Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.Bacillus thuringiensis subsp. israelensis has received considerable attention for mosquito control because of its specific and potent toxicity (15). B. thuringiensis subsp. israelensis-based microbial insecticides have been widely used as active components for integrated management of mosquitoes (11, 13, 33, 34). B. thuringiensis subsp. israelensis produces at least four major crystal toxins (Cry toxins), namely, Cry4Aa, Cry4Ba, Cry11Aa, and Cyt1Aa (5). Cry4Aa exhibits specific toxicity against Anopheles, Aedes, and Culex mosquito larvae (15, 27). The 130-kDa Cry4Aa protoxin is released from the protein crystal upon ingestion by susceptible mosquito larvae and is activated by gut proteases into two protease-resistant fragments with molecular masses of 20 and 45 kDa through intramolecular cleavage of a 60-kDa intermediate (39). The three-dimensional structure of Cry4Aa has been determined by X-ray crystallography at a resolution of 2.8 Å (6). The structure of Cry4Aa is similar to the structures of previously characterized Cry toxins (24, 26, 31) that are composed of three domains (domains I, II, and III). In general, domain I, which is located in the N-terminal region, is composed of seven amphipathic α-helices and is thought to participate in membrane insertion. Domain II, which consists of three antiparallel β-sheets, is a putative receptor binding domain (Fig. ​(Fig.1).1). In particular, the loops in domain II that are exposed on the surface of the toxin molecule vary significantly in length and amino acid sequence among Cry toxins (31) and are thought to be receptor binding sites. Domain III in the C-terminal region contains two antiparallel β-sheets that form a β-sandwich fold with a jellyroll topology (31). Domain III is assumed to be involved in structural integrity, membrane protein recognition, or both (23, 24, 30).Open in a separate windowFIG. 1.Three-dimensional structure of Cry4Aa domain II. The structure was generated with PyMOL software (8) using the Cry4Aa PDB code (6). The amino acid sequences and corresponding regions of loops 1, 2, and 3 are indicated by blue, red, and yellow, respectively. The amino acid sequences of β-strands adjacent to the loops are underlined.The insecticidal mechanism of Cry toxin involves multiple steps, including ingestion by susceptible insects, solubilization in the alkaline midgut juice, activation by trypsin-like midgut proteases, binding to specific receptors on midgut epithelial cells, and then insertion into the plasma membrane followed by the formation of cation-selective channels or pores (26, 31, 34, 41). According to the colloid-osmotic lysis model, these channels or pores allow ions and water to pass into the cells, resulting in destruction of the membrane potential, cell swelling, cell lysis, and eventual death of the host (20, 21). Thus, the mechanism seems to be very complicated and is affected by multiple factors. The binding of the toxin to the specific receptor is considered a vital step for specific insecticidal activity (35). In fact, modification of the receptor molecules has been reported for insects resistant to certain Cry toxins (12, 22, 36).In a search for the functional structures of Cry4Aa, we previously constructed various loop replacement mutants with mutations in the three major loops in domain II and showed that the replacement of loop 2 resulted in a significant loss of mosquitocidal activity. Replacement of loops 1 and 3 of Cry4Aa also affected mosquitocidal activity, but it did not eliminate it (17). In this study, to further characterize the loops, we constructed Cry4Aa mutants in which individual amino acids in the loops were replaced with alanine and analyzed the mutants to determine their mosquitocidal activity against Culex pipiens larvae. We also analyzed the structural integrity of the Cry4Aa mutant proteins subjected to proteolytic digestion and their binding affinity to brush border membrane vesicles (BBMV) prepared from C. pipiens larvae.     

The Arf-like GTPase Arl8 Mediates Delivery of Endocytosed Macromolecules to Lysosomes in Caenorhabditis elegans

Late endocytic organelles including lysosomes are highly dynamic acidic organelles. Late endosomes and lysosomes directly fuse for content mixing to form hybrid organelles, from which lysosomes are reformed. It is not fully understood how these processes are regulated and maintained. Here we show that the Caenorhabditis elegans ARL-8 GTPase is localized primarily to lysosomes and involved in late endosome-lysosome fusion in the macrophage-like coelomocytes. Loss of arl-8 results in an increase in the number of late endosomal/lysosomal compartments, which are smaller than wild type. In arl-8 mutants, late endosomal compartments containing endocytosed macromolecules fail to fuse with lysosomal compartments enriched in the aspartic protease ASP-1. Furthermore, loss of arl-8 strongly suppresses formation of enlarged late endosome-lysosome hybrid organelles caused by mutations of cup-5, which is the orthologue of human mucolipin-1. These findings suggest that ARL-8 mediates delivery of endocytosed macromolecules to lysosomes by facilitating late endosome-lysosome fusion.     

Functional characterization of IRESes by an inhibitor of the RNA helicase eIF4A

RNA helicases are molecular motors that are involved in virtually all aspects of RNA metabolism. Eukaryotic initiation factor (eIF) 4A is the prototypical member of the DEAD-box family of RNA helicases. It is thought to use energy from ATP hydrolysis to unwind mRNA structure and, in conjunction with other translation factors, it prepares mRNA templates for ribosome recruitment during translation initiation. In screening marine extracts for new eukaryotic translation initiation inhibitors, we identified the natural product hippuristanol. We show here that this compound is a selective and potent inhibitor of eIF4A RNA-binding activity that can be used to distinguish between eIF4A-dependent and -independent modes of translation initiation in vitro and in vivo. We also show that poliovirus replication is delayed when infected cells are exposed to hippuristanol. Our study demonstrates the feasibility of selectively targeting members of the DEAD-box helicase family with small-molecule inhibitors.     

Identification of TIFA as an adapter protein that links tumor necrosis factor receptor-associated factor 6 (TRAF6) to interleukin-1 (IL-1) receptor-associated kinase-1 (IRAK-1) in IL-1 receptor signaling

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals from members of the Toll/interleukin-1 (IL-1) receptor family by interacting with IL-1 receptor-associated kinase-1 (IRAK-1) after IRAK-1 is released from the receptor-MyD88 complex upon IL-1 stimulation. However, the molecular mechanisms underlying regulation of the IRAK-1/TRAF6 interaction are largely unknown. We have identified TIFA, a TRAF-interacting protein with a forkhead-associated (FHA) domain. The FHA domain is a motif known to bind directly to phosphothreonine and phosphoserine. In transient transfection assays, TIFA activates NFkappaBeta and c-Jun amino-terminal kinase. However, TIFA carrying a mutation that abolishes TRAF6 binding or mutations in the FHA domain that are known to abolish FHA domain binding to phosphopeptide fails to activate NFkappaBeta and c-Jun amino-terminal kinase. TIFA, when overexpressed, binds both TRAF6 and IRAK-1 and significantly enhances the IRAK-1/TRAF6 interaction. Furthermore, analysis of endogenous proteins indicates that TIFA associates with TRAF6 constitutively, whereas it associates with IRAK-1 in an IL-1 stimulation-dependent manner in vivo. Thus, TIFA is likely to mediate IRAK-1/TRAF6 interaction upon IL-1 stimulation.     

The rat-like hamster,Tscherskia triton,as a host for the parthenogenetic liver fluke Fasciola sp

Metacercariae of parthenogenetic Fasciola sp. triploid were inoculated into the rat-like hamster Tscherskia triton. Flukes at various stages of growth were found in the bile ducts of all 8 (50%) animals that survived from 42 to 90 days. The body length to width ratio ranged from 1.8 to 2.9, and flukes with the highest ratio were passed 68 days after inoculation. Our results indicate that T. triton is a suitable host for experimental infection when induced by a small number of metacercariae (less than 5) of Fasciola sp.     

The Novel α4B Murine α4 Integrin Protein Splicing Variant Inhibits α4 Protein-dependent Cell Adhesion

Integrins affect the motility of multiple cell types to control cell survival, growth, or differentiation, which are mediated by cell-cell and cell-extracellular matrix interactions. We reported previously that the α9 integrin splicing variant, SFα9, promotes WT α9 integrin-dependent adhesion. In this study, we introduced a new murine α4 integrin splicing variant, α4B, which has a novel short cytoplasmic tail. In inflamed tissues, the expression of α4B, as well as WT α4 integrin, was up-regulated. Cells expressing α4B specifically bound to VCAM-1 but not other α4 integrin ligands, such as fibronectin CS1 or osteopontin. The binding of cells expressing WT α4 integrin to α4 integrin ligands is inhibited by coexpression of α4B. Knockdown of α4B in metastatic melanoma cell lines results in a significant increase in lung metastasis. Expression levels of WT α4 integrin are unaltered by α4B, with α4B acting as a regulatory subunit for WT α4 integrin by a dominant-negative effect or inhibiting α4 integrin activation.     

Transport of maternal transthyretin to the fetus in the viviparous teleost Neoditrema ransonnetii (Perciformes,Embiotocidae)

Journal of Comparative Physiology B - The molecular basis of viviparity in non-mammalian species has not been widely studied. Neoditrema ransonnetii, a surfperch, is a matrotrophic teleost whose...