Small GTP binding proteins: Rab GTPases from the brain of Bombyx mori

From a mRNA of the brain of Bombyx mori, we isolated 8 cDNA clones (BRabs), each of which encodes a different member of Rab-protein family. Four of them have more than 80% amino acid identity to the corresponding members of Drosophila Rab proteins. The other 4 proteins show low sequence similarity to any of the known Rab proteins. However, all of them contain the region conserved in rab protein. Using RACE (Rapid Amplification of cDNA ends), the one full-length cDNA clone (BRab14) was isolated. The clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. After purification, the fusion protein was cut with protease to remove GST-Tag and applied to a glutathione S-Sepharose column. The protein bound [(3)H]-GDP with association constant of 1.02 x 10(11) M(-1). Further, the protein was phosphorylated by protein kinase. This result suggests that Rab protein in the brain of Bombyx mori binds GDP or GTP and its function is regulated by phosphorylation.     

Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.     

Phosphorylation of Rab proteins from the brain of Bombyx mori

Rab proteins play fundamental roles in the regulation of membrane traffic. Previously, from the brain of Bombyx mori we isolated two cDNA clones (BRab1 and BRab14), each of which encoded a different member of Rab-protein family and was expressed in Escherichia coli and purified using an affinity chromatography. In this study, one cDNA clone (BRab8) was isolated from a cDNA library from the brain of B. mori. The recombinant protein was expressed in E. coli and purified. Next, the phosphorylations of these three purified BRab proteins were examined, using mammalian protein kinases in vitro. Protein kinase C (PKC) phosphorylated BRab8 and BRab14 proteins. Protein kinase A faintly phosphorylated BRab8 and BRab14 proteins. Calcium/calmodulin-dependent protein kinase faintly phosphorylated BRab8 protein. Next, brains of B. mori were dissected and homogenized. The homogenate showed a calcium-dependent protein kinase activity of BRab8 and BRab14 proteins. So PKC from the brain of B. mori was partially purified by a sequence of chromatographies on DEAE-Cellulofine and affinity chromatography. This PKC phosphorylated BRab8 and BRab14 proteins. These results suggest that the function of Rab proteins in the brain of B. mori is regulated by calcium-dependent protein kinases.     

囊泡转运中的Rab小G蛋白与马达分子

Rab是小分子GTP结合蛋白Ras超家族中最大的亚家族,在囊泡运输的不同阶段发挥着调节作用.在与GTP结合后,Rab可募集特异的效应蛋白到膜上.近来发现,许多Rab可募集与微管和肌动蛋白相关的马达分子到靶膜,从而调节相应囊泡的转运.Rab所具有的分子开关特性,使其可在空间和时间上对囊泡转运进行调控.     

Emerging roles for Rab family GTPases in human cancer

Members of the Ras-associated binding (Rab) family of small GTPases function as molecular switches regulating vesicular transport in eukaryotes cells. Their pathophysiological roles in human malignancies are less well-known compared to members of Ras and Rho families. Several members of the Rab family have, however, been shown to be aberrantly expressed in various cancer tissues. Recent findings have also revealed, in particular, Rab25 as a determinant of tumor progression and aggressiveness of epithelial cancers. Rab25 associates with α5β1 integrin, and enhances tumor cell invasion by directing the localization of integrin-containing vesicles to the leading edge of matrix invading pseudopodia. We summarized here recent findings on Rab25 and other Rabs implicated to be involved in a variety of human cancers, and discussed plausible mechanisms of how dysregulation of Rab expression could be tumorigenic or tumor suppressive.     

Role of Rab family GTPases and their effectors in melanosomal logistics

Rab GTPases constitute a family of small GTPases that regulate a variety of membrane trafficking events in all eukaryotic cells by recruiting their specific effector molecules. Recent accumulating evidence indicates that members of the mammalian Rab small GTPase family are involved in certain physiological and pathological processes. In particular, functional impairments of specific Rab proteins, e.g. Rab38 and Rab27A, their regulators or their effectors cause pigmentation disorders in humans and coat colour variations in mice because such impairments cause defects in melanosomal logistics, i.e. defects in melanosome biogenesis and transport. Genetic and biochemical analyses of the gene products responsible for mammalian pigmentation disorders in the past decade have revealed that Rab-mediated endosomal transport systems and melanosome transport systems play crucial roles in the efficient darkening of mammalian hair and skin. In this article, we review current knowledge regarding melanosomal logistics, with particular focus on the roles of Rab small GTPases and their effectors.     

Determination of phosphorylated amino acid residues of Rab8 from Bombyx mori

The Rab family of small GTPases are key regulators of membrane trafficking. Partially purified Rab8 from Bombyx mori (BRab8) was phosphorylated by protein kinase C in mammalian cells in vitro. To determine which of the seven serines and four threonines are phosphorylated, we generated deletion and site-directed mutants of BRab8, inserted them in Escherichia coli, partially purified the encoded fusion proteins by affinity chromatography, and examined their phosphorylation by protein kinase C in vitro. We found that Ser-132 of BRab8 was specifically phosphorylated by protein kinase C. In addition, Western blotting using an antiserum against BRab8 and in-gel staining for phosphorylated proteins revealed that BRab8 is phosphorylated in vivo.     

Molecular evolution of the cecropin multigene family in silkworm Bombyx mori

Cecropins constitute one of the largest and most potent immune protein families found in insect species with diversified numbers and features. In view of the large number of cecropin proteins existing with much sequence variations among them, an overview of the multigene cecropin family in silkworm Bombyx mori was attempted in this study. Cecropin encodes an inducible 64 residue anti-bacterial peptide and was clustered into two groups; first group viz. A and second group including B, D, E and Enbocin. Cecropin A consisted of two sub-groups located on chromosome number 6 of B.mori genome. Cecropin B consisted of six sub-groups, cecropin D and E of one each and Enbocin of two. The second sub-group formed in tandem array of multigene family locus over a length of 78.62 kb on chromosome number 26 in B.mori genome and was organized in positive as well as opposite orientation. The results indicated that cecropin B genes were organized in a close cluster with the intergenic sequence ranging from 1366 bp to 23526 bp. Interestingly a distantly related cecropin E was also located within the cecropin B multigene locus. Similarly distant members like cecropin D and Enbocin were also located in the 3' region of cecropin B locus. The maximum intergenic region of 23526 bp observed between Cecropin D and Enbocin indicates that the two genes were distantly evolved. The phylogenetic analysis clearly indicates a positive correlation between the clusters and physical location on the chromosome, as the length of the intergenic region plays a major role to create newer cecropin families. EST database analysis suggests that most of the cecropin A members were expressed in the microbial fat body while, the cecropin B was equally expressed in fat body and other target tissues. The signal peptides were conserved in all the twelve paralogous gene sequences.     

Characterization of Argonaute family members in the silkworm,Bombyx mori

Abstract The Argonaute protein family is a highly conserved group of proteins, which have been implicated in RNA silencing in both plants and animals. Here, four members of the Argonaute family were systemically identified based on the genome sequence of Bombyx mori. Based on their sequence similarity, BmAgo1 and BmAgo2 belong to the Ago subfamily, while BmAgo3 and BmPiwi are in the Piwi subfamily. Phylogenetic analysis reveals that silkworm Argonaute family members are conserved in insects. Conserved amino acid residues involved in recognition of the 5′ end of the small RNA guide strand and of the conserved (aspartate, aspartate and histidine [DDH]) motif present in their PIWI domains suggest that these four Argonaute family members may have conserved slicer activities. The results of microarray expression analysis show that there is a low expression level for B. mori Argonaute family members in different tissues and different developmental stages, except for BmPiwi. All four B. mori Argonaute family members are upregulated upon infection with B. mori nucleopolyhedrovirus. The complete coding sequence of BmPiwi, the homolog of Drosophila piwi, was cloned and its expression occurred mainly in the area where spermatogonia and spermatocytes appear. Our results provide an overview of the B. mori Argonaute family members and suggest that they may have multiple roles. In addition, this is also the first report, to our knowledge, of the response of RNA silencing machinery to DNA virus infection in insects.     

Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.     

Rab14 from Bombyx mori (Lepidoptera: Bombycidae) shows ATPase activity

Rab GTPases are essential for vesicular transport, whereas adenosine triphosphate (ATP) is the most important and versatile of the activated carriers in the cell. But there are little reports to clarify the connection between ATP and Rab GTPases. A cDNA clone (Rab14) from Bombyx mori was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein bound to [³H]-GDP and [³⁵S]-GTPγS. Binding of [³⁵S]-GTPγS was inhibited by guanosine diphosphate (GDP), guanosine triphosphate (GTP) and ATP. Rab14 showed GTP- and ATP-hydrolysis activity. The Km value of Rab14 for ATP was lower than that for GTP. Human Rab14 also showed an ATPase activity. Furthermore, bound [³H]-GDP was exchanged efficiently with GTP and ATP. These results suggest that Rab14 is an ATPase as well as GTPase and gives Rab14 an exciting integrative function between cell metabolic status and membrane trafficking.     

Rab17 and Rab18, Small GTPases with Specificity for Polarized Epithelial Cells: Genetic Mapping in the Mouse

The Rab subfamily of small GTPases plays an important role in the regulation of membrane traffic in eukaryotic cells. While most Rab proteins are equally expressed in polarized and nonpolarized cells, Rab17 and Rab18 show epithelial cell specificity. Here we report the genetic mapping of Rab17 and Rab18 on mouse chromosomes 1 and 18, respectively. We also discuss some implications ofRab17andRab18mapping, including their candidacy for the mouse mutationsln(leaden),Tw(twirler), andax(ataxia).     

Structure-Function Analyses of the Interactions between Rab11 and Rab14 Small GTPases with Their Shared Effector Rab Coupling Protein (RCP)

Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis.