Pl-nectin,a discoidin family member,is a ligand for βC integrins in the sea urchin embryo

Pl-nectin is a component of the extracellular matrix that surrounds embryos of the sea urchin Paracentrotus lividus. Pl-nectin mediates adhesion of dissociated embryonic cells to substrates and interfering with ectodermic cells contacting Pl-nectin results in defects in skeleton growth and morphogenesis. Recently, we reported that Pl-nectin is a new member of the discoidin family, in agreement with the notion that many discoidin-containing proteins are involved in cell adhesion processes as integrin ligands. To better understand the molecular basis for the interaction of Pl-nectin with ectoderm, we investigated the hypothesis that Pl-nectin is an integrin ligand in sea urchin embryos. We show that in P. lividus embryos, βC-containing integrins localize to the apical surface of ectodermic cells, which are in contact with Pl-nectin. Immunoprecipitation experiments indicate that the two proteins are part of a complex in vivo and affinity chromatography indicates that βC-containing integrin receptors bind purified Pl-nectin. These data support a model in which ectodermic integrins binding to Pl-nectin mediate cellular adhesion to the hyaline layer. Regulated adhesion of cells to the hyaline layer is a critical component of several morphogenetic processes and the identification of the receptors and ligands involved provides new opportunities to investigate the underlying molecular mechanisms of ECM adhesion and morphogenesis.     

The cyclomodulin Cif of Photorhabdus luminescens inhibits insect cell proliferation and triggers host cell death by apoptosis

Cycle inhibiting factors (Cif) constitute a broad family of cyclomodulins present in bacterial pathogens of invertebrates and mammals. Cif proteins are thought to be type III effectors capable of arresting the cell cycle at G₂/M phase transition in human cell lines. We report here the first direct functional analysis of Cif_Pl, from the entomopathogenic bacterium Photorhabdus luminescens, in its insect host. The cif_Pl gene was expressed in P. luminescens cultures in vitro. The resulting protein was released into the culture medium, unlike the well characterized type III effector LopT. During locust infection, cif_Pl was expressed in both the hemolymph and the hematopoietic organ, but was not essential for P. luminescens virulence. Cif_Pl inhibited proliferation of the insect cell line Sf9, by blocking the cell cycle at the G₂/M phase transition. It also triggered host cell death by apoptosis. The integrity of the Cif_Pl catalytic triad is essential for the cell cycle arrest and pro-apoptotic activities of this protein. These results highlight, for the first time, the dual role of Cif in the control of host cell proliferation and apoptotic death in a non-mammalian cell line.     

Ail and PagC-Related Proteins in the Entomopathogenic Bacteria of Photorhabdus Genus

Among pathogenic Enterobacteriaceae, the proteins of the Ail/OmpX/PagC family form a steadily growing family of outer membrane proteins with diverse biological properties, potentially involved in virulence such as human serum resistance, adhesion and entry into eukaryotic culture cells. We studied the proteins Ail/OmpX/PagC in the bacterial Photorhabdus genus. The Photorhabdus bacteria form symbiotic complexes with nematodes of Heterorhabditis species, associations which are pathogenic to insect larvae. Our phylogenetic analysis indicated that in Photorhabdus asymbiotica and Photorhabdus luminescens only Ail and PagC proteins are encoded. The genomic analysis revealed that the Photorhabdus ail and pagC genes were present in a unique copy, except two ail paralogs from P. luminescens. These genes, referred to as ail1 _Pl and ail2 _Pl, probably resulted from a recent tandem duplication. Surprisingly, only ail1 _Pl expression was directly controlled by PhoPQ and low external Mg²⁺ conditions. In P. luminescens, the magnesium-sensing two-component regulatory system PhoPQ regulates the outer membrane barrier and is required for pathogenicity against insects. In order to characterize Ail functions in Photorhabdus, we showed that only ail2 _Pl and pagC _Pl had the ability, when expressed into Escherichia coli, to confer resistance to complement in human serum. However no effect in resistance to antimicrobial peptides was found. Thus, the role of Ail and PagC proteins in Photorhabdus life cycle is discussed.     

Laccase2 is required for cuticular pigmentation in stinkbugs

During the maturation of insect cuticle, protein-protein and protein-chitin crosslinkages are formed by the action of diphenoloxidases. Two types of diphenoloxidases, laccases and tyrosinases, are present in the insect cuticle. In coleopteran and hymenopteran insects, laccase2 gene has been identified as encoding an enzyme principally responsible for cuticular pigmentation and hardening, whereas biological roles of laccase genes in hemimetabolous insects remain to be established. Here we identified laccase2 genes from three hemipteran stinkbugs, Riptortus pedestris (Alydidae), Nysius plebeius (Lygaeidae) and Megacopta punctatissima (Plataspidae). In R. pedestris, laccase2 gene was highly expressed in epidermal tissues prior to molting. When the gene expression was suppressed by an RNA interference technique, cuticular pigmentation after molting were blocked depending on the dose of injected double-stranded RNA targeting the laccase2 gene. Similar results were obtained for N. plebeius and M. punctatissima. In all the stinkbug species, injecting 20 ng of double-stranded RNA was sufficient to prevent the cuticular maturation. These results indicate that laccase2 gene is generally required for cuticular pigmentation in different stinkbug families, highlighting its conserved biological function across diverse insect taxa.     

Molecular chaperone function of Arabidopsis thaliana phloem protein 2-A1, encodes a protein similar to phloem lectin

Although several phloem sap proteins have been identified from protein extracts of heat-treated Arabidopsis seedlings using FPLC gel filtration columns, many of the physiological roles played by these proteins remain to be elucidated. We functionally characterized a phloem protein 2-A1, which encodes a protein similar to phloem lectin. Using a bacterially expressed recombinant protein of AtPP2-A1, we found that it performs dual functions, showing both molecular chaperone activity and antifungal activity. mRNA expression of the AtPP2-1 gene was induced by diverse external stresses such as pathogens, and other signaling molecules, such as ethylene. These results suggest that the AtPP2-A1 molecular chaperone protein plays a critical role in the Arabidopsis defense system against diverse external stresses including fungal pathogenic attack and heat shock.     

cuticleDB: a relational database of Arthropod cuticular proteins

Background  

The insect exoskeleton or cuticle is a bi-partite composite of proteins and chitin that provides protective, skeletal and structural functions. Little information is available about the molecular structure of this important complex that exhibits a helicoidal architecture. Scores of sequences of cuticular proteins have been obtained from direct protein sequencing, from cDNAs, and from genomic analyses.     

Cloning,chromosome mapping and expression pattern of porcine PLIN and M6PRBP1 genes

The PAT proteins, named after the three PLIN/ADRP/TIP47 (PAT) proteins, PLIN for perilipin, ADRP for adipose differentiation-related protein and TIP47 for tail-interacting protein of 47 kDa, now officially named M6PRBP1 for mannose-6-phosphate receptor binding protein 1, is a set of intracellular lipid droplet binding proteins. They are localized in the outer membrane monolayer enveloping lipid droplets and are involved in the metabolism of intracellular lipid. This work describes the cloning and sequencing of porcine PLIN and M6PRBP1 cDNAs, the chromosome mapping of these two genes, as well as the expression pattern of porcine PAT genes. Sequence analysis shows that the porcine PLIN cDNA contains an open reading frame of 1551 bp encoding 516 amino acids and that the porcine M6PRBP1 cDNA contains a coding region of 1320 bp encoding 439 amino acids. Comparison of PLIN and M6PRBP1 amino-acid sequences among various species reveals that porcine and bovine proteins are the most conserved. Porcine PLIN and M6PRBP1 genes have been mapped to pig chromosomes 7 and 2, respectively, by radiation hybrid analysis using the IMpRH panel. Expression analyses in pig showed a high expression of PLIN mRNA in adipose tissue, M6PRBP1 mRNA in small intestine, kidney and spleen and ADRP mRNA in adipose tissue, lung and spleen.     

Molecular cloning of precursors for TEP-1 and TEP-2: The GGNG peptide-related peptides of a prosobranch gastropod,Thais clavigera

TEP (Thais excitatory peptide)-1 and TEP-2 are molluscan counterparts of annelidan GGNG-peptides, identified in a neogastropod, Thais clavigera (Morishita et al., 2006). We have cloned two cDNAs encoding TEP-1 and TEP-2 precursor protein, respectively, by the standard molecular cloning techniques. Predicted TEP-1 precursor protein consists of 161 amino acids, while predicted TEP-2 precursor protein has 118 amino acids. Only a single copy of TEP was found on the respective precursor. The semi-quantitative RT-PCR showed that expression of TEP-1 was high in sub-esophageal, pleural, pedal and visceral ganglia, while it was low in supra-esophageal ganglion. By contrast, expression level of TEP-2 was high in pedal and visceral ganglia. In situ hybridization visualized different subsets of TEP-1 and TEP-2 expressing neurons in Thais ganglia. For example, supra-esophageal ganglion contained many TEP-2 expressing neuron, but not TEP-1 expressing ones. These results suggest that expression of TEP-1 and TEP-2 is differently regulated in the Thais ganglia.     

Prohibitin Interacts with Envelope Proteins of White Spot Syndrome Virus and Prevents Infection in the Red Swamp Crayfish,Procambarus clarkii

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans.     

The Distribution of GYR- and YLP-Like Motifs in Drosophila Suggests a General Role in Cuticle Assembly and Other Protein-Protein Interactions

Background

Arthropod cuticle is composed predominantly of a self-assembling matrix of chitin and protein. Genes encoding structural cuticular proteins are remarkably abundant in arthropod genomes, yet there has been no systematic survey of conserved motifs across cuticular protein families.

Methodology/Principal Findings

Two short sequence motifs with conserved tyrosines were identified in Drosophila cuticular proteins that were similar to the GYR and YLP Interpro domains. These motifs were found in members of the CPR, Tweedle, CPF/CPFL, and (in Anopheles gambiae) CPLCG cuticular protein families, and the Dusky/Miniature family of cuticle-associated proteins. Tweedle proteins have a characteristic motif architecture that is shared with the Drosophila protein GCR1 and its orthologs in other species, suggesting that GCR1 is also cuticular. A resilin repeat, which has been shown to confer elasticity, matched one of the motifs; a number of other Drosophila proteins of unknown function exhibit a motif architecture similar to that of resilin. The motifs were also present in some proteins of the peritrophic matrix and the eggshell, suggesting molecular convergence among distinct extracellular matrices. More surprisingly, gene regulation, development, and proteolysis were statistically over-represented ontology terms for all non-cuticular matches in Drosophila. Searches against other arthropod genomes indicate that the motifs are taxonomically widespread.

Conclusions

This survey suggests a more general definition for GYR and YLP motifs and reveals their contribution to several types of extracellular matrix. They may define sites of protein interaction with DNA or other proteins, based on ontology analysis. These results can help guide experimental studies on the biochemistry of cuticle assembly.     

Post-translational modifications of the cuticular proteins of Hyalophora cecropia from different anatomical regions and metamorphic stages

Post-translational modifications are a conspicuous feature of the proteins of vertebrate extracellular matrices such as cartilage. Yet this feature remains virtually unexplored with insect cuticle, a situation this paper begins to remedy. Cuticular proteins were extracted from cuticles of Hyalophora cecropia and separated on isoelectrofocusing and 2D gels. Periodic acid-Schiff reagent stained several proteins from flexible cuticles and a few proteins from rigid cuticles, indicating that some proteins were glycosylated. Elucidation of the specific nature of this glycosylation came from probing electrophoretically separated cuticular proteins blotted onto nitrocellulose with biotinylated lectins. Most major cuticular proteins did not react; minor cuticular proteins and molecules which do not stain with Coomassie blue were found to bind lectins specific for mannose and N-acetylgalactosamine. Limited binding was also detected with lectins specific for N-acetylglucosamine, galactose and fucose. No sialic acid was detected using either lectins or neuraminidase digestion. The amount of glycosylation was greatest in proteins extracted from flexible cuticles. Although several proteins stained with Alcian blue indicating presence of sulfation, ³⁵S which had been incorporated at low levels in cuticular proteins corresponded to [³⁵S]methionine. No indication of the presence of mammalian-type glycosaminoglycans in insect cuticles was obtained after treatment with chondroitinase or nitrous acid. The functional significance of the modifications detected remains unknown. No evidence for phosphorylated proteins or lipoproteins was found.