell proteins, an engineered version of green fluorescent protein, and other proteins are included. The method combines an expression vector (pVP62K) that provides in vivo cleavage of an initial fusion protein, a factorial designed auto-induction medium that improves the performance of small-scale production, and rapid, automated metal affinity purification of His8-tagged proteins. For initial small-scale production screening, single colony transformants were grown overnight in 0.4 ml of auto-induction medium, produced proteins were purified using the Promega Maxwell 16, and purification results were analyzed by Caliper LC90 capillary electrophoresis. The yield of purified [U-15N]-His8-Tcl-1 was 7.5 microg/ml of culture medium, of purified [U-15N]-His8-GFP was 68 microg/ml, and of purified selenomethione-labeled AIA-GFP (His8 removed by treatment with TEV protease) was 172 microg/ml. The yield information obtained from a successful automated purification from 0.4 ml was used to inform the decision to scale-up for a second meso-scale (10-50 ml) cell growth and automated purification. 1H-15N NMR HSQC spectra of His8-Tcl-1 and of His8-GFP prepared from 50 ml cultures showed excellent chemical shift dispersion, consistent with well folded states in solution suitable for structure determination. Moreover, AIA-GFP obtained by proteolytic removal of the His8 tag was subjected to crystallization screening, and yielded crystals under several conditions. Single crystals were subsequently produced and optimized by the hanging drop method. The structure was solved by molecular replacement at a resolution of 1.7 A. This approach provides an efficient way to carry out several key target screening steps that are essential for successful operation of proteomics pipelines with eukaryotic proteins: examination of total expression, determination of proteolysis of fusion tags, quantification of the yield of purified protein, and suitability for structure determination.     

Catalytic properties of domain-exchanged chimeric proteins between firefly luciferase and Drosophila fatty Acyl-CoA synthetase CG6178

Firefly luciferase and fatty acyl-CoA synthetase are members of the acyl-CoA synthetase super family, which consists of a large N-terminal domain and a small C-terminal domain. Previously we found that firefly luciferase has fatty acyl-CoA synthetic activity, and also identified that the homolog of firefly luciferase in Drosophila melanogaster (CG6178) is a fatty acyl-CoA synthetase and is not a luciferase. In this study, we constructed chimeric proteins by exchanging the domain between Photinus pyralis luciferase (PpLase) and Drosophila CG6178, and determined luminescence and fatty acyl-CoA synthetic activities. A chimeric protein with the N-terminal domain of PpLase and the C-terminal domain of CG6178 (Pp/Dm) had luminescence activity, showing approximately 4% of the activity of wild-type luciferase. The Pp/Dm protein also had fatty acyl-CoA synthetic activity and the substrate specificity was similar to PpLase. In contrast, a chimeric protein with the N-terminal domain of CG6178 and the C-terminal of PpLase (Dm/Pp) had only fatty acyl-CoA synthetase activity, and the substrate specificity was similar to CG6178. These results suggest that the N-terminal domain of firefly luciferase is essential for substrate recognition, and that the C-terminal domain is indispensable but not specialized for the luminescence reaction.     

A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors

PIWI proteins and their bound PIWI-interacting RNAs (piRNAs) form the core of a gonad-specific small RNA silencing pathway that protects the animal genome against the deleterious activity of transposable elements. Recent studies linked the piRNA pathway to TUDOR biology as TUDOR domains of various proteins bind symmetrically methylated Arginine residues in PIWI proteins. We systematically analysed the Drosophila TUDOR protein family and identified four previously not characterized TUDOR domain-containing proteins (CG4771, CG14303, CG11133 and CG31755) as essential piRNA pathway factors. We characterized CG4771 (Vreteno) in detail and demonstrate a critical role for this protein in primary piRNA biogenesis. Vreteno physically and/or genetically interacts with the primary pathway components Piwi, Armitage, Yb and Zucchini. Vreteno also interacts with the Tdrd12 orthologues CG11133 (Brother of Yb) and CG31755 (Sister of Yb), which are essential for the primary piRNA pathway in the germline and probably replace the function of the related but soma-specific factor Yb.     

The SNMP/CD36 gene family in Diptera, Hymenoptera and Coleoptera: Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae, Aedes aegypti, Apis mellifera, and Tribolium castaneum

Sensory neuron membrane proteins (SNMPs) are membrane bound proteins initially identified in olfactory receptor neurons of Lepidoptera and are thought to play a role in odor detection; SNMPs belong to a larger gene family characterized by the human protein CD36. We have identified 12-14 candidate SNMP/CD36 homologs from each of the genomes of Drosophila melanogaster, D. pseudoobscura, Anopheles gambiae and Aedes aegypti (Diptera), eight candidate homologs from Apis mellifera (Hymenoptera), and 15 from Tribolium castaneum (Coleoptera). Analysis (sequence similarity and intron locations) suggests that the insect SNMP/CD36 genes fall into three major groups. Group 1 includes the previously characterized D. melanogaster emp (epithelial membrane protein). Group 2 includes the previously characterized D. melanogaster croquemort, ninaD, santa maria, and peste. Group 3 genes include the SNMPs, which fall into two subgroups referred to as SNMP1 and SNMP2. D. melanogaster SNMP1 (CG7000) shares both significant sequence similarity and five of its six intron insertion sites with the lepidopteran Bombyx mori SNMP1. The topological conservation of this gene family within the three major holometabolous lineages indicates that it predates the coleopteran and hymenoptera/dipera/lepidoptera split 300+ million years ago. The current state of knowledge of the characterized insect members of this gene family is discussed.     

A novel Takeout-like protein expressed in the taste and olfactory organs of the blowfly, Phormia regina

In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout-like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout-like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout-like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout-like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout-like protein expression were found between the sexes. Our results suggest that Phormia Takeout-like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems.     

Interaction of non-histone proteins with DNA and chromatin from Drosophila and mouse cells. Specificity, isolation and analysis of complexes.

The specificity of the binding of purified non-histone proteins to DNA has been investigated through two types of experiments. Using a nitrocellulose filter assay at a low protein/DNA ratio, the binding of mouse non-histone proteins to mouse DNA was twice as great as the binding of mouse non histone protein to Drosophila DNA. The reverse experiment using Drosophila non-histone protein confirmed the interpretation that some protein . DNA complexes were specific. Protein . DNA complexes isolated by gel filtration chromatography indicated that 20% or 10% of the non-histone protein was bound to homologous or heterologous DNA respectively. Purified non-histone proteins bound with lower efficiency (15%) than unpurified but with higher specificity to soluble chromatin than to naked DNA. This binding did not result from an exchange between chromatin non-histone proteins and purified non-histone proteins added in excess. DNA-bound and chromatin-bound proteins were analysed on polyacrylamide gels. Whereas no major qualitative differences were observed with DNA-bound proteins, some proteins bound to homologous mouse chromatin were different from those bound to heterologous Drosophila chromatin. These results suggest a possible role of DNA-bound non-histone proteins in the regulation of gene expression.     

Identification of the gene encoding bursicon, an insect neuropeptide responsible for cuticle sclerotization and wing spreading

To accommodate growth, insects must periodically replace their exoskeletons. After shedding the old cuticle, the new soft cuticle must sclerotize. Sclerotization has long been known to be controlled by the neuropeptide hormone bursicon, but its large size of 30 kDa has frustrated attempts to determine its sequence and structure. Using partial sequences obtained from purified cockroach bursicon, we identified the Drosophila melanogaster gene CG13419 as a candidate bursicon gene. CG13419 encodes a peptide with a predicted final molecular weight of 15 kDa, which likely functions as a dimer. This predicted bursicon protein belongs to the cystine knot family, which includes vertebrate transforming growth factor-beta (TGF-beta) and glycoprotein hormones. Point mutations in the bursicon gene cause defects in cuticle sclerotization and wing expansion behavior. Bioassays show that these mutants have decreased bursicon bioactivity. In situ hybridization and immunocytochemistry revealed that bursicon is co-expressed with crustacean cardioactive peptide (CCAP). Transgenic flies that lack CCAP neurons also lacked bursicon bioactivity. Our results indicate that CG13419 encodes bursicon, the last of the classic set of insect developmental hormones. It is the first member of the cystine knot family to have a defined function in invertebrates. Mutants show that the spectrum of bursicon actions is broader than formerly demonstrated.     

SARS冠状病毒核衣壳(N)蛋白不同区域的原核表达

利用大肠杆菌表达系统对SARS冠状病毒的核衣壳(N)蛋白全长及N末端或／和C末端缺失突变体进行了表达,共表达了39个重组蛋白,表达量在15％～30％之间。分别利用电洗脱或金属鳌合介质纯化重组蛋白,用蛋白印迹实验检测纯化蛋白对SARS病人恢复期血清的反应性,结果发现全长N蛋白活性最好,其余的末端缺失蛋白均无法达到同—：活性水平。由此说明N蛋白的完整性对于其优势表位的充分暴露是必要的。     

The human Dnmt2 has residual DNA-(cytosine-C5) methyltransferase activity

The human Dnmt2 protein is one member of a protein family conserved from Schizosaccharomyces pombe and Drosophila melanogaster to Mus musculus and Homo sapiens. It contains all of the amino acid motifs characteristic for DNA-(Cytosine-C5) methyltransferases, and its structure is very similar to prokaryotic DNA methyltransferases. Nevertheless, so far all attempts to detect catalytic activity of this protein have failed. We show here by two independent assay systems that the purified Dnmt2 protein has weak DNA methyltransferase activity. Methylation was observed at CG sites in a loose ttnCGga(g/a) consensus sequence, suggesting that Dnmt2 has a more specialized role than other mammalian DNA methyltransferases.     

Auto-induction medium for the production of [U-15N]- and [U-13C, U-15N]-labeled proteins for NMR screening and structure determination

Protocols have been developed and applied for the high-throughput production of [U-15N]- or [U-13C-, U-15N]-labeled proteins using the conditional methionine auxotroph Escherichia coli B834. The large-scale growth and expression uses a chemically defined auto-induction medium containing salts and trace metals, vitamins including vitamin B12, and glucose, glycerol, and lactose. The results from nine expression trials in 2-L of the auto-induction medium (500 mL in each of four polyethylene terephthalate beverage bottles) gave an average final optical density at 600 nm of approximately 5, an average wet cell mass yield of approximately 9.5 g L(-1), and an average yield of approximately 20 mg of labeled protein in the six instances in which proteolysis of the fusion protein was observed. Correlations between the cell mass recovered, the level of protein expression, and the relative amounts of glucose, glycerol, and lactose in the auto-induction medium were noted. Mass spectral analysis showed that the purified proteins contained both 15N and 13C at levels greater than 95%. 1H-15N heteronuclear single quantum correlation spectroscopy as well as 13C; 15N-edited spectroscopy showed that the purified [U-15N]- and [U-13C, U-15N]-labeled proteins were suitable for structure analysis.     

Regulation of flagellar motility by the conserved flagellar protein CG34110/Ccdc135/FAP50

Eukaryotic cilia and flagella are vital sensory and motile organelles. The calcium channel PKD2 mediates sensory perception on cilia and flagella, and defects in this can contribute to ciliopathic diseases. Signaling from Pkd2-dependent Ca2+ rise in the cilium to downstream effectors may require intermediary proteins that are largely unknown. To identify these proteins, we carried out genetic screens for mutations affecting Drosophila melanogaster sperm storage, a process mediated by Drosophila Pkd2. Here we show that a new mutation lost boys (lobo) encodes a conserved flagellar protein CG34110, which corresponds to vertebrate Ccdc135 (E = 6e-78) highly expressed in ciliated respiratory epithelia and sperm, and to FAP50 (E = 1e-28) in the Chlamydomonas reinhardtii flagellar proteome. CG34110 localizes along the fly sperm flagellum. FAP50 is tightly associated with the outer doublet microtubules of the axoneme and appears not to be a component of the central pair, radial spokes, dynein arms, or structures defined by the mbo waveform mutants. Phenotypic analyses indicate that both Pkd2 and lobo specifically affect sperm movement into the female storage receptacle. We hypothesize that the CG34110/Ccdc135/FAP50 family of conserved flagellar proteins functions within the axoneme to mediate Pkd2-dependent processes in the sperm flagellum and other motile cilia.     

Specific protein homeostatic functions of small heat‐shock proteins increase lifespan

During aging, oxidized, misfolded, and aggregated proteins accumulate in cells, while the capacity to deal with protein damage declines severely. To cope with the toxicity of damaged proteins, cells rely on protein quality control networks, in particular proteins belonging to the family of heat‐shock proteins (HSPs). As safeguards of the cellular proteome, HSPs assist in protein folding and prevent accumulation of damaged, misfolded proteins. Here, we compared the capacity of all Drosophila melanogaster small HSP family members for their ability to assist in refolding stress‐denatured substrates and/or to prevent aggregation of disease‐associated misfolded proteins. We identified CG14207 as a novel and potent small HSP member that exclusively assisted in HSP70‐dependent refolding of stress‐denatured proteins. Furthermore, we report that HSP67BC, which has no role in protein refolding, was the most effective small HSP preventing toxic protein aggregation in an HSP70‐independent manner. Importantly, overexpression of both CG14207 and HSP67BC in Drosophila leads to a mild increase in lifespan, demonstrating that increased levels of functionally diverse small HSPs can promote longevity in vivo.     

Tentative identification of a resilin gene in Drosophila melanogaster

A search of the Drosophila genome for gene products with similarities to the amino acid sequences of three tryptic peptides from locust (Schistocerca gregaria) resilin gave two positive results: gene products CG15920 and CG9036. In both conceptual translation products a 62-residue region is present, which is identical to the resilin peptides in 29 positions. Gene product CG15920 has an amino acid composition closely resembling that of resilins from various insect species, and it has an N-terminal signal peptide sequence indicating that it is an extracellular protein. The 62-residue region shows similarity to the RR-2 sequence, which is common for a number of matrix proteins from insect solid cuticle. The N- and C-terminal regions flanking the 62-residue in CG15920 are dominated by 18 repeats of a 15-residue sequence and 11 repeats of a 13-residue sequence, respectively. The structures of the repeats predict that the peptide chain will fold in an irregular, extended beta-spiral, resembling the structures suggested for mammalian elastin and spider flagelliform silk, two materials which, like resilin, possess long-range elasticity. Accordingly, we suggest that gene product CG15920 is a Drosophila resilin precursor.     

Uukuniemi virus S RNA segment: ambisense coding strategy, packaging of complementary strands into virions, and homology to members of the genus Phlebovirus.

We determined the complete nucleotide sequence of the small (S) RNA segment of Uukuniemi virus, the prototype of the Uukuvirus genus within the Bunyaviridae family. The RNA, which is 1,720 nucleotides long, contains two nonoverlapping open reading frames. The 5' end of one strand (complementary to the viral strand) encodes the nonstructural protein NSs (273 residues; molecular weight, 32,019), whereas the 5' end of the viral-sense strand encodes the nucleocapsid protein N (254 residues; molecular weight, 28,508). Thus, the S RNA uses an ambisense coding strategy previously described for the S segment of two phleboviruses and the arenaviruses. The localization of the N protein within the S RNA sequence was confirmed by amino-terminal sequence analysis of all five possible cyanogen bromide fragments obtained from purified N protein. Northern (RNA) blot analyses with strand-specific probes showed that the N and NSs proteins are translated from subgenomic mRNAs about 800 and 850 nucleotides long, respectively. These mRNAs are apparently transcribed from full-length S RNAs of opposite polarities. The two mRNA species were also detected in virus-infected cells. Interestingly, highly purified virions contained full-length S RNA copies of both polarities at a ratio of about 10:1. In contrast, virions contained exclusively negative-strand copies of the M RNA segment. The possible significance of these results for viral infection is discussed. The amino acid sequence of the N protein showed 35 and 32% homology (identity) with the N protein of Punta Toro and sandfly fever Sicilian viruses, two members of the Phlebovirus genus. The NSs proteins were much less related (about 15% identity). In addition, the extreme 5' and 3' ends of the S RNA, which are complementary to each other, also showed a high degree of conservation with the two phleboviruses. These results indicate that the uukuviruses and phleboviruses are evolutionarily related and suggest that the two genera could be merged into a single genus within the Bunyaviridae family.     

Two-step purification and N-terminal amino acid sequence analysis of the rat Mr 90,000 heat shock protein

A fast and efficient method for the isolation of the rat Mr approximately 90,000 heat shock protein is presented, comprising a two-step high-performance anion-exchange and gel-permeation column chromatography. The Mr approximately 90,000 protein was purified to electrophoretic homogeneity in high yield (up to 2 mg per 10g of normal rat liver) in 4-5 h. The purified protein was recognized on protein immunoblots by monospecific rabbit antibodies raised against the rat glucocorticoid receptor-associated Mr approximately 90,000 non-ligand-binding protein. The N-terminal sequence of the first 25 amino acids of the purified protein showed a high degree of similarity with Mr approximately 90,000 heat shock proteins from calf, human, Drosophila, and yeast.     

An endogenous Drosophila receptor for glycans bearing alpha 1,3-linked core fucose residues

The genome of Drosophila melanogaster encodes several proteins that are predicted to contain Ca(2+)-dependent, C-type carbohydrate-recognition domains. The CG2958 gene encodes a protein containing 359 amino acid residues. Analysis of the CG2958 sequence suggests that it consists of an N-terminal domain found in other Drosophila proteins, a middle segment that is unique, and a C-terminal C-type carbohydrate-recognition domain. Expression studies show that the full-length protein is a tetramer formed by noncovalent association of disulfide-linked dimers that are linked through cysteine residues in the N-terminal domain. The expressed protein binds to immobilized yeast invertase through the C-terminal carbohydrate-recognition domain. Competition binding studies using monosaccharides demonstrate that CG2958 interacts specifically with fucose and mannose. Fucose binds approximately 5-fold better than mannose. Blotting studies reveal that the best glycoprotein ligands are those that contain N-linked glycans bearing alpha1,3-linked fucose residues. Binding is enhanced by the additional presence of alpha1,6-linked fucose. It has previously been proposed that labeling of the Drosophila neural system by anti-horseradish peroxidase antibodies is a result of the presence of difucosylated N-linked glycans. CG2958 is a potential endogenous receptor for such neural-specific carbohydrate epitopes.     

Identification of Drosophila neuropeptide receptors by G protein-coupled receptors-beta-arrestin2 interactions

Activation of G protein-coupled receptors (GPCR) leads to the recruitment of beta-arrestins. By tagging the beta-arrestin molecule with a green fluorescent protein, we can visualize the activation of GPCRs in living cells. We have used this approach to de-orphan and study 11 GPCRs for neuropeptide receptors in Drosophila melanogaster. Here we verify the identities of ligands for several recently de-orphaned receptors, including the receptors for the Drosophila neuropeptides proctolin (CG6986), neuropeptide F (CG1147), corazonin (CG10698), dFMRF-amide (CG2114), and allatostatin C (CG7285 and CG13702). We also de-orphan CG6515 and CG7887 by showing these two suspected tachykinin receptor family members respond specifically to a Drosophila tachykinin neuropeptide. Additionally, the translocation assay was used to de-orphan three Drosophila receptors. We show that CG14484, encoding a receptor related to vertebrate bombesin receptors, responds specifically to allatostatin B. Furthermore, the pair of paralogous receptors CG8985 and CG13803 responds specifically to the FMRF-amide-related peptide dromyosuppressin. To corroborate the findings on orphan receptors obtained by the translocation assay, we show that dromyosuppressin also stimulated GTPgammaS binding and inhibited cAMP by CG8985 and CG13803. Together these observations demonstrate the beta-arrestin-green fluorescent protein translocation assay is an important tool in the repertoire of strategies for ligand identification of novel G protein-coupled receptors.