WorldWideScience

Sample records for rab escort protein

  1. Phylogeny and evolution of Rab7 and Rab9 proteins

    Directory of Open Access Journals (Sweden)

    Wyroba Elżbieta

    2009-05-01

    Full Text Available Abstract Background An important role in the evolution of intracellular trafficking machinery in eukaryotes played small GTPases belonging to the Rab family known as pivotal regulators of vesicle docking, fusion and transport. The Rab family is very diversified and divided into several specialized subfamilies. We focused on the VII functional group comprising Rab7 and Rab9, two related subfamilies, and analysed 210 sequences of these proteins. Rab7 regulates traffic from early to late endosomes and from late endosome to vacuole/lysosome, whereas Rab9 participates in transport from late endosomes to the trans-Golgi network. Results Although Rab7 and Rab9 proteins are quite small and show heterogeneous rates of substitution in different lineages, we found a phylogenetic signal and inferred evolutionary relationships between them. Rab7 proteins evolved before radiation of main eukaryotic supergroups while Rab9 GTPases diverged from Rab7 before split of choanoflagellates and metazoans. Additional duplication of Rab9 and Rab7 proteins resulting in several isoforms occurred in the early evolution of vertebrates and next in teleost fishes and tetrapods. Three Rab7 lineages emerged before divergence of monocots and eudicots and subsequent duplications of Rab7 genes occurred in particular angiosperm clades. Interestingly, several Rab7 copies were identified in some representatives of excavates, ciliates and amoebozoans. The presence of many Rab copies is correlated with significant differences in their expression level. The diversification of analysed Rab subfamilies is also manifested by non-conserved sequences and structural features, many of which are involved in the interaction with regulators and effectors. Individual sites discriminating different subgroups of Rab7 and Rab9 GTPases have been identified. Conclusion Phylogenetic reconstructions of Rab7 and Rab9 proteins were performed by a variety of methods. These Rab GTPases show diversification

  2. The trypanosome Rab-related proteins RabX1 and RabX2 play no role in intracellular trafficking but may be involved in fly infectivity.

    Directory of Open Access Journals (Sweden)

    Senthil Kumar A Natesan

    2009-09-01

    Full Text Available Rab GTPases constitute the largest subgroup of the Ras superfamily and are primarily involved in vesicle targeting. The full extent of Rab family function is unexplored. Several divergent Rab-like proteins are known but few have been characterized. In Trypanosoma brucei there are sixteen Rab genes, but RabX1, RabX2 and RabX3 are divergent within canonical sequence regions. Where known, trypanosome Rab functions are broadly conserved when orthologous relationships may be robustly established, but specific functions for RabX1, X2 and X3 have yet to be determined. RabX1 and RabX2 originated via tandem duplication and subcellular localization places RabX1 at the endoplasmic reticulum, while RabX2 is at the Golgi complex, suggesting distinct functions. We wished to determine whether RabX1 and RabX2 are involved in vesicle transport or other cellular processes.Using comparative genomics we find that RabX1 and RabX2 are restricted to trypanosomatids. Gene knockout indicates that RabX1 and RabX2 are non-essential. Simultaneous RNAi knockdown of both RabX1 and RabX2, while partial, was also non-lethal and may suggest non-redundant function, consistent with the distinct locations of the proteins. Analysis of the knockout cell lines unexpectedly failed to uncover a defect in exocytosis, endocytosis or in the morphology or location of multiple markers for the endomembrane system, suggesting that neither RabX1 nor RabX2 has a major role in intracellular transport. However, it was apparent that RabX1 and RabX2 knockout cells displayed somewhat enhanced survival within flies.RabX1 and RabX2, two members of the trypanosome Rab subfamily, were shown to have no major detectable role in intracellular transport, despite the localization of each gene product to highly specific endomembrane compartments. These data extend the functional scope of Rab proteins in trypanosomes to include non-canonical roles in differentiation-associated processes in protozoa.

  3. Rab23 is a flagellar protein in Trypanosoma brucei

    Directory of Open Access Journals (Sweden)

    Field Mark C

    2011-06-01

    Full Text Available Abstract Background Rab small GTPases are important mediators of membrane transport, and orthologues frequently retain similar locations and functions, even between highly divergent taxa. In metazoan organisms Rab23 is an important negative regulator of Sonic hedgehog signaling and is crucial for correct development and differentiation of cellular lineages by virtue of an involvement in ciliary recycling. Previously, we reported that Trypanosoma brucei Rab23 localized to the nuclear envelope 1, which is clearly inconsistent with the mammalian location and function. As T. brucei is unicellular the potential that Rab23 has no role in cell signaling was possible. Here we sought to further investigate the role(s of Rab23 in T. brucei to determine if Rab23 was an example of a Rab protein with divergent function in distinct taxa. Methods/major findings The taxonomic distribution of Rab23 was examined and compared with the presence of flagella/cilia in representative taxa. Despite evidence for considerable secondary loss, we found a clear correlation between a conventional flagellar structure and the presence of a Rab23 orthologue in the genome. By epitope-tagging, Rab23 was localized and found to be present at the flagellum throughout the cell cycle. However, RNAi knockdown did not result in a flagellar defect, suggesting that Rab23 is not required for construction or maintenance of the flagellum. Conclusions The location of Rab23 at the flagellum is conserved between mammals and trypanosomes and the Rab23 gene is restricted to flagellated organisms. These data may suggest the presence of a Rab23-mediated signaling mechanism in trypanosomes.

  4. Role of rab proteins in epithelial membrane traffic

    NARCIS (Netherlands)

    van Ijzendoorn, SCD; Mostov, KE; Hoekstra, D

    2003-01-01

    Small GTPase rab proteins play an important role in various aspects of membrane traffic, including cargo selection, vesicle budding, vesicle motility, tethering, docking, and fusion. Recent data suggest also that rabs, and their divalent effector proteins, organize organelle subdomains and as such

  5. Rab proteins: The key regulators of intracellular vesicle transport

    International Nuclear Information System (INIS)

    Bhuin, Tanmay; Roy, Jagat Kumar

    2014-01-01

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future

  6. Rab proteins: The key regulators of intracellular vesicle transport

    Energy Technology Data Exchange (ETDEWEB)

    Bhuin, Tanmay [Cell and Developmental Biology Unit, Department of Zoology, The University of Burdwan, Golapbag 713104 (India); Roy, Jagat Kumar, E-mail: jkroy@bhu.ac.in [Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221005 (India)

    2014-10-15

    Vesicular/membrane trafficking essentially regulates the compartmentalization and abundance of proteins within the cells and contributes in many signalling pathways. This membrane transport in eukaryotic cells is a complex process regulated by a large and diverse array of proteins. A large group of monomeric small GTPases; the Rabs are essential components of this membrane trafficking route. Most of the Rabs are ubiquitously expressed proteins and have been implicated in vesicle formation, vesicle motility/delivery along cytoskeleton elements and docking/fusion at target membranes through the recruitment of effectors. Functional impairments of Rabs affecting transport pathways manifest different diseases. Rab functions are accompanied by cyclical activation and inactivation of GTP-bound and GDP-bound forms between the cytosol and membranes which is regulated by upstream regulators. Rab proteins are characterized by their distinct sub-cellular localization and regulate a wide variety of endocytic, transcytic and exocytic transport pathways. Mutations of Rabs affect cell growth, motility and other biological processes. - Highlights: • Rab proteins regulate different signalling pathways. • Deregulation of Rabs is the fundamental causes of a variety of human diseases. • This paper gives potential directions in developing therapeutic targets. • This paper also gives ample directions for modulating pathways central to normal physiology. • These are the huge challenges for drug discovery and delivery in near future.

  7. α1B-Adrenergic Receptors Differentially Associate with Rab Proteins during Homologous and Heterologous Desensitization

    Science.gov (United States)

    Castillo-Badillo, Jean A.; Sánchez-Reyes, Omar B.; Alfonzo-Méndez, Marco A.; Romero-Ávila, M. Teresa; Reyes-Cruz, Guadalupe; García-Sáinz, J. Adolfo

    2015-01-01

    Internalization of G protein-coupled receptors can be triggered by agonists or by other stimuli. The process begins within seconds of cell activation and contributes to receptor desensitization. The Rab GTPase family controls endocytosis, vesicular trafficking, and endosomal fusion. Among their remarkable properties is the differential distribution of its members on the surface of various organelles. In the endocytic pathway, Rab 5 controls traffic from the plasma membrane to early endosomes, whereas Rab 4 and Rab 11 regulate rapid and slow recycling from early endosomes to the plasma membrane, respectively. Moreover, Rab 7 and Rab 9 regulate the traffic from late endosomes to lysosomes and recycling to the trans-Golgi. We explore the possibility that α1B-adrenergic receptor internalization induced by agonists (homologous) and by unrelated stimuli (heterologous) could involve different Rab proteins. This possibility was explored by Fluorescence Resonance Energy Transfer (FRET) using cells coexpressing α1B-adrenergic receptors tagged with the red fluorescent protein, DsRed, and different Rab proteins tagged with the green fluorescent protein. It was observed that when α1B-adrenergic receptors were stimulated with noradrenaline, the receptors interacted with proteins present in early endosomes, such as the early endosomes antigen 1, Rab 5, Rab 4, and Rab 11 but not with late endosome markers, such as Rab 9 and Rab 7. In contrast, sphingosine 1-phosphate stimulation induced rapid and transient α1B-adrenergic receptor interaction of relatively small magnitude with Rab 5 and a more pronounced and sustained one with Rab 9; interaction was also observed with Rab 7. Moreover, the GTPase activity of the Rab proteins appears to be required because no FRET was observed when dominant-negative Rab mutants were employed. These data indicate that α1B-adrenergic receptors are directed to different endocytic vesicles depending on the desensitization type (homologous vs

  8. Rab11-family of interacting protein 2 associates with chlamydial inclusions through its Rab-binding domain and promotes bacterial multiplication.

    Science.gov (United States)

    Leiva, Natalia; Capmany, Anahí; Damiani, María Teresa

    2013-01-01

    Chlamydia trachomatis, an obligate intracellular pathogen, survives within host cells in a special compartment named 'inclusion' and takes advantage of host vesicular transport pathways for its growth and replication. Rab GTPases are key regulatory proteins of intracellular trafficking. Several Rabs, among them Rab11 and Rab14, are implicated in chlamydial development. FIP2, a member of the Rab11-Family of Interacting Proteins, presents at the C-terminus a Rab-binding domain that interacts with both Rab11 and Rab14. In this study, we determined and characterized the recruitment of endogenous and GFP-tagged FIP2 to the chlamydial inclusions. The recruitment of FIP2 is specific since other members of the Rab11-Family of Interacting Proteins do not associate with the chlamydial inclusions. The Rab-binding domain of FIP2 is essential for its association. Our results indicate that FIP2 binds to Rab11 at the chlamydial inclusion membrane through its Rab-binding domain. The presence of FIP2 at the chlamydial inclusion favours the recruitment of Rab14. Furthermore, our results show that FIP2 promotes inclusion development and bacterial replication. In agreement, the silencing of FIP2 decreases the bacterial progeny. C. trachomatis likely recruits FIP2 to hijack host intracellular trafficking to redirect vesicles full of nutrients towards the inclusion. © 2012 Blackwell Publishing Ltd.

  9. Posttranslational modifications of Rab proteins cause effective displacement of GDP dissociation inhibitor.

    Science.gov (United States)

    Oesterlin, Lena K; Goody, Roger S; Itzen, Aymelt

    2012-04-10

    Intracellular vesicular trafficking is regulated by approximately 60 members of the Rab subfamily of small Ras-like GDP/GTP binding proteins. Rab proteins cycle between inactive and active states as well as between cytosolic and membrane bound forms. Membrane extraction/delivery and cytosolic distribution of Rabs is mediated by interaction with the protein GDP dissociation inhibitor (GDI) that binds to prenylated inactive (GDP-bound) Rab proteins. Because the Rab:GDP:GDI complex is of high affinity, the question arises of how GDI can be displaced efficiently from Rab protein in order to allow the necessary recruitment of the Rab to its specific target membrane. While there is strong evidence that DrrA, as a bacterially encoded GDP/GTP exchange factor, contributes to this event, we show here that posttranslational modifications of Rabs can also modulate the affinity for GDI and thus cause effective displacement of GDI from Rab:GDI complexes. These activities have been found associated with the phosphocholination and adenylylation activities of the enzymes AnkX and DrrA/SidM, respectively, from the pathogenic bacterium Legionella pneumophila. Both modifications occur after spontaneous dissociation of Rab:GDI complexes within their natural equilibrium. Therefore, the effective GDI displacement that is observed is caused by inhibition of reformation of Rab:GDI complexes. Interestingly, in contrast to adenylylation by DrrA, AnkX can covalently modify inactive Rabs with high catalytic efficiency even when GDP is bound to the GTPase and hence can inhibit binding of GDI to Rab:GDP complexes. We therefore speculate that human cells could employ similar mechanisms in the absence of infection to effectively displace Rabs from GDI.

  10. The Human Escort Protein Hep Binds to the ATPase Domain of Mitochondrial Hsp70 and Regulates ATP Hydrolysis*

    Science.gov (United States)

    Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J.

    2008-01-01

    Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8 ± 0.2 × 10-4 s-1) at 25 °C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain. PMID:18632665

  11. The human escort protein Hep binds to the ATPase domain of mitochondrial hsp70 and regulates ATP hydrolysis.

    Science.gov (United States)

    Zhai, Peng; Stanworth, Crystal; Liu, Shirley; Silberg, Jonathan J

    2008-09-19

    Hsp70 escort proteins (Hep) have been implicated as essential for maintaining the function of yeast mitochondrial hsp70 molecular chaperones (mtHsp70), but the role that escort proteins play in regulating mammalian chaperone folding and function has not been established. We present evidence that human mtHsp70 exhibits limited solubility due to aggregation mediated by its ATPase domain and show that human Hep directly enhances chaperone solubility through interactions with this domain. In the absence of Hep, mtHsp70 was insoluble when expressed in Escherichia coli, as was its isolated ATPase domain and a chimera having this domain fused to the peptide-binding domain of HscA, a soluble monomeric chaperone. In contrast, these proteins all exhibited increased solubility when expressed in the presence of Hep. In vitro studies further revealed that purified Hep regulates the interaction of mtHsp70 with nucleotides. Full-length mtHsp70 exhibited slow intrinsic ATP hydrolysis activity (6.8+/-0.2 x 10(-4) s(-1)) at 25 degrees C, which was stimulated up to 49-fold by Hep. Hep also stimulated the activity of the isolated ATPase domain, albeit to a lower maximal extent (11.5-fold). In addition, gel-filtration studies showed that formation of chaperone-escort protein complexes inhibited mtHsp70 self-association, and they revealed that Hep binding to full-length mtHsp70 and its isolated ATPase domain is strongest in the absence of nucleotides. These findings provide evidence that metazoan escort proteins regulate the catalytic activity and solubility of their cognate chaperones, and they indicate that both forms of regulation arise from interactions with the mtHsp70 ATPase domain.

  12. Rabies virus co-localizes with early (Rab5) and late (Rab7) endosomal proteins in neuronal and SH-SY5Y cells.

    Science.gov (United States)

    Ahmad, Waqas; Li, Yingying; Guo, Yidi; Wang, Xinyu; Duan, Ming; Guan, Zhenhong; Liu, Zengshan; Zhang, Maolin

    2017-06-01

    Rabies virus (RABV) is a highly neurotropic virus that follows clathrin-mediated endocytosis and pH-dependent pathway for trafficking and invasion into endothelial cells. Early (Rab5, EEA1) and late (Rab7, LAMP1) endosomal proteins play critical roles in endosomal sorting, maturity and targeting various molecular cargoes, but their precise functions in the early stage of RABV neuronal infection remain elusive. In this study, the relationship between enigmatic entry of RABV with these endosomal proteins into neuronal and SH-SY5Y cells was investigated. Immunofluorescence, TCID 50 titers, electron microscopy and western blotting were carried out to determine the molecular interaction of the nucleoprotein (N) of RABV with early or late endosomal proteins in these cell lines. The expression of N was also determined by down-regulating Rab5 and Rab7 in both cell lines through RNA interference. The results were indicative that N proficiently colocalized with Rab5/EEA1 and Rab7/LAMP1 in both cell lines at 24 and 48 h post-infection, while N titers significantly decreased in early infection of RABV. Down-regulation of Rab5 and Rab7 did not inhibit N expression, but it prevented productive infection via blocking the normal trafficking of RABV in a low pH environment. Ultrathin sections of cells studied by electron microscope also verified the close association of RABV with Rab5 and Rab7 in neurons. From the data it was concluded that primary entry of RABV strongly correlates with the kinetics of Rab-proteins present on early and late vesicles, which provides helpful clues to explain the early events of RABV in nerve cells.

  13. Disruption of endocytic trafficking protein Rab7 impairs invasiveness of cholangiocarcinoma cells.

    Science.gov (United States)

    Suwandittakul, Nantana; Reamtong, Onrapak; Molee, Pattamaporn; Maneewatchararangsri, Santi; Sutherat, Maleerat; Chaisri, Urai; Wongkham, Sopit; Adisakwattana, Poom

    2017-09-07

    Alterations and mutations of endo-lysosomal trafficking proteins have been associated with cancer progression. Identification and characterization of endo-lysosomal trafficking proteins in invasive cholangiocarcinoma (CCA) cells may benefit prognosis and drug design for CCA. To identify and characterize endo-lysosomal trafficking proteins in invasive CCA. A lysosomal-enriched fraction was isolated from a TNF-α induced invasive CCA cell line (KKU-100) and uninduced control cells and protein identification was performed with nano-LC MS/MS. Novel lysosomal proteins that were upregulated in invasive CCA cells were validated by real-time RT-PCR. We selected Rab7 for further studies of protein level using western blotting and subcellular localization using immunofluorescence. The role of Rab7 in CCA invasion was determined by siRNA gene knockdown and matrigel transwell assay. Rab7 mRNA and protein were upregulated in invasive CCA cells compared with non-treated controls. Immunofluorescence studies demonstrated that Rab7 was expressed predominantly in invasive CCA cells and was localized in the cytoplasm and lysosomes. Suppression of Rab7 translation significantly inhibited TNF-α-induced cell invasion compared to non-treated control (p= 0.044). Overexpression of Rab7 in CCA cells was associated with cell invasion, supporting Rab7 as a novel candidate for the development of diagnostic and therapeutic strategies for CCA.

  14. Rab GTPases Regulate Endothelial Cell Protein C Receptor-Mediated Endocytosis and Trafficking of Factor VIIa

    Science.gov (United States)

    Nayak, Ramesh C.; Keshava, Shiva; Esmon, Charles T.; Pendurthi, Usha R.; Rao, L. Vijaya Mohan

    2013-01-01

    Recent studies have established that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR). FVIIa binding to EPCR may promote the endocytosis of this receptor/ligand complex. Rab GTPases are known to play a crucial role in the endocytic and exocytic pathways of receptors or receptor/ligand complexes. The present study was undertaken to investigate the role of Rab GTPases in the intracellular trafficking of EPCR and FVIIa. CHO-EPCR cells and human umbilical vein endothelial cells (HUVEC) were transduced with recombinant adenoviral vectors to express wild-type, constitutively active, or dominant negative mutant of various Rab GTPases. Cells were exposed to FVIIa conjugated with AF488 fluorescent probe (AF488-FVIIa), and intracellular trafficking of FVIIa, EPCR, and Rab proteins was evaluated by immunofluorescence confocal microscopy. In cells expressing wild-type or constitutively active Rab4A, internalized AF488-FVIIa accumulated in early/sorting endosomes and its entry into the recycling endosomal compartment (REC) was inhibited. Expression of constitutively active Rab5A induced large endosomal structures beneath the plasma membrane where EPCR and FVIIa accumulated. Dominant negative Rab5A inhibited the endocytosis of EPCR-FVIIa. Expression of constitutively active Rab11 resulted in retention of accumulated AF488-FVIIa in the REC, whereas expression of a dominant negative form of Rab11 led to accumulation of internalized FVIIa in the cytoplasm and prevented entry of internalized FVIIa into the REC. Expression of dominant negative Rab11 also inhibited the transport of FVIIa across the endothelium. Overall our data show that Rab GTPases regulate the internalization and intracellular trafficking of EPCR-FVIIa. PMID:23555015

  15. Rab3 proteins involved in vesicle biogenesis and priming in embryonic mouse chromaffin cells

    DEFF Research Database (Denmark)

    Schonn, Jean-Sébastien; van Weering, Jan R T; Mohrmann, Ralf

    2010-01-01

    The four Rab3 paralogs A-D are involved in exocytosis, but their mechanisms of action are hard to study due to functional redundancy. Here we used a quadruple Rab3 knock-out (rab3a, rab3b, rab3c, rab3d null, here denoted ABCD(-/-)) mouse line to investigate Rab3 function in embryonic mouse adrena...

  16. Silent decision: HP1 protein escorts heterochromatic RNAs to their destiny

    Science.gov (United States)

    Ren, Jie; Martienssen, Robert A

    2012-01-01

    EMBO J advance online publication 06072012; doi:10.1016/j.molcel.2012.05.009 Heterochromatin is classically perceived to be refractory to transcription because of its compact structure. However, Keller et al (2012) now demonstrated that heterochromatic transcripts can accumulate even when heterochromatin is normally packaged. By tracking down the fate of these heterochromatic RNAs, they revealed a new post-transcriptional mechanism of silencing in heterochromatin that involves the dynamic turnover of HP1Swi6 between its free, chromatin-bound and RNA-bound forms. The latter form escorts heterochromatic RNA to degradation. PMID:22705945

  17. Targeting eukaryotic Rab proteins: a smart strategy for chlamydial survival and replication.

    Science.gov (United States)

    Damiani, María Teresa; Gambarte Tudela, Julián; Capmany, Anahí

    2014-09-01

    Chlamydia, an obligate intracellular bacterium which passes its entire lifecycle within a membrane-bound vacuole called the inclusion, has evolved a variety of unique strategies to establish an advantageous intracellular niche for survival. This review highlights the mechanisms by which Chlamydia subverts vesicular transport in host cells, particularly by hijacking the master controllers of eukaryotic trafficking, the Rab proteins. A subset of Rabs and Rab interacting proteins that control the recycling pathway or the biosynthetic route are selectively recruited to the chlamydial inclusion membrane. By interfering with Rab-controlled transport steps, this intracellular pathogen not only prevents its own degradation in the phagocytic pathway, but also creates a favourable intracellular environment for growth and replication. Chlamydia, a highly adapted and successful intracellular pathogen, has several redundant strategies to re-direct vesicles emerging from biosynthetic compartments that carry host molecules essential for bacterial development. Although current knowledge is limited, the latest findings have shed light on the role of Rab proteins in the course of chlamydial infections and could open novel opportunities for anti-chlamydial therapy. © 2014 John Wiley & Sons Ltd.

  18. Rab11 is phosphorylated by classical and novel protein kinase C isoenzymes upon sustained phorbol ester activation.

    Science.gov (United States)

    Pavarotti, Martín; Capmany, Anahí; Vitale, Nicolas; Colombo, María Isabel; Damiani, María Teresa

    2012-02-01

    Rab11 is a small GTPase that controls diverse intracellular trafficking pathways. However, the molecular machinery that regulates the participation of Rab11 in those different transport events is poorly understood. In resting cells, Rab11 localizes at the endocytic recycling compartment (ERC), whereas the different protein kinase C (PKC) isoforms display a cytosolic distribution. Sustained phorbol ester stimulation induces the translocation of the classical PKCα and PKCβII isoenzymes to the ERC enriched in Rab11, and results in transferrin recycling inhibition. In contrast, novel PKCε and atypical PKCζ isoenzymes neither redistribute to the perinucleus nor modify transferrin recycling transport after phorbol ester stimulation. Although several Rabs have been shown to be phosphorylated, there is to date no evidence indicating Rab11 as a kinase substrate. In this report, we show that Rab11 appears phosphorylated in vivo in phorbol ester-stimulated cells. A bioinformatic analysis of Rab11 allowed us to identify several high-probability Ser/Thr kinase phosphorylation sites. Our results demonstrate that classical PKC (PKCα and PKCβII but not PKCβI) directly phosphorylate Rab11 in vitro. In addition, novel PKCε and PKCη but not PKCδ isoenzymes also phosphorylate Rab11. Mass spectrometry analysis revealed that Ser 177 is the Rab11 residue to be phosphorylated in vitro by either PKCβII or PKCε. In agreement, the phosphomimetic mutant, Rab11 S177D, retains transferrin at the ERC in the absence of phorbol-12-myristate-13-acetate stimulus. This report shows for the first time that Rab11 is differentially phosphorylated by distinct PKC isoenzymes and that this post-translational modification might be a regulatory mechanism of intracellular trafficking. Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.

  19. Rab proteins in the brain and corpus allatum of Bombyx mori.

    Science.gov (United States)

    Uno, Tomohide; Furutani, Masayuki; Watanabe, Chihiro; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Yamagata, Hiroshi; Mizoguchi, Akira; Takeda, Makio

    2016-07-01

    In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.

  20. FOXC1 modulates MYOC secretion through regulation of the exocytic proteins RAB3GAP1, RAB3GAP2 and SNAP25.

    Directory of Open Access Journals (Sweden)

    Alexandra Rasnitsyn

    Full Text Available The neurodegenerative disease glaucoma is one of the leading causes of blindness in the world. Glaucoma is characterized by progressive visual field loss caused by retinal ganglion cell (RGC death. Both surgical glaucoma treatments and medications are available, however, they only halt glaucoma progression and are unable to reverse damage. Furthermore, many patients do not respond well to treatments. It is therefore important to better understand the mechanisms involved in glaucoma pathogenesis. Patients with Axenfeld-Rieger syndrome (ARS offer important insight into glaucoma progression. ARS patients are at 50% risk of developing early onset glaucoma and respond poorly to treatments, even when surgical treatments are combined with medications. Mutations in the transcription factor FOXC1 cause ARS. Alterations in FOXC1 levels cause ocular malformations and disrupt stress response in ocular tissues, thereby contributing to glaucoma progression. In this study, using biochemical and molecular techniques, we show that FOXC1 regulates the expression of RAB3GAP1, RAB3GAP2 and SNAP25, three genes with central roles in both exocytosis and endocytosis, responsible for extracellular trafficking. FOXC1 positively regulates RAB3GAP1 and RAB3GAP2, while either increase or decrease in FOXC1 levels beyond its normal range results in decreased SNAP25. In addition, we found that FOXC1 regulation of RAB3GAP1, RAB3GAP2 and SNAP25 affects secretion of Myocilin (MYOC, a protein associated with juvenile onset glaucoma and steroid-induced glaucoma. The present work reveals that FOXC1 is an important regulator of exocytosis and establishes a new link between FOXC1 and MYOC-associated glaucoma.

  1. Molecular characterization and expression analysis of a GTP-binding protein (MiRab5) in Mangifera indica.

    Science.gov (United States)

    Liu, Zhao-liang; Luo, Cong; Dong, Long; Van Toan, Can; Wei, Peng-xiao; He, Xin-hua

    2014-04-25

    The Rab family, the largest branch of Ras small GTPases, plays a crucial role in the vesicular transport in plants. The members of Rab family act as molecular switches that regulate the fusion of vesicles with target membranes through conformational changes. However, little is known about the Rab5 gene involved in fruit ripening and stress response. In this study, the MiRab5 gene was isolated from stress-induced Mangifera indica. The full-length cDNA sequence was 984bp and contained an open reading frame of 600bp, which encoded a 200 amino acid protein with a molecular weight of 21.83kDa and a theoretical isoelectric point of 6.99. The deduced amino acid sequence exhibited high homology with tomato (91% similarity) and contains all five characteristic Rab motifs. Real-time quantitative RT-PCR analysis demonstrated that MiRab5 was ubiquitously expressed in various mango tree tissues at different levels. The expression of MiRab5 was up-regulated during later stages of fruit ripening. Moreover, MiRab5 was generally up-regulated in response to various abiotic stresses (cold, salinity, and PEG treatments). Recombinant MiRab5 protein was successfully expressed and purified. SDS-PAGE and western blot analysis indicated that the expressed protein was recognized by the anti-6-His antibody. These results provide insights into the role of the MiRab5 gene family in fruit ripening and stress responses in the mango plant. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Directory of Open Access Journals (Sweden)

    Cecilia Bucci

    2014-10-01

    Full Text Available Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC and p75NTR, a member of the tumor necrosis factor (TNF receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways.

  3. The Role of Rab Proteins in Neuronal Cells and in the Trafficking of Neurotrophin Receptors

    Science.gov (United States)

    Bucci, Cecilia; Alifano, Pietro; Cogli, Laura

    2014-01-01

    Neurotrophins are a family of proteins that are important for neuronal development, neuronal survival and neuronal functions. Neurotrophins exert their role by binding to their receptors, the Trk family of receptor tyrosine kinases (TrkA, TrkB, and TrkC) and p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily. Binding of neurotrophins to receptors triggers a complex series of signal transduction events, which are able to induce neuronal differentiation but are also responsible for neuronal maintenance and neuronal functions. Rab proteins are small GTPases localized to the cytosolic surface of specific intracellular compartments and are involved in controlling vesicular transport. Rab proteins, acting as master regulators of the membrane trafficking network, play a central role in both trafficking and signaling pathways of neurotrophin receptors. Axonal transport represents the Achilles' heel of neurons, due to the long-range distance that molecules, organelles and, in particular, neurotrophin-receptor complexes have to cover. Indeed, alterations of axonal transport and, specifically, of axonal trafficking of neurotrophin receptors are responsible for several human neurodegenerative diseases, such as Huntington’s disease, Alzheimer’s disease, amyotrophic lateral sclerosis and some forms of Charcot-Marie-Tooth disease. In this review, we will discuss the link between Rab proteins and neurotrophin receptor trafficking and their influence on downstream signaling pathways. PMID:25295627

  4. Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

    OpenAIRE

    Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

    2006-01-01

    A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising ...

  5. Liver X receptors balance lipid stores in hepatic stellate cells through Rab18, a retinoid responsive lipid droplet protein.

    Science.gov (United States)

    O'Mahony, Fiona; Wroblewski, Kevin; O'Byrne, Sheila M; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S; Beaven, Simon W

    2015-08-01

    Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ(-/-) mice have increased lipid droplet (LD) size, but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ(-/-) and wild-type mice were profiled by gene array during in vitro activation. Lipid content was quantified by high-performance liquid chromatography and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with small interfering RNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ(-/-) HSCs have increased cholesterol and retinyl esters. The retinoid increase drives intrinsic retinoic acid receptor signaling, and activation occurs more rapidly in Lxrαβ(-/-) HSCs. We identify Rab18 as a novel retinoic acid-responsive, LD-associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 guanosine triphosphatase activity and isoprenylation are required for stellate cell LD loss and induction of activation markers. These phenomena are accelerated in Lxrαβ(-/-) HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards LD loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Retinoid and cholesterol metabolism are linked in stellate cells by the LD-associated protein Rab18. Retinoid overload helps explain the profibrotic phenotype of Lxrαβ(-/-) mice, and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. © 2015 by the American Association for the Study of Liver Diseases.

  6. Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

    Science.gov (United States)

    O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

    2014-01-01

    Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

  7. Bioinformatic and Comparative Localization of Rab Proteins Reveals Functional Insights into the Uncharacterized GTPases Ypt10p and Ypt11p†

    Science.gov (United States)

    Buvelot Frei, Stéphanie; Rahl, Peter B.; Nussbaum, Maria; Briggs, Benjamin J.; Calero, Monica; Janeczko, Stephanie; Regan, Andrew D.; Chen, Catherine Z.; Barral, Yves; Whittaker, Gary R.; Collins, Ruth N.

    2006-01-01

    A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance. PMID:16980630

  8. RAB10 Interacts with the Male Germ Cell-Specific GTPase-Activating Protein during Mammalian Spermiogenesis

    Directory of Open Access Journals (Sweden)

    Ying-Hung Lin

    2017-01-01

    Full Text Available According to recent estimates, 2%–15% of couples are sterile, and approximately half of the infertility cases are attributed to male reproductive factors. However, the reasons remain undefined in approximately 25% of male infertility cases, and most infertility cases exhibit spermatogenic defects. Numerous genes involved in spermatogenesis still remain unknown. We previously identified Male Germ Cells Rab GTPase-Activating Proteins (MGCRABGAPs through cDNA microarray analysis of human testicular tissues with spermatogenic defects. MGCRABGAP contains a conserved RABGAP catalytic domain, TBC (Tre2/Bub2/Cdc16. RABGAP family proteins regulate cellular function (e.g., cytoskeletal remodeling, vesicular trafficking, and cell migration by inactivating RAB proteins. MGCRABGAP is a male germ cell-specific protein expressed in elongating and elongated spermatids during mammalian spermiogenesis. The purpose of this study was to identify proteins that interact with MGCRABGAP during mammalian spermiogenesis using a proteomic approach. We found that MGCRABGAP exhibited GTPase-activating bioability, and several MGCRABGAP interactors, possible substrates (e.g., RAB10, RAB5C, and RAP1, were identified using co-immunoprecipitation (co-IP and nano liquid chromatography-mass spectrometry/mass spectrometry (nano LC-MS/MS. We confirmed the binding ability between RAB10 and MGCRABGAP via co-IP. Additionally, MGCRABGAP–RAB10 complexes were specifically colocalized in the manchette structure, a critical structure for the formation of spermatid heads, and were slightly expressed at the midpiece of mature spermatozoa. Based on these results, we propose that MGCRABGAP is involved in mammalian spermiogenesis by modulating RAB10.

  9. Online Female Escort Advertisements

    Directory of Open Access Journals (Sweden)

    James D. Griffith

    2016-05-01

    Full Text Available Female escorts represent an occupational group that charges a fee for sex, which can be regarded as an extreme form of short-term mating. The present study examined if the fees charged by escorts are related to traits typically associated with female short-term mate value. A total of 2,925 advertisements for female escorts offering sexual services in the United States were examined, as a customized software program was used to download all the advertisements from an online escort directory. The advertisement content was coded, and relationships between advertised physical characteristics and the hourly rate charged by female escorts were examined. The analyses showed that higher fees were associated with female escorts who advertised a waist-to-hip ratio near 0.7, lower weight and body mass index, younger age, and photographic displays of breast and buttocks nudity. The findings provide evidence that evolutionarily relevant traits associated with female short-term mate value are systematically related to fees charged for sexual services.

  10. Expression profile of Rab5, Rab7, tryptophan aspartate-containing coat protein, leprae lipoarabinomannan, and phenolic glycolipid-1 on the failure of the phagolysosome process in macrophages of leprosy patients as a viability marker of Mycobacterium leprae.

    Science.gov (United States)

    Prakoeswa, Cita Rosita Sigit; Wahyuni, Ratna; Iswahyudi; Adriaty, Dinar; Yusuf, Irawan; Sutjipto; Agusni, Indropo; Izumi, Shinzo

    2016-06-01

    Phagolysosome process in macrophage of leprosy patients' is important in the early phase of eliminating Mycobacterium leprae invasion. This study was to clarify the involvement of Rab5, Rab7, and trytophan aspartate-containing coat protein (TACO) from host macrophage and leprae lipoarabinomannan (Lep-LAM) and phenolic glycolipid-1 (PGL-1) from M. leprae cell wall as the reflection of phagolysosome process in relation to 16 subunit ribosomal RNA (16S rRNA) M. leprae as a marker of viability of M. leprae. Using a cross sectional design study, skin biopsies were obtained from 47 newly diagnosed, untreated leprosy at Dr Soetomo Hospital, Surabaya, Indonesia. RNA isolation and complementary DNA synthesis were performed. Samples were divided into two groups: 16S rRNA M. leprae-positive and 16S rRNA M. leprae-negative. The expressions of Rab5, Rab7, TACO, Lep-LAM, and PGL-1 were assessed with an immunohistochemistry technique. Using Mann-Whitney U analysis, a significant difference in the expression profile of Rab5, Rab7, Lep-LAM, and PGL-1 was found (p.05). Spearman analysis revealed that there was a significant correlation between the score of Rab5, Rab7, Lep-LAM, and PGL-1 and the score of 16S rRNA M. leprae (pleprae infection, Rab5, Rab7, and Lep-LAM play important roles in the failure of phagolysosome process via a membrane trafficking pathway, while PGL-1 plays a role via blocking lysosomal activities. These inventions might be used for the development of an early diagnostic device in the future. Copyright © 2016 Asian-African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.

  11. Rab27a regulates epithelial sodium channel (ENaC) activity through synaptotagmin-like protein (SLP-5) and Munc13-4 effector mechanism

    International Nuclear Information System (INIS)

    Saxena, Sunil K.; Horiuchi, Hisanori; Fukuda, Mitsunori

    2006-01-01

    Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins

  12. Munc13-4 Is a Rab11-binding Protein That Regulates Rab11-positive Vesicle Trafficking and Docking at the Plasma Membrane.

    Science.gov (United States)

    Johnson, Jennifer L; He, Jing; Ramadass, Mahalakshmi; Pestonjamasp, Kersi; Kiosses, William B; Zhang, Jinzhong; Catz, Sergio D

    2016-02-12

    The small GTPase Rab11 and its effectors control trafficking of recycling endosomes, receptor replenishment and the up-regulation of adhesion and adaptor molecules at the plasma membrane. Despite recent advances in the understanding of Rab11-regulated mechanisms, the final steps mediating docking and fusion of Rab11-positive vesicles at the plasma membrane are not fully understood. Munc13-4 is a docking factor proposed to regulate fusion through interactions with SNAREs. In hematopoietic cells, including neutrophils, Munc13-4 regulates exocytosis in a Rab27a-dependent manner, but its possible regulation of other GTPases has not been explored in detail. Here, we show that Munc13-4 binds to Rab11 and regulates the trafficking of Rab11-containing vesicles. Using a novel Time-resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, we demonstrate that Munc13-4 binds to Rab11a but not to dominant negative Rab11a. Immunoprecipitation analysis confirmed the specificity of the interaction between Munc13-4 and Rab11, and super-resolution microscopy studies support the interaction of endogenous Munc13-4 with Rab11 at the single molecule level in neutrophils. Vesicular dynamic analysis shows the common spatio-temporal distribution of Munc13-4 and Rab11, while expression of a calcium binding-deficient mutant of Munc13-4 significantly affected Rab11 trafficking. Munc13-4-deficient neutrophils showed normal endocytosis, but the trafficking, up-regulation, and retention of Rab11-positive vesicles at the plasma membrane was significantly impaired. This correlated with deficient NADPH oxidase activation at the plasma membrane in response to Rab11 interference. Our data demonstrate that Munc13-4 is a Rab11-binding partner that regulates the final steps of Rab11-positive vesicle docking at the plasma membrane. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Akt regulates the subcellular localization of the Rab27a-binding protein JFC1 by phosphorylation.

    Science.gov (United States)

    Johnson, Jennifer L; Pacquelet, Sandrine; Lane, William S; Eam, Boreth; Catz, Sergio D

    2005-08-01

    Here, we show that the Rab27a-binding protein JFC1/Slp1 (synaptotagmin-like protein) is regulated by Akt-mediated phosphorylation. Using the phosphatase and tensin homolog-null LNCaP cells and the phosphatidylinositol 3-kinase inhibitor LY294002, we show that the phosphorylation of endogenous JFC1 is dependent on the phosphatidylinositol 3-kinase/Akt pathway. JFC1 was phosphorylated in cells expressing a constitutively active Akt, confirming that it is an Akt substrate in vivo. Direct phosphorylation of JFC1 by Akt was confirmed in vitro. Using microcapillary high-performance liquid chromatography tandem mass spectrometry, we identified five Akt-phosphorylation sites in JFC1. By mutagenesis analysis and subsequent immunoprecipitation (IP), we established that Akt phosphorylates JFC1 at serine 241. JFC1 and Rab27a colocalize in the proximity of the plasma membrane in LNCaP cells. The interaction was confirmed by IP analysis and was abolished by the point mutation W83S in JFC1. Phosphorylation did not alter the ability of JFC1 to bind to Rab27a. Instead, phosphorylation by Akt dramatically decreased when JFC1 was bound to Rab27a. Finally, we show that as a consequence of in vivo phosphorylation, JFC1 dissociates from the membrane, promoting JFC1 redistribution to the cytosol. Our results suggest that Akt regulates JFC1/Slp1 function by phosphorylation and may have implications on Rab27a-containing vesicle secretion.

  14. Rab5 Enhances Classical Swine Fever Virus Proliferation and Interacts with Viral NS4B Protein to Facilitate Formation of NS4B Related Complex

    Directory of Open Access Journals (Sweden)

    Jihui Lin

    2017-08-01

    Full Text Available Classical swine fever virus (CSFV is a fatal pig pestivirus and causes serious financial losses to the pig industry. CSFV NS4B protein is one of the most important viral replicase proteins. Rab5, a member of the small Rab GTPase family, is involved in infection and replication of numerous viruses including hepatitis C virus and dengue virus. Until now, the effects of Rab5 on the proliferation of CSFV are poorly defined. In the present study, we showed that Rab5 could enhance CSFV proliferation by utilizing lentivirus-mediated constitutive overexpression and eukaryotic plasmid transient overexpression approaches. On the other hand, lentivirus-mediated short hairpin RNA knockdown of Rab5 dramatically inhibited virus production. Co-immunoprecipitation, glutathione S-transferase pulldown and laser confocal microscopy assays further confirmed the interaction between Rab5 and CSFV NS4B protein. In addition, intracellular distribution of NS4B-Red presented many granular fluorescent signals (GFS in CSFV infected PK-15 cells. Inhibition of basal Rab5 function with Rab5 dominant negative mutant Rab5S34N resulted in disruption of the GFS. These results indicate that Rab5 plays a critical role in facilitating the formation of the NS4B related complexes. Furthermore, it was observed that NS4B co-localized with viral NS3 and NS5A proteins in the cytoplasm, suggesting that NS3 and NS5A might be components of the NS4B related complex. Taken together, these results demonstrate that Rab5 positively modulates CSFV propagation and interacts with NS4B protein to facilitate the NS4B related complexes formation.

  15. Cloning and molecular characterization of the salt-regulated jojoba ScRab cDNA encoding a small GTP-binding protein.

    Science.gov (United States)

    Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

    2002-10-01

    Salt stress results in a massive change in gene expression. An 837 bp cDNA designated ScRab was cloned from shoot cultures of the salt tolerant jojoba (Simmondsia chinesis). The cloned cDNA encodes a full length 200 amino acid long polypeptide that bears high homology to the Rab subfamily of small GTP binding proteins, particularly, the Rab5 subfamily. ScRab expression is reduced in shoots grown in the presence of salt compared to shoots from non-stressed cultures. His6-tagged ScRAB protein was expressed in E. coli, and purified to homogeneity. The purified protein bound radiolabelled GTP. The unlabelled guanine nucleotides GTP, GTP gamma S and GDP but not ATP, CTP or UTP competed with GTP binding.

  16. The Rab-GTPase-activating protein TBC1D1 regulates skeletal muscle glucose metabolism

    DEFF Research Database (Denmark)

    Szekeres, Ferenc; Chadt, Alexandra; Tom, Robby Z

    2012-01-01

    The Rab-GTPase-activating protein TBC1D1 has emerged as a novel candidate involved in metabolic regulation. Our aim was to determine whether TBC1D1 is involved in insulin as well as energy-sensing signals controlling skeletal muscle metabolism. TBC1D1-deficient congenic B6.SJL-Nob1.10 (Nob1.10(SJL...... be explained partly by a 50% reduction in GLUT4 protein, since proximal signaling at the level of Akt, AMPK, and acetyl-CoA carboxylase (ACC) was unaltered. Paradoxically, in vivo insulin-stimulated 2-deoxyglucose uptake was increased in EDL and tibialis anterior muscle from TBC1D1-deficient mice......)) and wild-type littermates were studied. Glucose and insulin tolerance, glucose utilization, hepatic glucose production, and tissue-specific insulin-mediated glucose uptake were determined. The effect of insulin, AICAR, or contraction on glucose transport was studied in isolated skeletal muscle. Glucose...

  17. Contrasting patterns in the evolution of the Rab GTPase family in Archaeplastida

    Directory of Open Access Journals (Sweden)

    Romana Petrželková

    2014-12-01

    Full Text Available Rab GTPases are a vast group of proteins serving a role of master regulators in membrane trafficking in eukaryotes. Previous studies delineated some 23 Rab and Rab-like paralogs ancestral for eukaryotes and mapped their current phylogenetic distribution, but the analyses relied on a limited sampling of the eukaryotic diversity. Taking advantage of the recent growth of genome and transcriptome resources for phylogenetically diverse plants and algae, we reanalyzed the evolution of the Rab family in eukaryotes with the primary plastid, collectively constituting the presumably monophyletic supergroup Archaeplastida. Our most important novel findings are as follows: (i the ancestral set of Rabs in Archaeplastida included not only the paralogs Rab1, Rab2, Rab5, Rab6, Rab7, Rab8, Rab11, Rab18, Rab23, Rab24, Rab28, IFT27, and RTW (=Rabl2, as suggested previously, but also Rab14 and Rab34, because Rab14 exists in glaucophytes and Rab34 is present in glaucophytes and some green algae; (ii except in embryophytes, Rab gene duplications have been rare in Archaeplastida. Most notable is the independent emergence of divergent, possibly functionally novel, in-paralogs of Rab1 and Rab11 in several archaeplastidial lineages; (iii recurrent gene losses have been a significant factor shaping Rab gene complements in archaeplastidial species; for example, the Rab21 paralog was lost at least six times independently within Archaeplastida, once in the lineage leading to the “core” eudicots; (iv while the glaucophyte Cyanophora paradoxa has retained the highest number of ancestral Rab paralogs among all archaeplastidial species studied so far, rhodophytes underwent an extreme reduction of the Rab gene set along their stem lineage, resulting in only six paralogs (Rab1, Rab2, Rab6, Rab7, Rab11, and Rab18 present in modern red algae. Especially notable is the absence of Rab5, a virtually universal paralog essential for the endocytic pathway, suggesting that endocytosis

  18. Rab5 GTPase controls chromosome alignment through Lamin disassembly and relocation of the NuMA-like protein Mud to the poles during mitosis

    Science.gov (United States)

    Capalbo, Luisa; D'Avino, Pier Paolo; Archambault, Vincent; Glover, David M.

    2011-01-01

    The small GTPase Rab5 is a conserved regulator of membrane trafficking; it regulates the formation of early endosomes, their transport along microtubules, and the fusion to the target organelles. Although several members of the endocytic pathway were recently implicated in spindle organization, it is unclear whether Rab5 has any role during mitosis. Here, we describe that Rab5 is required for proper chromosome alignment during Drosophila mitoses. We also found that Rab5 associated in vivo with nuclear Lamin and mushroom body defect (Mud), the Drosophila counterpart of nuclear mitotic apparatus protein (NuMA). Consistent with this finding, Rab5 was required for the disassembly of the nuclear envelope at mitotic entry and the accumulation of Mud at the spindle poles. Furthermore, Mud depletion caused chromosome misalignment defects that resembled the defects of Rab5 RNAi cells, and double-knockdown experiments indicated that the two proteins function in a linear pathway. Our results indicate a role for Rab5 in mitosis and reinforce the emerging view of the contributions made by cell membrane dynamics to spindle function. PMID:21987826

  19. A Small Ras-like protein Ray/Rab1c modulates the p53-regulating activity of PRPK

    International Nuclear Information System (INIS)

    Abe, Yasuhito; Takeuchi, Takashi; Imai, Yoshinori; Murase, Ryuichi; Kamei, Yoshiaki; Fujibuchi, Taketsugu; Matsumoto, Suguru; Ueda, Norifumi; Ogasawara, Masahito; Shigemoto, Kazuhiro; Kito, Katsumi

    2006-01-01

    PRPK phosphorylates serine-15 residue of p53 and enhances transcriptional activity. PRPK possesses a bipartite nuclear localization signal and localizes in nucleus when over-expressed in cells. However, intrinsic PRPK localizes mainly in the cytosol in situ. While studying the mechanisms in the distribution of intrinsic PRPK, we identified a PRPK binding protein, an ubiquitously expressed Small Ras-like GTPase, Rab1c, also named Ray or Rab35. The over-expressed Ray was distributed in the nucleus, cytosol, and cell membrane. Both Ray wild type and GTP-restrictively binding mutant Ray-Q67L, but not guanine nucleotide unstable binding mutant Ray-N120I, partially distributed the over-expressed PRPK to the cytosol and also suppressed the PRPK-induced p53-transcriptional activity profoundly. A Small Ras-like GTPase protein Ray was thus indicated to modulate p53 transcriptional activity of PRPK

  20. Antagonistic control of lysosomal fusion by Rab14 and the Lyst-related protein LvsB

    OpenAIRE

    Kypri, Elena; Falkenstein, Kristin; De Lozanne, Arturo

    2013-01-01

    While loss of the protein Lyst causes abnormal lysosomes in patients with Chediak-Higashi Syndrome, the contribution of Lyst to lysosome biology is not known. Previously we found that the Dictyostelium ortholog of Lyst, LvsB, is a cytosolic protein that associates with lysosomes and post-lysosomes to prevent their inappropriate fusion. Here we provide three lines of evidence that indicate that LvsB contributes to lysosome function by antagonizing the function of DdRab14, a protein that promot...

  1. A RabGAP regulates life-cycle duration via trimeric G-protein cascades in Dictyostelium discoideum.

    Directory of Open Access Journals (Sweden)

    Hidekazu Kuwayama

    Full Text Available BACKGROUND: The life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified. METHODOLOGY/PRINCIPAL FINDINGS: Using yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3-deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.

  2. Identification and biochemical analysis of Slac2-c/MyRIP as a Rab27A-, myosin Va/VIIa-, and actin-binding protein.

    Science.gov (United States)

    Kuroda, Taruho S; Fukuda, Mitsunori

    2005-01-01

    Slac2-c/MyRIP is a specific Rab27A-binding protein that contains an N-terminal synaptotagmin-like protein (Slp) homology domain (SHD, a newly identified GTP-Rab27A-binding motif), but in contrast to the Slp family proteins, it lacks C-terminal tandem C2 domains. In vitro Slac2-c simultaneously directly interacts with both Rab27A and an actin-based motor protein, myosin Va, via its N-terminal SHD and middle region, respectively, consistent with the fact that the overall structure of Slac2-c is similar to that of Slac2-a/melanophilin, a linker protein between Rab27A and myosin Va in the melanosome transport in melanocytes. Unlike Slac2-a, however, the middle region of Slac2-c interacts with two types of myosins, myosin Va and myosin VIIa. In addition, the most C-terminal part of both Slac2-a and Slac2-c functions as an actin-binding domain: it directly interacts with globular and fibrous actin in vitro, and the actin-binding domain of Slac2-a and Slac2-c colocalizes with actin filaments when it is expressed in living cells (i.e., PC12 cells and mouse melanocytes). In this chapter we describe the methods that have been used to analyze the protein-protein interactions of Slac2-c, specifically with Rab27A, myosin Va/VIIa, and actin.

  3. RAB-5 and RAB-10 cooperate to regulate neuropeptide release in Caenorhabditis elegans

    Science.gov (United States)

    Sasidharan, Nikhil; Sumakovic, Marija; Hannemann, Mandy; Hegermann, Jan; Liewald, Jana F.; Olendrowitz, Christian; Koenig, Sabine; Grant, Barth D.; Rizzoli, Silvio O.; Gottschalk, Alexander; Eimer, Stefan

    2012-01-01

    Neurons secrete neuropeptides from dense core vesicles (DCVs) to modulate neuronal activity. Little is known about how neurons manage to differentially regulate the release of synaptic vesicles (SVs) and DCVs. To analyze this, we screened all Caenorhabditis elegans Rab GTPases and Tre2/Bub2/Cdc16 (TBC) domain containing GTPase-activating proteins (GAPs) for defects in DCV release from C. elegans motoneurons. rab-5 and rab-10 mutants show severe defects in DCV secretion, whereas SV exocytosis is unaffected. We identified TBC-2 and TBC-4 as putative GAPs for RAB-5 and RAB-10, respectively. Multiple Rabs and RabGAPs are typically organized in cascades that confer directionality to membrane-trafficking processes. We show here that the formation of release-competent DCVs requires a reciprocal exclusion cascade coupling RAB-5 and RAB-10, in which each of the two Rabs recruits the other’s GAP molecule. This contributes to a separation of RAB-5 and RAB-10 domains at the Golgi–endosomal interface, which is lost when either of the two GAPs is inactivated. Taken together, our data suggest that RAB-5 and RAB-10 cooperate to locally exclude each other at an essential stage during DCV sorting. PMID:23100538

  4. ARHGEF10 directs the localization of Rab8 to Rab6-positive executive vesicles.

    Science.gov (United States)

    Shibata, Satoshi; Kawanai, Tsubasa; Hara, Takayuki; Yamamoto, Asuka; Chaya, Taro; Tokuhara, Yasunori; Tsuji, Chinami; Sakai, Manabu; Tachibana, Taro; Inagaki, Shinobu

    2016-10-01

    The function of ARHGEF10, a known guanine nucleotide exchange factor (GEF) for RhoA with proposed roles in various diseases, is poorly understood. To understand the precise function of this protein, we raised a monoclonal antibody against ARHGEF10 and determined its localization in HeLa cells. ARHGEF10 was found to localize to vesicles containing Rab6 (of which there are three isoforms, Rab6a, Rab6b and Rab6c), Rab8 (of which there are two isoforms, Rab8a and Rab8b), and/or the secretion marker neuropeptide Y (NPY)-Venus in a Rab6-dependent manner. These vesicles were known to originate from the Golgi and contain secreted or membrane proteins. Ectopic expression of an N-terminal-truncated ARHGEF10 mutant led to the generation of large vesicle-like structures containing both Rab6 and Rab8. Additionally, small interfering (si)RNA-mediated knockdown of ARHGEF10 impaired the localization of Rab8 to these exocytotic vesicles. Furthermore, the invasiveness of MDA-MB231 cells was markedly decreased by knockdown of ARHGEF10, as well as of Rab8. From these results, we propose that ARHGEF10 acts in exocytosis and tumor invasion in a Rab8-dependent manner. © 2016. Published by The Company of Biologists Ltd.

  5. Small G proteins in insulin action: Rab and Rho families at the crossroads of signal transduction and GLUT4 vesicle traffic.

    Science.gov (United States)

    Ishikura, S; Koshkina, A; Klip, A

    2008-01-01

    Insulin stimulates glucose uptake into muscle and adipose tissues through glucose transporter 4 (GLUT4). GLUT4 cycles between the intracellular compartments and the plasma membrane. GLUT4 traffic-regulating insulin signals are largely within the insulin receptor-insulin receptor substrate-phosphatidylinositol 3-kinase (IR-IRS-PI3K) axis. In muscle cells, insulin signal bifurcates downstream of the PI3K into one arm leading to the activation of the Ser/Thr kinases Akt and atypical protein kinase C, and another leading to the activation of Rho family protein Rac1 leading to actin remodelling. Activated Akt inactivates AS160, a GTPase-activating protein for Rab family small G proteins. Here we review the roles of Rab and Rho proteins, particularly Rab substrates of AS160 and Rac1, in insulin-stimulated GLUT4 traffic. We discuss: (1) how distinct steps in GLUT4 traffic may be regulated by discrete Rab proteins, and (2) the importance of Rac1 activation in insulin-induced actin remodelling in muscle cells, a key element for the net gain in surface GLUT4.

  6. Translocation of the ABC transporter ABCD4 from the endoplasmic reticulum to lysosomes requires the escort protein LMBD1.

    Science.gov (United States)

    Kawaguchi, Kosuke; Okamoto, Takumi; Morita, Masashi; Imanaka, Tsuneo

    2016-07-26

    We previously demonstrated that ABCD4 does not localize to peroxisomes but rather, the endoplasmic reticulum (ER), because it lacks the NH2-terminal hydrophilic region required for peroxisomal targeting. It was recently reported that mutations in ABCD4 result in a failure to release vitamin B12 from lysosomes. A similar phenotype is caused by mutations in LMBRD1, which encodes the lysosomal membrane protein LMBD1. These findings suggested to us that ABCD4 translocated from the ER to lysosomes in association with LMBD1. In this report, it is demonstrated that ABCD4 interacts with LMBD1 and then localizes to lysosomes, and this translocation depends on the lysosomal targeting ability of LMBD1. Furthermore, endogenous ABCD4 was localized to both lysosomes and the ER, and its lysosomal localization was disturbed by knockout of LMBRD1. To the best of our knowledge, this is the first report demonstrating that the subcellular localization of the ABC transporter is determined by its association with an adaptor protein.

  7. The Herpes Simplex Virus Protein pUL31 Escorts Nucleocapsids to Sites of Nuclear Egress, a Process Coordinated by Its N-Terminal Domain.

    Directory of Open Access Journals (Sweden)

    Christina Funk

    2015-06-01

    Full Text Available Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary

  8. Golgi coiled-coil proteins contain multiple binding sites for Rab family G proteins

    NARCIS (Netherlands)

    Sinka, Rita; Gillingham, Alison K.; Kondylis, Vangelis; Munro, Sean

    2008-01-01

    Vesicles and other carriers destined for the Golgi apparatus must be guided to the correct cisternae. Golgins, long coiled-coil proteins that localize to particular Golgi subdomains via their C termini, are candidate regulators of vesicle sorting. In this study, we report that the GRIP domain

  9. The Prenylated Rab GTPase Receptor PRA1.F4 Contributes to Protein Exit from the Golgi Apparatus.

    Science.gov (United States)

    Lee, Myoung Hui; Yoo, Yun-Joo; Kim, Dae Heon; Hanh, Nguyen Hong; Kwon, Yun; Hwang, Inhwan

    2017-07-01

    Prenylated Rab acceptor1 (PRA1) functions in the recruitment of prenylated Rab proteins to their cognate organelles. Arabidopsis ( Arabidopsis thaliana ) contains a large number of proteins belonging to the AtPRA1 family. However, their physiological roles remain largely unknown. Here, we investigated the physiological role of AtPRA1.F4, a member of the AtPRA1 family. A T-DNA insertion knockdown mutant of AtPRA1.F4 , atpra1.f4 , was smaller in stature than parent plants and possessed shorter roots, whereas transgenic plants overexpressing HA:AtPRA1.F4 showed enhanced development of secondary roots and root hairs. However, both overexpression and knockdown plants exhibited increased sensitivity to high-salt stress, lower vacuolar Na + /K + -ATPase and plasma membrane ATPase activities, lower and higher pH in the vacuole and apoplast, respectively, and highly vesiculated Golgi apparatus. HA:AtPRA1.F4 localized to the Golgi apparatus and assembled into high-molecular-weight complexes. atpra1.f4 plants displayed a defect in vacuolar trafficking, which was complemented by low but not high levels of HA : AtPRA1.F4 Overexpression of HA:AtPRA1.F4 also inhibited protein trafficking at the Golgi apparatus, albeit differentially depending on the final destination or type of protein: trafficking of vacuolar proteins, plasma membrane proteins, and trans-Golgi network (TGN)-localized SYP61 was strongly inhibited; trafficking of TGN-localized SYP51 was slightly inhibited; and trafficking of secretory proteins and TGN-localized SYP41 was negligibly or not significantly inhibited. Based on these results, we propose that Golgi-localized AtPRA1.F4 is involved in the exit of many but not all types of post-Golgi proteins from the Golgi apparatus. Additionally, an appropriate level of AtPRA1.F4 is crucial for its function at the Golgi apparatus. © 2017 American Society of Plant Biologists. All Rights Reserved.

  10. Rab11 family expression in the human placenta: Localization at the maternal-fetal interface

    Science.gov (United States)

    Artemiuk, Patrycja A.; Hanscom, Sara R.; Lindsay, Andrew J.; Wuebbolt, Danielle; Breathnach, Fionnuala M.; Tully, Elizabeth C.; Khan, Amir R.; McCaffrey, Mary W.

    2017-01-01

    Rab proteins are a family of small GTPases involved in a variety of cellular processes. The Rab11 subfamily in particular directs key steps of intracellular functions involving vesicle trafficking of the endosomal recycling pathway. This Rab subfamily works through a series of effector proteins including the Rab11-FIPs (Rab11 Family-Interacting Proteins). While the Rab11 subfamily has been well characterized at the cellular level, its function within human organ systems is still being explored. In an effort to further study these proteins, we conducted a preliminary investigation of a subgroup of endosomal Rab proteins in a range of human cell lines by Western blotting. The results from this analysis indicated that Rab11a, Rab11c(Rab25) and Rab14 were expressed in a wide range of cell lines, including the human placental trophoblastic BeWo cell line. These findings encouraged us to further analyse the localization of these Rabs and their common effector protein, the Rab Coupling Protein (RCP), by immunofluorescence microscopy and to extend this work to normal human placental tissue. The placenta is a highly active exchange interface, facilitating transfer between mother and fetus during pregnancy. As Rab11 proteins are closely involved in transcytosis we hypothesized that the placenta would be an interesting human tissue model system for Rab investigation. By immunofluorescence microscopy, Rab11a, Rab11c(Rab25), Rab14 as well as their common FIP effector RCP showed prominent expression in the placental cell lines. We also identified the expression of these proteins in human placental lysates by Western blot analysis. Further, via fluorescent immunohistochemistry, we noted abundant localization of these proteins within key functional areas of primary human placental tissues, namely the outer syncytial layer of placental villous tissue and the endothelia of fetal blood vessels. Overall these findings highlight the expression of the Rab11 family within the human

  11. RABL6A, a Novel RAB-Like Protein, Controls Centrosome Amplification and Chromosome Instability in Primary Fibroblasts

    Science.gov (United States)

    Zhang, Xuefeng; Hagen, Jussara; Muniz, Viviane P.; Smith, Tarik; Coombs, Gary S.; Eischen, Christine M.; Mackie, Duncan I.; Roman, David L.; Van Rheeden, Richard; Darbro, Benjamin; Tompkins, Van S.; Quelle, Dawn E.

    2013-01-01

    RABL6A (RAB-like 6 isoform A) is a novel protein that was originally identified based on its association with the Alternative Reading Frame (ARF) tumor suppressor. ARF acts through multiple p53-dependent and p53-independent pathways to prevent cancer. How RABL6A functions, to what extent it depends on ARF and p53 activity, and its importance in normal cell biology are entirely unknown. We examined the biological consequences of RABL6A silencing in primary mouse embryo fibroblasts (MEFs) that express or lack ARF, p53 or both proteins. We found that RABL6A depletion caused centrosome amplification, aneuploidy and multinucleation in MEFs regardless of ARF and p53 status. The centrosome amplification in RABL6A depleted p53−/− MEFs resulted from centrosome reduplication via Cdk2-mediated hyperphosphorylation of nucleophosmin (NPM) at threonine-199. Thus, RABL6A prevents centrosome amplification through an ARF/p53-independent mechanism that restricts NPM-T199 phosphorylation. These findings demonstrate an essential role for RABL6A in centrosome regulation and maintenance of chromosome stability in non-transformed cells, key processes that ensure genomic integrity and prevent tumorigenesis. PMID:24282525

  12. Loss-of-function of the ciliopathy protein Cc2d2a disorganizes the vesicle fusion machinery at the periciliary membrane and indirectly affects Rab8-trafficking in zebrafish photoreceptors.

    Science.gov (United States)

    Ojeda Naharros, Irene; Gesemann, Matthias; Mateos, José M; Barmettler, Gery; Forbes, Austin; Ziegler, Urs; Neuhauss, Stephan C F; Bachmann-Gagescu, Ruxandra

    2017-12-01

    Ciliopathies are human disorders caused by dysfunction of primary cilia, ubiquitous organelles involved in transduction of environmental signals such as light sensation in photoreceptors. Concentration of signal detection proteins such as opsins in the ciliary membrane is achieved by RabGTPase-regulated polarized vesicle trafficking and by a selective barrier at the ciliary base, the transition zone (TZ). Dysfunction of the TZ protein CC2D2A causes Joubert/Meckel syndromes in humans and loss of ciliary protein localization in animal models, including opsins in retinal photoreceptors. The link between the TZ and upstream vesicle trafficking has been little explored to date. Moreover, the role of the small GTPase Rab8 in opsin-carrier vesicle (OCV) trafficking has been recently questioned in a mouse model. Using correlative light and electron microscopy and live imaging in zebrafish photoreceptors, we provide the first live characterization of Rab8-mediated trafficking in photoreceptors in vivo. Our results support a possibly redundant role for both Rab8a/b paralogs in OCV trafficking, based on co-localization of Rab8 and opsins in vesicular structures, and joint movement of Rab8-tagged particles with opsin. We further investigate the role of the TZ protein Cc2d2a in Rab8-mediated trafficking using cc2d2a zebrafish mutants and identify a requirement for Cc2d2a in the latest step of OCV trafficking, namely vesicle fusion. Progressive accumulation of opsin-containing vesicles in the apical portion of photoreceptors lacking Cc2d2a is caused by disorganization of the vesicle fusion machinery at the periciliary membrane with mislocalization and loss of the t-SNAREs SNAP25 and Syntaxin3 and of the exocyst component Exoc4. We further observe secondary defects on upstream Rab8-trafficking with cytoplasmic accumulation of Rab8. Taken together, our results support participation of Rab8 in OCV trafficking and identify a novel role for the TZ protein Cc2d2a in fusion of incoming

  13. Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

    Science.gov (United States)

    Liu, Ou; Grant, Barth D.

    2015-01-01

    The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

  14. Crystal structure of inactive form of Rab3B

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wei [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Shen, Yang [Structural Genomics Consortium, University of Toronto, 101 College St., Toronto, Ontario, Canada M5G 1L7 (Canada); Jiao, Ronghong [Department of Function Inspection, Hebei Provincial People' s Hospital, Shijiazhuang 050051 (China); Liu, Yanli; Deng, Lingfu [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China); Qi, Chao, E-mail: qichao@mail.ccnu.edu.cn [Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Science, Huazhong Normal University, Wuhan 430079 (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer This is the first structural information of human Rab3B. Black-Right-Pointing-Pointer To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. Black-Right-Pointing-Pointer The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 A resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  15. Crystal structure of inactive form of Rab3B

    International Nuclear Information System (INIS)

    Zhang, Wei; Shen, Yang; Jiao, Ronghong; Liu, Yanli; Deng, Lingfu; Qi, Chao

    2012-01-01

    Highlights: ► This is the first structural information of human Rab3B. ► To provides a structural basis for the GDP/GTP switch in controlling the activity of Rab3. ► The charge distribution of Rab3B indicates its unique roles in vesicular trafficking. -- Abstract: Rab proteins are the largest family of ras-related GTPases in eukaryotic cells. They act as directional molecular switches at membrane trafficking, including vesicle budding, cargo sorting, transport, tethering, and fusion. Here, we generated and crystallized the Rab3B:GDP complex. The structure of the complex was solved to 1.9 Å resolution and the structural base comparison with other Rab3 members provides a structural basis for the GDP/GTP switch in controlling the activity of small GTPase. The comparison of charge distribution among the members of Rab3 also indicates their different roles in vesicular trafficking.

  16. Immune function of a Rab-related protein by modulating the JAK-STAT signaling pathway in the silkworm, Bombyx mori.

    Science.gov (United States)

    Chen, Chen; Eldein, Salah; Zhou, Xiaosan; Sun, Yu; Gao, Jin; Sun, Yuxuan; Liu, Chaoliang; Wang, Lei

    2018-01-01

    The Rab-family GTPases mainly regulate intracellular vesicle transport, and play important roles in the innate immune response in invertebrates. However, the function and signal transduction of Rab proteins in immune reactions remain unclear in silkworms. In this study, we analyzed a Rab-related protein of silkworm Bombyx mori (BmRABRP) by raising antibodies against its bacterially expressed recombinant form. Tissue distribution analysis showed that BmRABRP mRNA and protein were high expressed in the Malpighian tubule and fat body, respectively. However, among the different stages, only the fourth instar larvae and pupae showed significant BmRABRP levels. After challenge with four pathogenic microorganisms (Escherichia coli, BmNPV, Beauveria bassiana, Micrococcus luteus), the expression of BmRABRP mRNA in the fat body was significantly upregulated. In contrast, the BmRABRP protein was significantly upregulated after infection with BmNPV, while it was downregulated by E. coli, B. bassiana, and M. luteus. A specific dsRNA was used to explore the immune function and relationship between BmRABRP and the JAK-STAT signaling pathway. After BmRABRP gene interference, significant reduction in the number of nodules and increased mortality suggested that BmRABRP plays an important role in silkworm's response to bacterial challenge. In addition, four key genes (BmHOP, BmSTAT, BmSOCS2, and BmSOCS6) of the JAK-STAT signaling pathway showed significantly altered expressions after BmRABRP silencing. BmHOP and BmSOCS6 expressions were significantly decreased, while BmSTAT and BmSOCS2 were significantly upregulated. Our results suggested that BmRABRP is involved in the innate immune response against pathogenic microorganisms through the JAK-STAT signaling pathway in silkworm. © 2017 Wiley Periodicals, Inc.

  17. Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae.

    Science.gov (United States)

    Chen, Ming-Chyuan; Cheng, Ying-Min; Sung, Ping-Jyun; Kuo, Cham-En; Fang, Lee-Shing

    2003-08-29

    The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.

  18. The Dictyostelium Bcr/Abr-related protein DRG regulates both Rac- and Rab-dependent pathways

    OpenAIRE

    Knetsch, Menno L.W.; Schäfers, Nicole; Horstmann, Heinz; Manstein, Dietmar J.

    2001-01-01

    Dictyostelium discoideum DdRacGap1 (DRG) contains both Rho-GEF and Rho-GAP domains, a feature it shares with mammalian Bcr and Abr. To elucidate the physiological role of this multifunctional protein, we characterized the enzymatic activity of recombinant DRG fragments in vitro, created DRG-null cells, and studied the function of the protein in vivo by analysing the phenotypic changes displayed by DRG-depleted cells and DRG-null cells complemented with DRG or DRG fragments. Our results show t...

  19. Rab20 regulates phagosome maturation in RAW264 macrophages during Fc gamma receptor-mediated phagocytosis.

    Directory of Open Access Journals (Sweden)

    Youhei Egami

    Full Text Available Rab20, a member of the Rab GTPase family, is known to be involved in membrane trafficking, however its implication in FcγR-mediated phagocytosis is unclear. We examined the spatiotemporal localization of Rab20 during phagocytosis of IgG-opsonized erythrocytes (IgG-Es in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused Rab20, it was shown that Rab20 was transiently associated with the phagosomal membranes. During the early stage of phagosome formation, Rab20 was not localized on the membranes of phagocytic cups, but was gradually recruited to the newly formed phagosomes. Although Rab20 was colocalized with Rab5 to some extent, the association of Rab20 with the phagosomes persisted even after the loss of Rab5 from the phagosomal membranes. Then, Rab20 was colocalized with Rab7 and Lamp1, late endosomal/lysosomal markers, on the internalized phagosomes. Moreover, our analysis of Rab20 mutant expression revealed that the maturation of phagosomes was significantly delayed in cells expressing the GDP-bound mutant Rab20-T19N. These data suggest that Rab20 is an important component of phagosome and regulates the phagosome maturation during FcγR-mediated phagocytosis.

  20. Roles of the outer membrane protein AsmA of Salmonella enterica in the control of marRAB expression and invasion of epithelial cells.

    Science.gov (United States)

    Prieto, Ana I; Hernández, Sara B; Cota, Ignacio; Pucciarelli, M Graciela; Orlov, Yuri; Ramos-Morales, Francisco; García-del Portillo, Francisco; Casadesús, Josep

    2009-06-01

    A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S. enterica, suggesting that lack of AsmA activates expression of the marRAB operon. Hence, asmA mutations may enhance bile resistance by inducing gene expression changes in the marRAB-controlled Mar regulon. In silico analysis of AsmA structure predicted the existence of one transmembrane domain. Biochemical analysis of subcellular fractions revealed that the asmA gene of S. enterica encodes a protein of approximately 70 kDa located in the outer membrane. Because AsmA is unrelated to known transport and/or efflux systems, we propose that activation of marRAB in asmA mutants may be a consequence of envelope reorganization. Competitive infection of BALB/c mice with asmA(+) and asmA isogenic strains indicated that lack of AsmA attenuates Salmonella virulence by the oral route but not by the intraperitoneal route. Furthermore, asmA mutants showed a reduced ability to invade epithelial cells in vitro.

  1. Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

    Science.gov (United States)

    Fu, Yulong; Zhu, Jun-Yi; Zhang, Fujian; Richman, Adam; Zhao, Zhanzheng; Han, Zhe

    2017-06-01

    The Drosophila nephrocyte is a critical component of the fly renal system and bears structural and functional homology to podocytes and proximal tubule cells of the mammalian kidney. Investigations of nephrocyte cell biological processes are fundamental to understanding the insect renal system. Nephrocytes are highly active in endocytosis and vesicle trafficking. Rab GTPases regulate endocytosis and trafficking but specific functions of nephrocyte Rabs remain undefined. We analyzed Rab GTPase expression and function in Drosophila nephrocytes and found that 11 out of 27 Drosophila Rabs were required for normal activity. Rabs 1, 5, 7, 11 and 35 were most important. Gene silencing of the nephrocyte-specific Rab5 eliminated all intracellular vesicles and the specialized plasma membrane structures essential for nephrocyte function. Rab7 silencing dramatically increased clear vacuoles and reduced lysosomes. Rab11 silencing increased lysosomes and reduced clear vacuoles. Our results suggest that Rab5 mediates endocytosis that is essential for the maintenance of functionally critical nephrocyte plasma membrane structures and that Rabs 7 and 11 mediate alternative downstream vesicle trafficking pathways leading to protein degradation and membrane recycling, respectively. Elucidating molecular pathways underlying nephrocyte function has the potential to yield important insights into human kidney cell physiology and mechanisms of cell injury that lead to disease. The Drosophila nephrocyte is emerging as a useful in vivo model system for molecular target identification and initial testing of therapeutic approaches in humans.

  2. The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking.

    Directory of Open Access Journals (Sweden)

    Ruxandra Bachmann-Gagescu

    2015-10-01

    Full Text Available Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary

  3. LRRK2 mediated Rab8a phosphorylation promotes lipid storage.

    Science.gov (United States)

    Yu, Miao; Arshad, Muhammad; Wang, Wenmin; Zhao, Dongyu; Xu, Li; Zhou, Linkang

    2018-02-27

    Several mutations in leucine rich repeat kinase 2 (LRRK2) gene have been associated with pathogenesis of Parkinson's disease (PD), a neurodegenerative disorder marked by resting tremors, and rigidity, leading to Postural instability. It has been revealed that mutations that lead to an increase of kinase activity of LRRK2 protein are significantly associated with PD pathogenesis. Recent studies have shown that some Rab GTPases, especially Rab8, serve as substrates of LRRK2 and undergo phosphorylation in its switch II domain upon interaction. Current study was performed in order to find out the effects of the phosphorylation of Rab8 and its mutants on lipid metabolism and lipid droplets growth. The phosphorylation status of Rab8a was checked by phos-tag gel. Point mutant construct were generated to investigate the function of Rab8a. 3T3L1 cells were transfected with indicated plasmids and the lipid droplets were stained with Bodipy. Fluorescent microscopy experiments were performed to examine the sizes of lipid droplets. The interactions between Rab8a and Optineurin were determined by immunoprecipitation and western blot. Our assays demonstrated that Rab8a was phosphorylated by mutated LRRK2 that exhibits high kinase activity. Phosphorylation of Rab8a on amino acid residue T72 promoted the formation of large lipid droplets. T72D mutant of Rab8a had higher activity to promote the formation of large lipid droplets compared with wild type Rab8a, with increase in average diameter of lipid droplets from 2.10 μm to 2.46 μm. Moreover, phosphorylation of Rab8a weakened the interaction with its effector Optineurin. Y1699C mutated LRRK2 was able to phosphorylate Rab8a and phosphorylation of Rab8a on site 72 plays important role in the fusion and enlargement of lipid droplets. Taken together, our study suggests an indirect relationship between enhanced lipid storage capacity and PD pathogenesis.

  4. A Global Overview of Male Escort Websites.

    Science.gov (United States)

    Kumar, Navin; Minichiello, Victor; Scott, John; Harrington, Taylor

    2017-01-01

    This article details a preliminary dataset of global male escort sites to give insight into the scale of the online market. We conducted a content analysis of 499 Web sites and also measured traffic to these sites. Our analysis examined the structural characteristics of escort services, geographical and regulatory contexts, and resilience of such services. Results suggest that most sites are independent and not affiliated to escort agencies, and the majority cater to male escorts soliciting male clients, with a number of sites for female clientele and couples. These Web sites are dispersed globally, with Asian, European, and South American countries the major hubs in the market and a small number of large multinational sites based in the United States and Europe figuring as a major presence in markets. Although still subject to high levels of regulation in many parts of the world, the data suggest that male escorting is becoming more visible in diverse cultural contexts as measured by the number of Web sites appearing in public spaces.

  5. Maize DRE-binding proteins DBF1 and DBF2 are involved in rab17 regulation through the drought-responsive element in an ABA-dependent pathway.

    Science.gov (United States)

    Kizis, Dimosthenis; Pagès, Montserrat

    2002-06-01

    The abscisic acid-responsive gene rab17 of maize is expressed during late embryogenesis, and is induced by ABA and desiccation in embryo and vegetative tissues. ABRE and DRE cis-elements are involved in regulation of the gene by ABA and drought. Using yeast one-hybrid screening, we isolated two cDNAs encoding two new DRE-binding proteins, designated DBF1 and DBF2, that are members of the AP2/EREBP transcription factor family. Analysis of mRNA accumulation profiles showed that DBF1 is induced during maize embryogenesis and after desiccation, NaCl and ABA treatments in plant seedlings, whereas the DBF2 mRNA is not induced. DNA-binding preferences of DBFs were analysed by electrophoretic mobility shift assays, and showed that both DBF1 and DBF2 bound to the wild-type DRE2 element, but not to the DRE2 mutant or to the DRE1 element which differs only in a single nucleotide. Transactivation activity using particle bombardment showed that DBF1 functioned as activator of DRE2-dependent transcription of rab17 promoter by ABA, whereas DBF2 overexpression had a repression action downregulating not only the basal promoter activity, but also the ABA effect. These results show that ABA plays a role in the regulation of DBF activity, and suggests the existence of an ABA-dependent pathway for the regulation of genes through the C-repeat/DRE element.

  6. Rab7a modulates ER stress and ER morphology.

    Science.gov (United States)

    Mateus, Duarte; Marini, Elettra Sara; Progida, Cinzia; Bakke, Oddmund

    2018-05-01

    The Endoplasmic Reticulum (ER) is a membranous organelle with diverse structural and functional domains. Peripheral ER includes interconnected tubules, and dense tubular arrays called "ER matrices" together with bona fide flat cisternae. Transitions between these states are regulated by membrane-associated proteins and cytosolic factors. Recently, the small GTPases Rab10 and Rab18 were reported to control ER shape by regulating ER dynamics and fusion. Here, we present evidence that another Rab protein, Rab7a, modulates the ER morphology by controlling the ER homeostasis and ER stress. Indeed, inhibition of Rab7a expression by siRNA or expression of the dominant negative mutant Rab7aT22 N, leads to enlargement of sheet-like ER structures and spreading towards the cell periphery. Notably, such alterations are ascribable neither to a direct modulation of the ER shaping proteins Reticulon-4b and CLIMP63, nor to interactions with Protrudin, a Rab7a-binding protein known to affect the ER organization. Conversely, depletion of Rab7a leads to basal ER stress, in turn causing ER membrane expansion. Both ER enlargement and basal ER stress are reverted in rescue experiments by Rab7a re-expression, as well as by the ER chemical chaperone tauroursodeoxycholic acid (TUDCA). Collectively, these findings reveal a new role of Rab7a in ER homeostasis, and indicate that genetic and pharmacological ER stress manipulation may restore ER morphology in Rab7a silenced cells. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. 28 CFR 570.45 - Violation of escorted trip.

    Science.gov (United States)

    2010-07-01

    ... 28 Judicial Administration 2 2010-07-01 2010-07-01 false Violation of escorted trip. 570.45 Section 570.45 Judicial Administration BUREAU OF PRISONS, DEPARTMENT OF JUSTICE COMMUNITY PROGRAMS AND RELEASE COMMUNITY PROGRAMS Escorted Trips § 570.45 Violation of escorted trip. (a) Staff shall process as...

  8. Rab7: roles in membrane trafficking and disease.

    Science.gov (United States)

    Zhang, Ming; Chen, Li; Wang, Shicong; Wang, Tuanlao

    2009-06-01

    The endocytosis pathway controls multiple cellular and physiological events. The lysosome is the destination of newly synthesized lysosomal hydrolytic enzymes. Internalized molecules or particles are delivered to the lysosome for degradation through sequential transport along the endocytic pathway. The endocytic pathway is also emerging as a signalling platform, in addition to the well-known role of the plasma membrane for signalling. Rab7 is a late endosome-/lysosome-associated small GTPase, perhaps the only lysosomal Rab protein identified to date. Rab7 plays critical roles in the endocytic processes. Through interaction with its partners (including upstream regulators and downstream effectors), Rab7 participates in multiple regulation mechanisms in endosomal sorting, biogenesis of lysosome [or LRO (lysosome-related organelle)] and phagocytosis. These processes are closely related to substrates degradation, antigen presentation, cell signalling, cell survival and microbial pathogen infection. Consistently, mutations or dysfunctions of Rab7 result in traffic disorders, which cause various diseases, such as neuropathy, cancer and lipid metabolism disease. Rab7 also plays important roles in microbial pathogen infection and survival, as well as in participating in the life cycle of viruses. Here, we give a brief review on the central role of Rab7 in endosomal traffic and summarize the studies focusing on the participation of Rab7 in disease pathogenesis. The underlying mechanism governed by Rab7 and its partners will also be discussed.

  9. Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori.

    Science.gov (United States)

    Uno, Tomohide; Furutani, Masayuki; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Mizoguchi, Akira; Hiragaki, Susumu; Takeda, Makio

    2017-09-01

    Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori. © 2017 Wiley Periodicals, Inc.

  10. Family-wide characterization of the DENN domain Rab GDP-GTP exchange factors.

    Science.gov (United States)

    Yoshimura, Shin-ichiro; Gerondopoulos, Andreas; Linford, Andrea; Rigden, Daniel J; Barr, Francis A

    2010-10-18

    A key requirement for Rab function in membrane trafficking is site-specific activation by GDP-GTP exchange factors (GEFs), but the majority of the 63 human Rabs have no known GEF. We have performed a systematic characterization of the 17 human DENN domain proteins and demonstrated that they are specific GEFs for 10 Rabs. DENND1A/1B localize to clathrin patches at the plasma membrane and activate Rab35 in an endocytic pathway trafficking Shiga toxin to the trans-Golgi network. DENND2 GEFs target to actin filaments and control Rab9-dependent trafficking of mannose-6-phosphate receptor to lysosomes. DENND4 GEFs target to a tubular membrane compartment adjacent to the Golgi, where they activate Rab10, which suggests a function in basolateral polarized sorting in epithelial cells that compliments the non-DENN GEF Sec2 acting on Rab8 in apical sorting. DENND1C, DENND3, DENND5A/5B, MTMR5/13, and MADD activate Rab13, Rab12, Rab39, Rab28, and Rab27A/27B, respectively. Together, these findings provide a basis for future studies on Rab regulation and function.

  11. Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

    International Nuclear Information System (INIS)

    Mesa, Rosana; Magadan, Javier; Barbieri, Alejandro; Lopez, Cecilia; Stahl, Philip D.; Mayorga, Luis S.

    2005-01-01

    The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN)

  12. The GTPase Rab43 Controls the Anterograde ER-Golgi Trafficking and Sorting of GPCRs

    Directory of Open Access Journals (Sweden)

    Chunman Li

    2017-10-01

    Full Text Available G-protein-coupled receptors (GPCRs constitute the largest superfamily of cell-surface signaling proteins. However, mechanisms underlying their surface targeting and sorting are poorly understood. Here, we screen the Rab family of small GTPases in the surface transport of multiple GPCRs. We find that manipulation of Rab43 function significantly alters the surface presentation and signaling of all GPCRs studied without affecting non-GPCR membrane proteins. Rab43 specifically regulates the transport of nascent GPCRs from the endoplasmic reticulum (ER to the Golgi. More interestingly, Rab43 directly interacts with GPCRs in an activation-dependent fashion. The Rab43-binding domain identified in the receptors effectively converts non-GPCR membrane protein transport into a Rab43-dependent pathway. These data reveal a crucial role for Rab43 in anterograde ER-Golgi transport of nascent GPCRs, as well as the ER sorting of GPCR members by virtue of its ability to interact directly.

  13. Identification and characterization of Iporin as a novel interaction partner for rab1

    Directory of Open Access Journals (Sweden)

    Konczal Magdalena

    2005-03-01

    Full Text Available Abstract Background The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport. Results Here, we report the characterization of Iporin, which is similar to KIAA0375 as a novel rab1-interacting protein. It was initially identified by yeast two-hybrid screening experiments with the active mutant of rab1b (rab1b Q67R as bait. Iporin contains a SH3 domain and two polyproline stretches, which are known to play a role in protein/protein interactions. In addition, Iporin encloses a RUN domain, which seems to be a major part of the rab1binding domain (R1BD. Iporin is ubiquitously expressed and immunofluorescence staining displays a cytosolic punctual distribution. Interestingly, we also show that Iporin interacts with another rab1 interacting partner, the GM130 protein. Conclusion Our results demonstrate that Iporin is a potential new interacting partner of rab1. Iporin is different from already identified rab1 interacting proteins concerning protein structure and cellular localization. We conclude that Iporin might function as a link between the targeting of ER derived vesicles, triggered by the rab1 GTPase and a signaling pathway regulated by molecules containing SH3 and/or poly-proline regions. The characterization of this novel intermolecular relation could help to elucidate how vesicles find their way from ER to the Golgi apparatus.

  14. Rab3a-Bound CD63 Is Degraded and Rab3a-Free CD63 Is Incorporated into HIV-1 Particles

    Directory of Open Access Journals (Sweden)

    Yoshinao Kubo

    2017-08-01

    Full Text Available CD63, a member of the tetraspanin family, is involved in virion production by human immunodeficiency virus type 1 (HIV-1, but its mechanism is unknown. In this study, we showed that a small GTP-binding protein, Rab3a, interacts with CD63. When Rab3a was exogenously expressed, the amounts of CD63 decreased in cells. The Rab3a-mediated reduction of CD63 was suppressed by lysosomal and proteasomal inhibitors. The amount of CD63 was increased by reducing the endogenous Rab3a level using a specific shRNA. These results indicate that Rab3a binds to CD63 to induce the degradation of CD63. Rab3a is thought to be involved in exocytosis, but we found that another function of Rab3a affects the fate of CD63 in lysosomes. CD63 interacted with Rab3a and was incorporated into HIV-1 particles. However, Rab3a was not detected in HIV-1 virions, thereby indicating that Rab3a-free CD63, but not Rab3a-bound CD63, is incorporated into HIV-1 particles. Overexpression or silencing of Rab3a moderately reduced HIV-1 virion formation. Overexpression of Rab3a decreased CD63 levels, but did not affect the incorporation of CD63 into HIV-1 particles. This study showed that Rab3a binds to CD63 to induce the degradation of CD63, and only Rab3a-free CD63 is incorporated into HIV-1 particles.

  15. Identification and characterization of a member of Rab subfamily, Rab8, from Clonorchis sinensis.

    Science.gov (United States)

    Liang, Pei; He, Lei; Yu, Jinyun; Xie, Zhizhi; Chen, Xueqing; Mao, Qiang; Liang, Chi; Huang, Yan; Lu, Gang; Yu, Xinbing

    2015-05-01

    The Rabs act as a binary molecular switch that utilizes the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins. It regulates a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, and intracellular membrane trafficking in eukaryotes. The Rab8 from Clonorchis sinensis (CsRab8) was composed of 199 amino acids. The deduced amino acid sequence shared above 50% identities with other species from trematode, tapeworm, mammal, insecta, nematode, and reptile, respectively. The homologous analysis of sequences showed the conservative domains: G1 box (GDSGVGKS), G2 box (T), G3 box (DTAG), G4 box (GNKCDL), and G5 box. In addition, the structure modeling had also shown other functional domains: GTP/Mg(2+) binding sites, switch I region, and switch II region. A phylogenic tree analysis indicated that the CsRab8 was clustered with the Rab from Schistosoma japonicum, and trematode and tapeworm came from the same branch, which was different from an evolutional branch built by other species, such as mammal animal, insecta, nematode, and reptile. The recombinant CsRab8 protein was expressed in Escherichia coli and the purified protein was a soluble molecule by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis. CsRab8 was identified as a component of excretory/secretory products of C. sinensis by western blot analysis. The transcriptional level of CsRab8 at metacercaria stage was the highest at the four stages and higher by 56.49-folds than that at adult worm, 1.23-folds than that at excysted metacercaria, and 2.69-folds than that at egg stage. Immunohistochemical localization analysis showed that CsRab8 was specifically distributed in the tegument, vitellarium, eggs, and testicle of adult worms, and detected on the vitellarium and tegument of metacercaria. Combined with the results, CsRab8 is indispensable for survival and development of parasites, especially for regulating

  16. Rice Rab11 is required for JA-mediated defense signaling

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Min Ji [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of); Lee, Yun mi [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Son, Young Sim [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Im, Chak Han [Eco-Friendliness Research Department, Gyeongsangnam-do Agricultural Research and Extension Services, Jinju 660-360 (Korea, Republic of); Yi, Young Byung [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); Rim, Yeong Gil [Systems and Synthetic Agrobiotech Center, Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Bahk, Jeong Dong, E-mail: jdbahk@gnu.ac.kr [Division of Applied Life Sciences (BK21), Graduate School of Gyeongsang National University, Jinju 660-701 (Korea, Republic of); Heo, Jae Bok, E-mail: jbheo72@dau.ac.kr [Department of Molecular Biotechnology, Dong-A University, Busan 604-714 (Korea, Republic of); BK21 Center for Silver-Bio Industrialization, Dong-A University, Busan 604-714 (Korea, Republic of)

    2013-05-17

    Highlights: •OsRab11 interacts with OsOPR8. •OsOPR8 is localized in the cytosol and peroxisome. •OsRab11 enhances the NADPH consumption by OsOPR8. •Transgenic Arabidopsis overexpressing OsRab11 represents a pathogen-resistant phenotype. -- Abstract: Rab proteins play an essential role in regulating vesicular transport in eukaryotic cells. Previously, we characterized OsRab11, which in concert with OsGAP1 and OsGDI3 regulates vesicular trafficking from the trans-Golgi network (TGN) to the plasma membrane or vacuole. To further elucidate the physiological function of OsRab11 in plants, we performed yeast two-hybrid screens using OsRab11 as bait. OsOPR8 was isolated and shown to interact with OsRab11. A co-immunoprecipitation assay confirmed this interaction. The green fluorescent protein-OsOPR8 fusion product was targeted to the cytoplasm and peroxisomes of protoplasts from Arabidopsis thaliana. OsOPR8 exhibited NADPH-dependent reduction activity when 2-cyclohexen-1-one (CyHE) and 12-oxo-phytodienoic acid (OPDA) were supplied as possible substrates. Interestingly, NADPH oxidation by OsOPR8 was increased when wild-type OsRab11 or the constitutively active form of OsRab11 (Q78L) were included in the reaction mix, but not when the dominant negative form of OsRab11 (S28N) was included. OsRab11 was expressed broadly in plants and both OsRab11 and OsOPR8 were induced by jasmonic acid (JA) and elicitor treatments. Overexpressed OsRab11 transgenic plants showed resistance to pathogens through induced expression of JA-responsive genes. In conclusion, OsRab11 may be required for JA-mediated defense signaling by activating the reducing activity of OsOPR8.

  17. Role of Rab family GTPases and their effectors in melanosomal logistics.

    Science.gov (United States)

    Ohbayashi, Norihiko; Fukuda, Mitsunori

    2012-04-01

    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.

  18. High Rab27A expression indicates favorable prognosis in CRC.

    Science.gov (United States)

    Shi, Chuanbing; Yang, Xiaojun; Ni, Yijiang; Hou, Ning; Xu, Li; Zhan, Feng; Zhu, Huijun; Xiong, Lin; Chen, Pingsheng

    2015-06-13

    Rab27A is a peculiar member in Rab family and has been suggested to play essential roles in the development of human cancers. However, the association between Rab27A expression and clinicopathological characteristics of colorectal cancer (CRC) has not been elucidated yet. One-step quantitative real-time polymerase chain reaction (qPCR) test with 18 fresh-frozen CRC samples and immunohistochemistry (IHC) analysis in 112 CRC cases were executed to evaluate the relationship between Rab27A expression and the clinicopathological features of CRC. Cox regression and Kaplan-Meier survival analyses were performed to identify the prognostic factors for 112 CRC patients. The results specified that the expression levels of Rab27A mRNA and protein were significantly higher in CRC tissues than that in matched non-cancerous tissues, in both qPCR test (p = 0.029) and IHC analysis (p = 0.020). The IHC data indicated that the Rab27A protein expression in CRC was statistically correlated with lymph node metastasis (p = 0.022) and TNM stage (p = 0.026). Cox multi-factor analysis and Kaplan-Meier method suggested Rab27A protein expression (p = 0.012) and tumor differentiation (p = 0.004) were significantly associated with the overall survival of CRC patients. The data indicated the differentiate expression of Rab27A in CRC tissues and matched non-cancerous tissues. Rab27A may be used as a valuable prognostic biomarker for CRC patients.

  19. A novel missense mutation (G43S) in the switch I region of Rab27A causing Griscelli syndrome

    DEFF Research Database (Denmark)

    Westbroek, W.; Tuchman, M.; Tinloy, B.

    2008-01-01

    The autosomal recessive Griscelli syndrome type II (GSII) is caused by mutations in the RAB27A gene. Typical clinical features include immunological impairment, silver-gray scalp hair, eyelashes and eyebrows and hypomelanosis of the skin. Rabs help determine the specificity of membrane trafficking......-immunoprecipitation studies showed that Rab27A(G43S) fails to interact with its effector Melanophilin, indicating that the switch I region functions in the recruitment of Rab effector proteins Udgivelsesdato: 2008/6...

  20. Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

    Science.gov (United States)

    Ailion, Michael; Hannemann, Mandy; Dalton, Susan; Pappas, Andrea; Watanabe, Shigeki; Hegermann, Jan; Liu, Qiang; Han, Hsiao-Fen; Gu, Mingyu; Goulding, Morgan Q.; Sasidharan, Nikhil; Schuske, Kim; Hullett, Patrick; Eimer, Stefan; Jorgensen, Erik M.

    2014-01-01

    Summary Peptide neuromodulators are released from a unique organelle: the dense-core vesicle. Dense-core vesicles are generated at the trans-Golgi, and then sort cargo during maturation before being secreted. To identify proteins that act in this pathway, we performed a genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We identified two conserved Rab2-binding proteins: RUND-1, a RUN domain protein, and CCCP-1, a coiled-coil protein. RUND-1 and CCCP-1 colocalize with RAB-2 at the Golgi, and rab-2, rund-1 and cccp-1 mutants have similar defects in sorting soluble and transmembrane dense-core vesicle cargos. RUND-1 also interacts with the Rab2 GAP protein TBC-8 and the BAR domain protein RIC-19, a RAB-2 effector. In summary, a new pathway of conserved proteins controls the maturation of dense-core vesicles at the trans-Golgi network. PMID:24698274

  1. The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity.

    Directory of Open Access Journals (Sweden)

    Matthias Stein

    Full Text Available Rab GTPases constitute the largest subfamily of the Ras protein superfamily. Rab proteins regulate organelle biogenesis and transport, and display distinct binding preferences for effector and activator proteins, many of which have not been elucidated yet. The underlying molecular recognition motifs, binding partner preferences and selectivities are not well understood.Comparative analysis of the amino acid sequences and the three-dimensional electrostatic and hydrophobic molecular interaction fields of 62 human Rab proteins revealed a wide range of binding properties with large differences between some Rab proteins. This analysis assists the functional annotation of Rab proteins 12, 14, 26, 37 and 41 and provided an explanation for the shared function of Rab3 and 27. Rab7a and 7b have very different electrostatic potentials, indicating that they may bind to different effector proteins and thus, exert different functions. The subfamily V Rab GTPases which are associated with endosome differ subtly in the interaction properties of their switch regions, and this may explain exchange factor specificity and exchange kinetics.We have analysed conservation of sequence and of molecular interaction fields to cluster and annotate the human Rab proteins. The analysis of three dimensional molecular interaction fields provides detailed insight that is not available from a sequence-based approach alone. Based on our results, we predict novel functions for some Rab proteins and provide insights into their divergent functions and the determinants of their binding partner selectivity.

  2. Rab4GTPase modulates CFTR function by impairing channel expression at plasma membrane

    International Nuclear Information System (INIS)

    Saxena, Sunil K.; Kaur, Simarna; George, Constantine

    2006-01-01

    Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the disruption of biosynthesis or the function of a membrane cAMP-activated chloride channel, CFTR. CFTR regulatory mechanisms include recruitment of channel proteins to the cell surface from intracellular pools and by protein-protein interactions. Rab proteins are small GTPases involved in regulated trafficking controlling vesicle docking and fusion. Rab4 controls recycling events from endosome to the plasma membrane, fusion, and degradation. The colorectal cell line HT-29 natively expresses CFTR and responds to cAMP stimulation with an increase in CFTR-mediated currents. Rab4 over-expression in HT-29 cells inhibits both basal and cAMP-stimulated CFTR-mediated currents. GTPase-deficient Rab4Q67L and GDP locked Rab4S22N both inhibit channel activity, which appears characteristically different. Active status of Rab4 was confirmed by GTP overlay assay, while its expression was verified by Western blotting. The pull-down and immunoprecipitation experiments suggest that Rab4 physically interacts with CFTR through protein-protein interaction. Biotinylation with cell impermeant NHS-Sulfo-SS-Biotin implies that Rab4 impairs CFTR expression at cell surface. The enhanced cytosolic CFTR indicates that Rab4 expression restrains CFTR appearance at the cell membrane. The study suggests that Rab4 regulates the channel through multiple mechanisms that include protein-protein interaction, GTP/GDP exchange, and channel protein trafficking. We propose that Rab4 is a dynamic molecule with a significant role in CFTR function

  3. Molecular cloning, sequence characterization and expression pattern of Rab18 gene from watermelon (Citrullus lanatus).

    Science.gov (United States)

    Xinli, Xiao; Lei, Peng

    2015-03-04

    The complete mRNA sequence of watermelon Rab18 gene was amplified through the rapid amplification of cDNA ends (RACE) method. The full-length mRNA was 1010 bp containing a 645 bp open reading frame, which encodes a protein of 214 amino acids. Sequence analysis revealed that watermelon Rab18 protein shares high homology with the Rab18 of cucumber (99%), muskmelon (98%), Morus notabilis (90%), tomato (89%), wine grape (89%) and potato (88%). Phylogenetic analysis revealed that watermelon Rab18 gene has a closer genetic relationship with Rab18 gene of cucumber and muskmelon. Tissue expression profile analysis indicated that watermelon Rab18 gene was highly expressed in root, stem and leaf, moderately expressed in flower and weakly expressed in fruit.

  4. Rab1A is required for assembly of classical swine fever virus particle.

    Science.gov (United States)

    Lin, Jihui; Wang, Chengbao; Liang, Wulong; Zhang, Jing; Zhang, Longxiang; Lv, Huifang; Dong, Wang; Zhang, Yanming

    2018-01-15

    Rab1A belongs to the small Rab GTPase family and is involved in the lifecycle of numerous viruses. Here, knockdown of Rab1A inhibited CSFV growth. Further study revealed that Rab1A depletion decreased intracellular and extracellular CSFV titers, but did not affect intracellular virus genome copies and E2 protein expression within a virus lifecycle, which suggested that Rab1A is required for CSFV particle assembly rather than for genome replication or virion release. This was proofed by blocking the spread of virus using neutralizing antibodies, through which the negative effects of Rab1A knockdown on multi-cycle replication of CSFV were eliminated. Moreover, co-immunoprecipitation and confocal microscopy assays showed that Rab1A bound to CSFV NS5A protein, indicating that Rab1A and viral NS5A proteins may work cooperatively during CSFV particle assembly. In conclusion, this study demonstrated for the first time that Rab1A is required for CSFV particle assembly and binds to viral particle assembly-related NS5A protein. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. TBC-8, a Putative RAB-2 GAP, Regulates Dense Core Vesicle Maturation in Caenorhabditis elegans

    Science.gov (United States)

    Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M.; Koenig, Sabine; Eimer, Stefan

    2012-01-01

    Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2–specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation. PMID:22654674

  6. TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

    Science.gov (United States)

    Hannemann, Mandy; Sasidharan, Nikhil; Hegermann, Jan; Kutscher, Lena M; Koenig, Sabine; Eimer, Stefan

    2012-01-01

    Dense core vesicles (DCVs) are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs). We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

  7. TBC-8, a putative RAB-2 GAP, regulates dense core vesicle maturation in Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Mandy Hannemann

    Full Text Available Dense core vesicles (DCVs are thought to be generated at the late Golgi apparatus as immature DCVs, which subsequently undergo a maturation process through clathrin-mediated membrane remodeling events. This maturation process is required for efficient processing of neuropeptides within DCVs and for removal of factors that would otherwise interfere with DCV release. Previously, we have shown that the GTPase, RAB-2, and its effector, RIC-19, are involved in DCV maturation in Caenorhabditis elegans motoneurons. In rab-2 mutants, specific cargo is lost from maturing DCVs and missorted into the endosomal/lysosomal degradation route. Cargo loss could be prevented by blocking endosomal delivery. This suggests that RAB-2 is involved in retention of DCV components during the sorting process at the Golgi-endosomal interface. To understand how RAB-2 activity is regulated at the Golgi, we screened for RAB-2-specific GTPase activating proteins (GAPs. We identified a potential RAB-2 GAP, TBC-8, which is exclusively expressed in neurons and which, when depleted, shows similar DCV maturation defects as rab-2 mutants. We could demonstrate that RAB-2 binds to its putative GAP, TBC-8. Interestingly, TBC-8 also binds to the RAB-2 effector, RIC-19. This interaction appears to be conserved as TBC-8 also interacted with the human ortholog of RIC-19, ICA69. Therefore, we propose that a dynamic ON/OFF cycling of RAB-2 at the Golgi induced by the GAP/effector complex is required for proper DCV maturation.

  8. RAB1A promotes Vaccinia virus replication by facilitating the production of intracellular enveloped virions

    Energy Technology Data Exchange (ETDEWEB)

    Pechenick Jowers, Tali; Featherstone, Rebecca J.; Reynolds, Danielle K.; Brown, Helen K. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); James, John; Prescott, Alan [Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland (United Kingdom); Haga, Ismar R. [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom); Beard, Philippa M., E-mail: pip.beard@roslin.ed.ac.uk [The Roslin Institute and Royal (Dick) School of Veterinary Studies, University of Edinburgh, Roslin, Midlothian EH25 9RG, Scotland (United Kingdom)

    2015-01-15

    Vaccinia virus (VACV) is a large double-stranded DNA virus with a complex cytoplasmic replication cycle that exploits numerous cellular proteins. This work characterises the role of a proviral cellular protein, the small GTPase RAB1A, in VACV replication. Using siRNA, we identified RAB1A as required for the production of extracellular enveloped virions (EEVs), but not intracellular mature virions (IMVs). Immunofluorescence and electron microscopy further refined the role of RAB1A as facilitating the wrapping of IMVs to become intracellular enveloped virions (IEVs). This is consistent with the known function of RAB1A in maintenance of ER to Golgi transport. VACV can therefore be added to the growing list of viruses which require RAB1A for optimal replication, highlighting this protein as a broadly proviral host factor. - Highlights: • Characterisation of the role of the small GTPase RAB1A in VACV replication. • RAB1A is not required for production of the primary virion form (IMV). • RAB1A is required for production of processed virion forms (IEVs, CEVs and EEVs). • Consistent with known role of RAB1A in ER to Golgi transport.

  9. Rab7b at the intersection of intracellular trafficking and cell migration.

    Science.gov (United States)

    Distefano, Marita Borg; Kjos, Ingrid; Bakke, Oddmund; Progida, Cinzia

    2015-01-01

    Rab proteins are small GTPases essential for controlling and coordinating intracellular traffic. The small GTPase Rab7b regulates the retrograde transport from late endosomes toward the Trans-Golgi Network (TGN), and is important for the proper trafficking of several receptors such as Toll-like receptors (TLRs) and sorting receptors. We recently identified the actin motor protein myosin II as a new interaction partner for Rab7b, and found that Rab7b transport is dependent on myosin II. Interestingly, we also discovered that Rab7b influences the phosphorylation state of myosin II by controlling the activation status of the small GTPase RhoA. Consequently, Rab7b is important for the remodeling of actin filaments in processes such as stress fiber formation, cell adhesion, polarization and cell migration. Our finding that Rab7b can control actomyosin reorganization reveals yet another important role for Rab proteins, in addition to their already established role as master regulators of intracellular transport. Here we discuss our findings and speculate how they can explain the importance of Rab7b in dendritic cells (DCs).

  10. The functional interplay of Rab11, FIP3 and Rho proteins on the endosomal recycling pathway controls cell shape and symmetry.

    Science.gov (United States)

    Bouchet, Jérôme; McCaffrey, Mary W; Graziani, Andrea; Alcover, Andrés

    2018-07-04

    Several families of small GTPases regulate a variety of fundamental cellular processes, encompassing growth factor signal transduction, vesicular trafficking and control of the cytoskeleton. Frequently, their action is hierarchical and complementary, but much of the detail of their functional interactions remains to be clarified. It is well established that Rab family members regulate a variety of intracellular vesicle trafficking pathways. Moreover, Rho family GTPases are pivotal for the control of the actin and microtubule cytoskeleton. However, the interplay between these 2 types of GTPases has been rarely reported. We discuss here our recent findings showing that Rab11, a key regulator of endosomal recycling, and Rac1, a central actin cytoskeleton regulator involved in lamellipodium formation and cell migration, interplay on endosomes through the Rab11 effector FIP3. In the context of the rapidly reactive T lymphocytes, Rab11-Rac1 endosomal functional interplay is important to control cell shape changes and cell symmetry during lymphocyte spreading and immunological synapse formation and ultimately modulate T cell activation.

  11. SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling.

    Directory of Open Access Journals (Sweden)

    Rannar Airik

    Full Text Available Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired Hedgehog (Hh signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1, of the endosomal sorting complex (RABEP2, ERC1, and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14 at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2 in this process. Together, this study identifies several centrosome-associated proteins as novel SDCCAG8 interaction partners, and provides new insights into the function of SDCCAG8 at this structure.

  12. RAB-10-Dependent Membrane Transport Is Required for Dendrite Arborization

    Science.gov (United States)

    Zou, Wei; Yadav, Smita; DeVault, Laura; Jan, Yuh Nung; Sherwood, David R.

    2015-01-01

    Formation of elaborately branched dendrites is necessary for the proper input and connectivity of many sensory neurons. Previous studies have revealed that dendritic growth relies heavily on ER-to-Golgi transport, Golgi outposts and endocytic recycling. How new membrane and associated cargo is delivered from the secretory and endosomal compartments to sites of active dendritic growth, however, remains unknown. Using a candidate-based genetic screen in C. elegans, we have identified the small GTPase RAB-10 as a key regulator of membrane trafficking during dendrite morphogenesis. Loss of rab-10 severely reduced proximal dendritic arborization in the multi-dendritic PVD neuron. RAB-10 acts cell-autonomously in the PVD neuron and localizes to the Golgi and early endosomes. Loss of function mutations of the exocyst complex components exoc-8 and sec-8, which regulate tethering, docking and fusion of transport vesicles at the plasma membrane, also caused proximal dendritic arborization defects and led to the accumulation of intracellular RAB-10 vesicles. In rab-10 and exoc-8 mutants, the trans-membrane proteins DMA-1 and HPO-30, which promote PVD dendrite stabilization and branching, no longer localized strongly to the proximal dendritic membranes and instead were sequestered within intracellular vesicles. Together these results suggest a crucial role for the Rab10 GTPase and the exocyst complex in controlling membrane transport from the secretory and/or endosomal compartments that is required for dendritic growth. PMID:26394140

  13. SDCCAG8 interacts with rab effector proteins rabep2 and erc1 and is required for hedgehog signaling

    DEFF Research Database (Denmark)

    Airik, Rannar; Schueler, M.; Airik, M.

    2016-01-01

    Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage......1), and with non-muscle myosin motor proteins (MYH9, MYH10, MYH14) at the centrosome. Furthermore, we show that RABEP2 localization at the centrosome is regulated by SDCCAG8. siRNA mediated RABEP2 knockdown in hTERT-RPE1 cells leads to defective ciliogenesis, indicating a critical role for RABEP2...

  14. Protein: FBA6 [TP Atlas

    Lifescience Database Archive (English)

    Full Text Available FBA6 vesicular transport RAB11FIP3 ARFO1, KIAA0665 RAB11FIP3 Rab11 family-interacting pr...otein 3 Arfophilin-1, EF hands-containing Rab-interacting protein, MU-MB-17.148 9606 Homo sapiens O75154 9727 2HV8 2D7C 9727 21790911 ...

  15. Role of Rab5 in the formation of macrophage-derived foam cell.

    Science.gov (United States)

    Chan, Lokwern; Hong, Jin; Pan, Junjie; Li, Jian; Wen, Zhichao; Shi, Haiming; Ding, Jianping; Luo, Xinping

    2017-09-12

    Foam cells play a key role in the occurrence and pathogenesis of atherosclerosis. Its formation starts with the ingestion of oxidized low-density lipoprotein (oxLDL). The process is associated with Ras related protein in brain 5 (Rab5) which plays a critical role in regulating endocytosis and early endosomal trafficking. Base on this, we presumed that Rab5 might participate in the maturation of foam cell. The aim of this study is to investigate the effect of Rab5 on macrophage cholesterol during the evolvement of macrophage when induced by oxLDL to the formation of foam cell. Immunohistochemistry was performed to analyze the distribution of macrophages and Rab5 in atherosclerotic plaque. RNA inteference study and transfection of inactive mutant (GFP-Rab5-S34N) and active mutant (GFP-Rab5-Q79L) in U937-derived macrophage were utilized to investigate the impact of Rab5 on the process of macrophage cholesterol, which could be detected by oil red O staining, determination of intracellular lipid content, filipin staining, nile red staining and the costaining of early endosome antigen-1 (EEA-1) and 1,1'-dioctadecyl-3,3,3',3'-tetramethylin dicarbocyanine (Dil)-labelled oxLDL (Dil-oxLDL). Rab5 was found abundantly localized in macrophage rich areas of human atherosclerotic lesions. On the foam cell study, the expression of Rab5 was increased after the incubation of oxLDL. The inteference study indicated the depletion of Rab5 led to the decreases of oil red O staining areas, total cholesterol and cholesterol esters in U937-derived marophages. Moreover, the fluorescence intensity of filipin and nile red staining were lower in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. The confocal study demonstrated less Dil-oxLDL was internalized in GFP-Rab5-S34N as compared with GFP-Rab5-Q79L; the result showed also the decrease in colocalization of internalized Dil-oxLDL and EEA-1 for GFP-Rab5-S34N as compared with GFP-Rab5-Q79L. Rab5 plays an important role in modulating the

  16. Recognition and stabilization of geranylgeranylated human Rab5 by the GDP Dissociation Inhibitor (GDI).

    Science.gov (United States)

    Edler, Eileen; Stein, Matthias

    2017-10-25

    The small GTPase Rab5 is the key regulator of early endosomal fusion. It is post-translationally modified by covalent attachment of two geranylgeranyl (GG) chains to adjacent cysteine residues of the C-terminal hypervariable region (HVR). The GDP dissociation inhibitor (GDI) recognizes membrane-associated Rab5(GDP) and serves to release it into the cytoplasm where it is kept in a soluble state. A detailed new structural and dynamic model for human Rab5(GDP) recognition and binding with human GDI at the early endosome membrane and in its dissociated state is presented. In the cytoplasm, the GDI protein accommodates the GG chains in a transient hydrophobic binding pocket. In solution, two different binding modes of the isoprenoid chains inserted into the hydrophobic pocket of the Rab5(GDP):GDI complex can be identified. This equilibrium between the two states helps to stabilize the protein-protein complex in solution. Interprotein contacts between the Rab5 switch regions and characteristic patches of GDI residues from the Rab binding platform (RBP) and the C-terminus coordinating region (CCR) reveal insight on the formation of such a stable complex. GDI binding to membrane-anchored Rab5(GDP) is initially mediated by the solvent accessible switch regions of the Rab-specific RBP. Formation of the membrane-associated Rab5(GDP):GDI complex induces a GDI reorientation to establish additional interactions with the Rab5 HVR. These results allow to devise a detailed structural model for the process of extraction of GG-Rab5(GDP) by GDI from the membrane and the dissociation from targeting factors and effector proteins prior to GDI binding.

  17. SDCCAG8 Interacts with RAB Effector Proteins RABEP2 and ERC1 and Is Required for Hedgehog Signaling

    DEFF Research Database (Denmark)

    Airik, Rannar; Schueler, Markus; Airik, Merlin

    2016-01-01

    Recessive mutations in the SDCCAG8 gene cause a nephronophthisis-related ciliopathy with Bardet-Biedl syndrome-like features in humans. Our previous characterization of the orthologous Sdccag8gt/gt mouse model recapitulated the retinal-renal disease phenotypes and identified impaired DNA damage...... response signaling as an underlying disease mechanism in the kidney. However, several other phenotypic and mechanistic features of Sdccag8gt/gt mice remained unexplored. Here we show that Sdccag8gt/gt mice exhibit developmental and structural abnormalities of the skeleton and limbs, suggesting impaired...... Hedgehog (Hh) signaling. Indeed, cell culture studies demonstrate the requirement of SDCCAG8 for ciliogenesis and Hh signaling. Using an affinity proteomics approach, we demonstrate that SDCCAG8 interacts with proteins of the centriolar satellites (OFD1, AZI1), of the endosomal sorting complex (RABEP2, ERC...

  18. Rab32 is important for autophagy and lipid storage in Drosophila.

    Directory of Open Access Journals (Sweden)

    Chao Wang

    Full Text Available Lipids are essential components of all organisms. Within cells, lipids are mainly stored in a specific type of organelle, called the lipid droplet. The molecular mechanisms governing the dynamics of lipid droplets have been little explored. The protein composition of lipid droplets has been analyzed in numerous proteomic studies, and a large number of lipid droplet-associated proteins have been identified, including Rab small GTPases. Rab proteins are known to participate in many intracellular membranous events; however, their exact role in lipid droplets is largely unexplored. Here we systematically investigate the roles of Drosophila Rab family proteins in lipid storage in the larval adipose tissue, fat body. Rab32 and several other Rabs were found to affect the size of lipid droplets as well as lipid levels. Further studies showed that Rab32 and Rab32 GEF/Claret may be involved in autophagy, consequently affecting lipid storage. Loss-of-function mutants of several components in the autophagy pathway result in similar effects on lipid storage. These results highlight the potential functions of Rabs in regulating lipid metabolism.

  19. Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

    DEFF Research Database (Denmark)

    Aligianis, Irene A; Johnson, Colin A; Gissen, Paul

    2005-01-01

    Warburg Micro syndrome (WARBM1) is a severe autosomal recessive disorder characterized by developmental abnormalities of the eye and central nervous system and by microgenitalia. We identified homozygous inactivating mutations in RAB3GAP, encoding RAB3 GTPase activating protein, a key regulator...

  20. GTP- and GDP-Dependent Rab27a Effectors in Pancreatic Beta-Cells.

    Science.gov (United States)

    Yamaoka, Mami; Ishizaki, Toshimasa; Kimura, Toshihide

    2015-01-01

    Small guanosine triphosphatases (GTPases) participate in a wide variety of cellular functions including proliferation, differentiation, adhesion, and intracellular transport. Conventionally, only the guanosine 5'-triphosphate (GTP)-bound small GTPase interacts with effector proteins, and the resulting downstream signals control specific cellular functions. Therefore, the GTP-bound form is regarded as active, and the focus has been on searching for proteins that bind the GTP form to look for their effectors. The Rab family small GTPase Rab27a is highly expressed in some secretory cells and is involved in the control of membrane traffic. The present study reviews recent progress in our understanding of the roles of Rab27a and its effectors in pancreatic beta-cells. In the basal state, GTP-bound Rab27a controls insulin secretion at pre-exocytic stages via its GTP-dependent effectors. We previously identified novel guanosine 5'-diphosphate (GDP)-bound Rab27-interacting proteins. Interestingly, GDP-bound Rab27a controls endocytosis of the secretory membrane via its interaction with these proteins. We also demonstrated that the insulin secretagogue glucose converts Rab27a from its GTP- to GDP-bound forms. Thus, GTP- and GDP-bound Rab27a regulate pre-exocytic and endocytic stages in membrane traffic, respectively. Since the physiological importance of GDP-bound GTPases has been largely overlooked, we consider that the investigation of GDP-dependent effectors for other GTPases is necessary for further understanding of cellular function.

  1. A role for Na+,K+-ATPase α1 in regulating Rab27a localisation on melanosomes.

    Directory of Open Access Journals (Sweden)

    Antonia E G Booth

    Full Text Available The mechanism(s by which Rab GTPases are specifically recruited to distinct intracellular membranes remains elusive. Here we used Rab27a localisation onto melanosomes as a model to investigate Rab targeting. We identified the α1 subunit of Na+,K+-ATPase (ATP1a1 as a novel Rab27a interacting protein in melanocytes and showed that this interaction is direct with the intracellular M4M5 loop of ATP1a1 and independent of nucleotide bound status of the Rab. Knockdown studies in melanocytes revealed that ATP1a1 plays an essential role in Rab27a-dependent melanosome transport. Specifically, expression of ATP1a1, like the Rab27a GDP/GTP exchange factor (Rab3GEP, is essential for targeting and activation of Rab27a to melanosomes. Finally, we showed that the ability of Rab27a mutants to target to melanosomes correlates with the efficiency of their interaction with ATP1a1. Altogether these studies point to a new role for ATP1a1 as a regulator of Rab27a targeting and activation.

  2. Down-regulation of Rab5 decreases characteristics associated with maintenance of cell transformation

    International Nuclear Information System (INIS)

    Silva, Patricio; Soto, Nicolás; Díaz, Jorge; Mendoza, Pablo; Díaz, Natalia; Quest, Andrew F.G.; Torres, Vicente A.

    2015-01-01

    The early endosomal protein Rab5 is highly expressed in tumor samples, although a causal relationship between Rab5 expression and cell transformation has not been established. Here, we report the functional effects of targeting endogenous Rab5 with specific shRNA sequences in different tumor cell lines. Rab5 down-regulation in B16-F10 cells decreased tumor formation by subcutaneous injection into C57/BL6 mice. Accordingly, Rab5 targeting in B16-F10 and A549, but not MDA-MB-231 cells was followed by decreased cell proliferation, increased apoptosis and decreased anchorage-independent growth. These findings suggest that Rab5 expression is required to maintain characteristics associated with cell transformation. - Highlights: • Rab5 is important to the maintenance of cell transformation characteristics. • Down-regulation of Rab5 decreases cell proliferation and increases apoptosis in different cancer cells. • Rab5 is required for anchorage-independent growth and tumorigenicity in-vivo

  3. The Vici Syndrome Protein EPG5 Is a Rab7 Effector that Determines the Fusion Specificity of Autophagosomes with Late Endosomes/Lysosomes.

    Science.gov (United States)

    Wang, Zheng; Miao, Guangyan; Xue, Xue; Guo, Xiangyang; Yuan, Chongzhen; Wang, Zhaoyu; Zhang, Gangming; Chen, Yingyu; Feng, Du; Hu, Junjie; Zhang, Hong

    2016-09-01

    Mutations in the human autophagy gene EPG5 cause the multisystem disorder Vici syndrome. Here we demonstrated that EPG5 is a Rab7 effector that determines the fusion specificity of autophagosomes with late endosomes/lysosomes. EPG5 is recruited to late endosomes/lysosomes by direct interaction with Rab7 and the late endosomal/lysosomal R-SNARE VAMP7/8. EPG5 also binds to LC3/LGG-1 (mammalian and C. elegans Atg8 homolog, respectively) and to assembled STX17-SNAP29 Qabc SNARE complexes on autophagosomes. EPG5 stabilizes and facilitates the assembly of STX17-SNAP29-VAMP7/8 trans-SNARE complexes, and promotes STX17-SNAP29-VAMP7-mediated fusion of reconstituted proteoliposomes. Loss of EPG5 activity causes abnormal fusion of autophagosomes with various endocytic vesicles, in part due to elevated assembly of STX17-SNAP25-VAMP8 complexes. SNAP25 knockdown partially suppresses the autophagy defect caused by EPG5 depletion. Our study reveals that EPG5 is a Rab7 effector involved in autophagosome maturation, providing insight into the molecular mechanism underlying Vici syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Vesicular Trafficking Defects, Developmental Abnormalities, and Alterations in the Cellular Death Process Occur in Cell Lines that Over-Express Dictyostelium GTPase, Rab2, and Rab2 Mutants

    Directory of Open Access Journals (Sweden)

    Katherine Maringer

    2014-08-01

    Full Text Available Small molecular weight GTPase Rab2 has been shown to be a resident of pre-Golgi intermediates and required for protein transport from the ER to the Golgi complex, however, the function of Rab2 in Dictyostelium has yet to be fully characterized. Using cell lines that over-express DdRab2, as well as cell lines over-expressing constitutively active (CA, and dominant negative (DN forms of the GTPase, we report a functional role in vesicular transport specifically phagocytosis, and endocytosis. Furthermore, Rab2 like other GTPases cycles between an active GTP-bound and an inactive GDP-bound state. We found that this GTP/GDP cycle for DdRab2 is crucial for normal Dictyostelium development and cell–cell adhesion. Similar to Rab5 and Rab7 in C. elegans, we found that DdRab2 plays a role in programmed cell death, possibly in the phagocytic removal of apoptotic corpses.

  5. Structural plasticity mediates distinct GAP-dependent GTP hydrolysis mechanisms in Rab33 and Rab5.

    Science.gov (United States)

    Majumdar, Soneya; Acharya, Abhishek; Prakash, Balaji

    2017-12-01

    The classical GTP hydrolysis mechanism, as seen in Ras, employs a catalytic glutamine provided in cis by the GTPase and an arginine supplied in trans by a GTPase activating protein (GAP). The key idea emergent from a large body of research on small GTPases is that GTPases employ a variety of different hydrolysis mechanisms; evidently, these variations permit diverse rates of GTPase inactivation, crucial for temporal regulation of different biological processes. Recently, we unified these variations and argued that a steric clash between active site residues (corresponding to positions 12 and 61 of Ras) governs whether a GTPase utilizes the cis-Gln or the trans-Gln (from the GAP) for catalysis. As the cis-Gln encounters a steric clash, the Rab GTPases employ the so-called dual finger mechanism where the interacting GAP supplies a trans-Gln for catalysis. Using experimental and computational methods, we demonstrate how the cis-Gln of Rab33 overcomes the steric clash when it is stabilized by a residue in the vicinity. In effect, this demonstrates how both cis-Gln- and trans-Gln-mediated mechanisms could operate in the same GTPase in different contexts, i.e. depending on the GAP that regulates its action. Interestingly, in the case of Rab5, which possesses a higher intrinsic GTP hydrolysis rate, a similar stabilization of the cis-Gln appears to overcome the steric clash. Taken together with the mechanisms seen for Rab1, it is evident that the observed variations in Rab and their GAP partners allow structural plasticity, or in other words, the choice of different catalytic mechanisms. © 2017 Federation of European Biochemical Societies.

  6. Membrane localization and dynamics of geranylgeranylated Rab5 hypervariable region.

    Science.gov (United States)

    Edler, Eileen; Schulze, Eric; Stein, Matthias

    2017-08-01

    The small GTPase Rab5 is a key regulator of endosomal trafficking processes and a marker for the early endosome. The C-terminal hypervariable region (HVR) of Rab5 is post-translationally modified at residues Cys 212 and Cys 213 to accommodate two geranylgeranyl anchors (C20 carbon chain length) in order to associate Rab5 with the membrane. The structural role of the HVR regarding protein-early endosome membrane recruitment is not resolved due to its high degree of flexibility and lack of crystallographic information. Here, full-atomistic and coarse-grained molecular dynamics simulations of the truncated Rab5 HVR 206-215 in three model membranes of increasing complexity (pure phospholipid bilayer, ternary membrane with cholesterol, six-component early endosome) were performed. Specific electrostatic interactions between the HVR 206-215 Arg 209 residue and the phosphate group of the inositol ring of PI(3)P were detected. This shows that PI(3)P acts as a first contact site of protein recruitment to the early endosome. The free energy change of HVR 206-215 extraction from the bilayer was largest for the physiological negatively charged membrane. 5μs coarse-grained simulations revealed an active recruitment of PI(3)P to the HVR 206-215 supporting the formation of Rab5- and PI(3)P enriched signaling platforms. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

    Directory of Open Access Journals (Sweden)

    Anahí Capmany

    2010-11-01

    Full Text Available Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

  8. Chlamydia trachomatis intercepts Golgi-derived sphingolipids through a Rab14-mediated transport required for bacterial development and replication.

    Science.gov (United States)

    Capmany, Anahí; Damiani, María Teresa

    2010-11-22

    Chlamydia trachomatis are obligate intracellular bacteria that survive and replicate in a bacterial-modified phagosome called inclusion. As other intracellular parasites, these bacteria subvert the phagocytic pathway to avoid degradation in phagolysosomes and exploit trafficking pathways to acquire both energy and nutrients essential for their survival. Rabs are host proteins that control intracellular vesicular trafficking. Rab14, a Golgi-related Rab, controls Golgi to endosomes transport. Since Chlamydia establish a close relationship with the Golgi apparatus, the recruitment and participation of Rab14 on inclusion development and bacteria growth were analyzed. Time course analysis revealed that Rab14 associated with inclusions by 10 h post infection and was maintained throughout the entire developmental cycle. The recruitment was bacterial protein synthesis-dependent but independent of microtubules and Golgi integrity. Overexpression of Rab14 dominant negative mutants delayed inclusion enlargement, and impaired bacteria replication as determined by IFU. Silencing of Rab14 by siRNA also decreased bacteria multiplication and infectivity. By electron microscopy, aberrant bacteria were observed in cells overexpressing the cytosolic negative Rab14 mutant. Our results showed that Rab14 facilitates the delivery of sphingolipids required for bacterial development and replication from the Golgi to chlamydial inclusions. Novel anti-chlamydial therapies could be developed based on the knowledge of how bacteria subvert host vesicular transport events through Rabs manipulation.

  9. Rab3A, a possible marker of cortical granules, participates in cortical granule exocytosis in mouse eggs

    Energy Technology Data Exchange (ETDEWEB)

    Bello, Oscar Daniel; Cappa, Andrea Isabel; Paola, Matilde de; Zanetti, María Natalia [Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina); Fukuda, Mitsunori [Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578 (Japan); Fissore, Rafael A. [Department of Veterinary and Animal Sciences, University of Massachusetts Amherst, 661 North Pleasant Street, Amherst, MA 01003 (United States); Mayorga, Luis S. [Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina); Michaut, Marcela A., E-mail: mmichaut@gmail.com [Instituto de Histología y Embriología, CONICET – Universidad Nacional de Cuyo, Av. Libertador 80, 5500 Mendoza (Argentina); Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Cuyo (Argentina)

    2016-09-10

    Fusion of cortical granules with the oocyte plasma membrane is the most significant event to prevent polyspermy. This particular exocytosis, also known as cortical reaction, is regulated by calcium and its molecular mechanism is still not known. Rab3A, a member of the small GTP-binding protein superfamily, has been implicated in calcium-dependent exocytosis and is not yet clear whether Rab3A participates in cortical granules exocytosis. Here, we examine the involvement of Rab3A in the physiology of cortical granules, particularly, in their distribution during oocyte maturation and activation, and their participation in membrane fusion during cortical granule exocytosis. Immunofluorescence and Western blot analysis showed that Rab3A and cortical granules have a similar migration pattern during oocyte maturation, and that Rab3A is no longer detected after cortical granule exocytosis. These results suggested that Rab3A might be a marker of cortical granules. Overexpression of EGFP-Rab3A colocalized with cortical granules with a Pearson correlation coefficient of +0.967, indicating that Rab3A and cortical granules have almost a perfect colocalization in the egg cortical region. Using a functional assay, we demonstrated that microinjection of recombinant, prenylated and active GST-Rab3A triggered cortical granule exocytosis, indicating that Rab3A has an active role in this secretory pathway. To confirm this active role, we inhibited the function of endogenous Rab3A by microinjecting a polyclonal antibody raised against Rab3A prior to parthenogenetic activation. Our results showed that Rab3A antibody microinjection abolished cortical granule exocytosis in parthenogenetically activated oocytes. Altogether, our findings confirm that Rab3A might function as a marker of cortical granules and participates in cortical granule exocytosis in mouse eggs. - Highlights: • Rab3A has a similar migration pattern to cortical granules in mouse oocytes. • Rab3A can be a marker of

  10. Apical transport of influenza A virus ribonucleoprotein requires Rab11-positive recycling endosome.

    Directory of Open Access Journals (Sweden)

    Fumitaka Momose

    Full Text Available Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs. Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM. However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD of Rab11 family interacting proteins (Rab11-FIPs. Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

  11. Rab27 GTPases Distribute Extracellular Nanomaps for Invasive Growth and Metastasis: Implications for Prognosis and Treatment

    Directory of Open Access Journals (Sweden)

    Olivier De Wever

    2013-05-01

    Full Text Available The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.

  12. Enhanced water stress tolerance of transgenic maize plants over-expressing LEA Rab28 gene.

    Science.gov (United States)

    Amara, Imen; Capellades, Montserrat; Ludevid, M Dolors; Pagès, Montserrat; Goday, Adela

    2013-06-15

    Late Embryogenesis Abundant (LEA) proteins participate in plant stress responses and contribute to the acquisition of desiccation tolerance. In this report Rab28 LEA gene has been over-expressed in maize plants under a constitutive maize promoter. The expression of Rab28 transcripts led to the accumulation and stability of Rab28 protein in the transgenic plants. Native Rab28 protein is localized to nucleoli in wild type maize embryo cells; here we find by whole-mount immunocytochemistry that in root cells of Rab28 transgenic and wild-type plants the protein is also associated to nucleolar structures. Transgenic plants were tested for stress tolerance and resulted in sustained growth under polyethyleneglycol (PEG)-mediated dehydration compared to wild-type controls. Under osmotic stress transgenic seedlings showed increased leaf and root areas, higher relative water content (RWC), reduced chlorophyll loss and lower Malondialdehyde (MDA) production in relation to wild-type plants. Moreover, transgenic seeds exhibited higher germination rates than wild-type seeds under water deficit. Overall, our results highlight the presence of transgenic Rab28 protein in nucleolar structures and point to the potential of group 5 LEA Rab28 gene as candidate to enhance stress tolerance in maize plants. Copyright © 2013 Elsevier GmbH. All rights reserved.

  13. Wheat TaRab7 GTPase is part of the signaling pathway in responses to stripe rust and abiotic stimuli.

    Directory of Open Access Journals (Sweden)

    Furong Liu

    Full Text Available Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance.

  14. Escort entropies and divergences and related canonical distribution

    International Nuclear Information System (INIS)

    Bercher, J.-F.

    2011-01-01

    We discuss two families of two-parameter entropies and divergences, derived from the standard Renyi and Tsallis entropies and divergences. These divergences and entropies are found as divergences or entropies of escort distributions. Exploiting the nonnegativity of the divergences, we derive the expression of the canonical distribution associated to the new entropies and a observable given as an escort-mean value. We show that this canonical distribution extends, and smoothly connects, the results obtained in nonextensive thermodynamics for the standard and generalized mean value constraints. -- Highlights: → Two-parameter entropies are derived from q-entropies and escort distributions. → The related canonical distribution is derived. → This connects and extends known results in nonextensive statistics.

  15. IL4/PGE2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent

    International Nuclear Information System (INIS)

    Wainszelbaum, Marisa J.; Proctor, Brandon M.; Pontow, Suzanne E.; Stahl, Philip D.; Barbieri, M. Alejandro

    2006-01-01

    The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E 2 (PGE 2 ) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR-mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells

  16. RAB-10 Regulates Dendritic Branching by Balancing Dendritic Transport

    Science.gov (United States)

    Taylor, Caitlin A.; Yan, Jing; Howell, Audrey S.; Dong, Xintong; Shen, Kang

    2015-01-01

    The construction of a large dendritic arbor requires robust growth and the precise delivery of membrane and protein cargoes to specific subcellular regions of the developing dendrite. How the microtubule-based vesicular trafficking and sorting systems are regulated to distribute these dendritic development factors throughout the dendrite is not well understood. Here we identify the small GTPase RAB-10 and the exocyst complex as critical regulators of dendrite morphogenesis and patterning in the C. elegans sensory neuron PVD. In rab-10 mutants, PVD dendritic branches are reduced in the posterior region of the cell but are excessive in the distal anterior region of the cell. We also demonstrate that the dendritic branch distribution within PVD depends on the balance between the molecular motors kinesin-1/UNC-116 and dynein, and we propose that RAB-10 regulates dendrite morphology by balancing the activity of these motors to appropriately distribute branching factors, including the transmembrane receptor DMA-1. PMID:26633194

  17. 78 FR 50335 - Double Hull Tanker Escorts on the Waters of Prince William Sound, Alaska

    Science.gov (United States)

    2013-08-19

    ...-AB96 Double Hull Tanker Escorts on the Waters of Prince William Sound, Alaska AGENCY: Coast Guard, DHS... mandates two tug escorts for double hull tankers over 5,000 gross tons transporting oil in bulk in PWS. The... tug escort requirements apply to certain double hull tankers. DATES: This interim rule is effective...

  18. Extensive in silico analysis of Mimivirus coded Rab GTPase homolog suggests a possible role in virion membrane biogenesis

    Directory of Open Access Journals (Sweden)

    Amrutraj eZade

    2015-09-01

    Full Text Available Rab GTPases are the key regulators of intracellular membrane trafficking in eukaryotes. Many viruses and intracellular bacterial pathogens have evolved to hijack the host Rab GTPase functions, mainly through activators and effector proteins, for their benefit. Acanthamoeba polyphaga mimivirus (APMV is one of the largest viruses and belongs to the monophyletic clade of nucleo-cytoplasmic large DNA viruses (NCLDV. The inner membrane lining is integral to the APMV virion structure. APMV assembly involves extensive host membrane modifications, like vesicle budding and fusion, leading to the formation of a membrane sheet that is incorporated into the virion. Intriguingly, APMV and all group I members of the Mimiviridae family code for a putative Rab GTPase protein. APMV is the first reported virus to code for a Rab GTPase (encoded by R214 gene. Our thorough in silico analysis of the subfamily specific (SF region of Mimiviridae Rab GTPase sequences suggests that they are related to Rab5, a member of the group II Rab GTPases, of lower eukaryotes. Because of their high divergence from the existing three isoforms, A, B and C of the Rab5-family, we suggest that Mimiviridae Rabs constitute a new isoform, Rab5D. Phylogenetic analysis indicated probable horizontal acquisition from a lower eukaryotic ancestor followed by selection and divergence. Furthermore, interaction network analysis suggests that vps34 (a Class III P13K homolog, coded by APMV L615, Atg-8 and dynamin (host proteins are recruited by APMV Rab GTPase during capsid assembly. Based on these observations, we hypothesize that APMV Rab plays a role in the acquisition of inner membrane during virion assembly.

  19. Rab22a enhances CD147 recycling and is required for lung cancer cell migration and invasion.

    Science.gov (United States)

    Zhou, Yang; Wu, Bo; Li, Jiang-Hua; Nan, Gang; Jiang, Jian-Li; Chen, Zhi-Nan

    2017-08-01

    Rab22a is a member of the Ras-related small GTPase family, which plays a key role in regulating the recycling of cargo proteins entering cells through clathrin-independent endocytosis (CIE). Rab22a is overexpressed in different cancer types, including liver cancer, malignant melanoma, ovarian cancer and osteosarcoma. However, its oncogenic role remains unknown. In this study, we found that silencing of Rab22a suppressed the migration and invasion of lung cancer cells. Furthermore, Rab22a interacts with CD147, and knockdown of Rab22a blocks CD147 recycling and promotes CD147 degradation. Taken together, our findings indicate that Rab22a enhances recycling of CD147, which is required for lung cancer cell migration and invasion,and targeting CD147 recycling may be a rational strategy for lung cancer therapy. Copyright © 2017. Published by Elsevier Inc.

  20. Structural basis for the recruitment and activation of the Legionella phospholipase VipD by the host GTPase Rab5

    Science.gov (United States)

    Lucas, María; Gaspar, Andrew H.; Pallara, Chiara; Rojas, Adriana Lucely; Fernández-Recio, Juan; Machner, Matthias P.; Hierro, Aitor

    2014-01-01

    A challenge for microbial pathogens is to assure that their translocated effector proteins target only the correct host cell compartment during infection. The Legionella pneumophila effector vacuolar protein sorting inhibitor protein D (VipD) localizes to early endosomal membranes and alters their lipid and protein composition, thereby protecting the pathogen from endosomal fusion. This process requires the phospholipase A1 (PLA1) activity of VipD that is triggered specifically on VipD binding to the host cell GTPase Rab5, a key regulator of endosomes. Here, we present the crystal structure of VipD in complex with constitutively active Rab5 and reveal the molecular mechanism underlying PLA1 activation. An active site-obstructing loop that originates from the C-terminal domain of VipD is repositioned on Rab5 binding, thereby exposing the catalytic pocket within the N-terminal PLA1 domain. Substitution of amino acid residues located within the VipD–Rab5 interface prevented Rab5 binding and PLA1 activation and caused a failure of VipD mutant proteins to target to Rab5-enriched endosomal structures within cells. Experimental and computational analyses confirmed an extended VipD-binding interface on Rab5, explaining why this L. pneumophila effector can compete with cellular ligands for Rab5 binding. Together, our data explain how the catalytic activity of a microbial effector can be precisely linked to its subcellular localization. PMID:25114243

  1. Pratt and Whitney ESCORT derivative for mars surface power

    International Nuclear Information System (INIS)

    Feller, Gerald J.; Joyner, Russell

    1999-01-01

    The purpose of this paper is to address the applicability of a common reactor system design from the Pratt and Whitney ESCORT nuclear thermal rocket engine concept to support current NASA mars surface-based power requirements. The ESCORT is a bimodal engine capable of supporting a wide range of propulsive thermal and vehicle electrical power requirements. The ESCORT engine is powered by a fast-spectrum beryllium-reflected CERMET-fueled nuclear reactor. In addition to an expander cycle propulsive mode, the ESCORT is capable of operating in an electrical power mode. In this mode, the reactor is used to heat a mixture of helium and xenon to drive a closed-loop Brayton cycle in order to generate electrical energy. Recent Design Reference Mission requirements (DRM) from NASA Johnson Space Center and NASA Lewis Research Center studies in 1997 and 1998 have detailed upgraded requirements for potential mars transfer missions. The current NASA DRM requires a nuclear thermal propulsion system capable of delivering total mission requirements of 200170 N (45000 lbf) thrust and 50 kWe of spacecraft electrical power. Additionally, these requirements detailed a surface power system capable of providing approximately 160 kW of electrical energy over an approximate 10 year period within a given weight and volume envelope. Current NASA studies use a SP-100 reactor (0.8 MT) and a NERVA derivative (1.6 MT) as baseline systems. A mobile power cart of approximate dimensions 1.7 mx4.5 mx4.4 m has been conceptualized to transport the reactor power system on the Mars Surface. The 63.25 cm diameter and 80.25 cm height of the ESCORT and its 1.3 MT of weight fit well within the current weight and volume target range of the NASA DRM requirements. The modifications required to the ESCORT reactor system to support this upgraded electrical power requirements along with operation in the Martian atmospheric conditions are addressed in this paper. Sufficient excess reactivity and burnup capability

  2. Characterization of a Rab11-like GTPase, EhRab11, of Entamoeba histolytica.

    Science.gov (United States)

    McGugan, Glen C; Temesvari, Lesly A

    2003-07-01

    The Entamoeba histolytica Rab11 family of small molecular weight GTPases consists of three members, EhRab11, EhRab11B, and EhRab11C. The functions of these Rabs in Entamoeba have not been determined. Therefore, as an approach to elucidate the role of the Rab11 family of GTPases in Entamoeba, immunofluorescence microscopy was undertaken to define the subcellular localization of one member of this family, EhRab11. Under conditions of growth, EhRab11 displayed a punctate pattern in the cytoplasm of trophozoites. EhRab11 did not colocalize with markers for the Golgi apparatus, endoplasmic reticulum, pinosomes, phagosomes, or compartments formed by receptor-mediated endocytosis, suggesting that this Rab may not play a role in vesicle trafficking between these organelles. Under conditions of iron and serum starvation, EhRab11 was translocated to the periphery of the cell. The altered cellular localization was accompanied by multinucleation of the cells as well as the acquisition of detergent resistance by the cells, features that are characteristic of Entamoeba cysts. The translocation of EhRab11 to the periphery of the cell during iron and serum starvation was specific as the subcellular localizations of two other Rab GTPases, EhRab7 and EhRabA, were not altered under the same conditions. In addition, the formation of multinucleated cells by inhibition of cytokinesis was not sufficient to induce the translocation of EhRab11 to the cell periphery. Taken together, the data suggest that iron and serum starvation may induce encystation in E. histolytica and that EhRab11 may play a role in this process. Moreover, these studies are the first to describe a putative role for a Rab GTPase in encystation in Entamoeba sp.

  3. Revealing localization and regulation of GTPase PmRab7 in lymphoid cells of Penaeus monodon after WSSV infection

    Directory of Open Access Journals (Sweden)

    Amrendra Kumar

    2016-10-01

    Full Text Available Objective: To identify white spot syndrome virus (WSSV entry into the host-cells of the cultured shrimp Penaeus monodon, we have attempted to localize PmRab7 (Ras-related in brain which is playing a vital role in the WSSV internalization. Methods: In this study, we have cloned PmRab7 and expressed in Escherichia coli, further purified rPmRab7 was used for antibody production, isolation of lysosomal sub-cellular fractions and western blot against lysosomal protein. Moreover, high fold-change in PmRab7 regulation with increasing copy number of WSSV has been studied by using real-time PCR. Results: 651 bp amplicon size gene was successfully amplified, ligated amplicon with pTZ T-tail vector confirmed by colony PCR and retriction enzyme digestion on agarose gel. Subcloned (pRSET-B 651 bp gene transformed successfully in Rosetta and after 6 h of induction expressed rPmRab7 was on SDS page, furthermore soluble fraction of rPmRab7 (26 kDa was purified by ni-NTA column. AntiPmRab7 antibody was received by Merk Pvt. Ltd., and western blot analysis revealed that PmRab7 is present in the lysosomal sub-cellular fraction. Copy number of WSSV was increased 5 fold in 24 h and 20 fold in 72 h of infection and subsequently transcrtipt of PmRab7 was Ct = 1.0 to Ct = 8.5. Conclusions: Presence of PmRab7 on lysosome clearly indicating PmRab7 participating in lysosomal maturation, other hand WSSV may follow the same route of entry. WSSV internalization has directly linked with regulation of PmRab7.

  4. rab3 mediates cortical granule exocytosis in the sea urchin egg.

    Science.gov (United States)

    Conner, S; Wessel, G M

    1998-11-15

    Egg activation at fertilization in the sea urchin results in the exocytosis of approximately 15,000 cortical granules that are docked at the plasma membrane. Previously, we reported that several integral membrane proteins modeled in the SNARE hypothesis, synaptotagmin, VAMP, and syntaxin, in addition to a small GTPase of the ras superfamily, rab3, were present on cortical granules (Conner, S., Leaf, D., and Wessel, G., Mol. Reprod. Dev. 48, 1-13, 1997). Here we report that rab3 is associated with cortical granules throughout oogenesis, during cortical granule translocation, and while docked at the egg plasma membrane. Following cortical granule exocytosis, however, rab3 reassociates with a different population of vesicles, at least some of which are of endocytic origin. Because of its selective association with cortical granules in eggs and oocytes, we hypothesize that rab3 functions in cortical granule exocytosis. To test this hypothesis, we used a strategy of interfering with rab3 function by peptide competition with its effector domain, a conserved region within specific rab types. We first identified the effector domain sequence in Lytechinus variegatus eggs and find the sequence 94% identical to the effector domain of rab3 in Stronglocentrotus purpuratus. Then, with synthetic peptides to different regions of the rab3 protein, we find that cortical granule exocytosis is inhibited in eggs injected with effector domain peptides, but not with peptides from the hypervariable region or with a scrambled effector peptide. Additionally, effector-peptide-injected eggs injected with IP3 are blocked in their ability to exocytose cortical granules, suggesting that the inhibition is directly on the membrane fusion event and not the result of interference with the signal transduction mechanism leading to calcium release. We interpret these results to mean that rab3 functions in the regulation of cortical granule exocytosis following vesicle docking. Copyright 1998 Academic

  5. Cellular vacuoles induced by Mycoplasma pneumoniae CARDS toxin originate from Rab9-associated compartments.

    Directory of Open Access Journals (Sweden)

    Coreen Johnson

    Full Text Available Recently, we identified an ADP-ribosylating and vacuolating cytotoxin in Mycoplasma pneumoniae designated Community Acquired Respiratory Distress Syndrome (CARDS toxin. In this study we show that vacuoles induced by recombinant CARDS (rCARDS toxin are acidic and derive from the endocytic pathway as determined by the uptake of neutral red and the fluid-phase marker, Lucifer yellow, respectively. Also, we demonstrate that the formation of rCARDS toxin-associated cytoplasmic vacuoles is inhibited by the vacuolar ATPase inhibitor, bafilomycin A1, and the ionophore, monensin. To examine the ontogeny of these vacuoles, we analyzed the distribution of endosomal and lysosomal membrane markers during vacuole formation and observed the enrichment of the late endosomal GTPase, Rab9, around rCARDS toxin-induced vacuoles. Immunogold-labeled Rab9 and overexpression of green fluorescent-tagged Rab9 further confirmed vacuolar association. The late endosomal- and lysosomal-associated membrane proteins, LAMP1 and LAMP2, also localized to the vacuolar membranes, while the late endosomal protein, Rab7, and early endosomal markers, Rab5 and EEA1, were excluded. HeLa cells expressing dominant-negative (DN Rab9 exhibited markedly reduced vacuole formation in the presence of rCARDS toxin, in contrast to cells expressing DN-Rab7, highlighting the importance of Rab9 function in rCARDS toxin-induced vacuolation. Our findings reveal the unique Rab9-association with rCARDS toxin-induced vacuoles and its possible relationship to the characteristic histopathology that accompanies M. pneumoniae infection.

  6. The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses.

    Science.gov (United States)

    Takahama, Michihiro; Fukuda, Mitsunori; Ohbayashi, Norihiko; Kozaki, Tatsuya; Misawa, Takuma; Okamoto, Toru; Matsuura, Yoshiharu; Akira, Shizuo; Saitoh, Tatsuya

    2017-09-19

    Cyclic GMP-AMP synthase (cGAS) is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5), in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING), the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  7. The RAB2B-GARIL5 Complex Promotes Cytosolic DNA-Induced Innate Immune Responses

    Directory of Open Access Journals (Sweden)

    Michihiro Takahama

    2017-09-01

    Full Text Available Cyclic GMP-AMP synthase (cGAS is a cytosolic DNA sensor that induces the IFN antiviral response. However, the regulatory mechanisms that mediate cGAS-triggered signaling have not been fully explored. Here, we show the involvement of a small GTPase, RAB2B, and its effector protein, Golgi-associated RAB2B interactor-like 5 (GARIL5, in the cGAS-mediated IFN response. RAB2B-deficiency affects the IFN response induced by cytosolic DNA. Consistent with this, RAB2B deficiency enhances replication of vaccinia virus, a DNA virus. After DNA stimulation, RAB2B colocalizes with stimulator of interferon genes (STING, the downstream signal mediator of cGAS, on the Golgi apparatus. The GTP-binding activity of RAB2B is required for its localization on the Golgi apparatus and for recruitment of GARIL5. GARIL5 deficiency also affects the IFN response induced by cytosolic DNA and enhances replication of vaccinia virus. These findings indicate that the RAB2B-GARIL5 complex promotes IFN responses against DNA viruses by regulating the cGAS-STING signaling axis.

  8. Classifying the molecular functions of Rab GTPases in membrane trafficking using deep convolutional neural networks.

    Science.gov (United States)

    Le, Nguyen-Quoc-Khanh; Ho, Quang-Thai; Ou, Yu-Yen

    2018-06-13

    Deep learning has been increasingly used to solve a number of problems with state-of-the-art performance in a wide variety of fields. In biology, deep learning can be applied to reduce feature extraction time and achieve high levels of performance. In our present work, we apply deep learning via two-dimensional convolutional neural networks and position-specific scoring matrices to classify Rab protein molecules, which are main regulators in membrane trafficking for transferring proteins and other macromolecules throughout the cell. The functional loss of specific Rab molecular functions has been implicated in a variety of human diseases, e.g., choroideremia, intellectual disabilities, cancer. Therefore, creating a precise model for classifying Rabs is crucial in helping biologists understand the molecular functions of Rabs and design drug targets according to such specific human disease information. We constructed a robust deep neural network for classifying Rabs that achieved an accuracy of 99%, 99.5%, 96.3%, and 97.6% for each of four specific molecular functions. Our approach demonstrates superior performance to traditional artificial neural networks. Therefore, from our proposed study, we provide both an effective tool for classifying Rab proteins and a basis for further research that can improve the performance of biological modeling using deep neural networks. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Spontaneous CD8 T cell responses against the melanocyte differentiation antigen RAB38/NY-MEL-1 in melanoma patients.

    Science.gov (United States)

    Walton, Senta M; Gerlinger, Marco; de la Rosa, Olga; Nuber, Natko; Knights, Ashley; Gati, Asma; Laumer, Monika; Strauss, Laura; Exner, Carolin; Schäfer, Niklaus; Urosevic, Mirjana; Dummer, Reinhard; Tiercy, Jean-Marie; Mackensen, Andreas; Jaeger, Elke; Lévy, Frédéric; Knuth, Alexander; Jäger, Dirk; Zippelius, Alfred

    2006-12-01

    The melanocyte differentiation Ag RAB38/NY-MEL-1 was identified by serological expression cloning (SEREX) and is expressed in the vast majority of melanoma lesions. The immunogenicity of RAB38/NY-MEL-1 has been corroborated previously by the frequent occurrence of specific Ab responses in melanoma patients. To elucidate potential CD8 T cell responses, we applied in vitro sensitization with overlapping peptides spanning the RAB38/NY-MEL-1 protein sequence and the reverse immunology approach. The identified peptide RAB38/NY-MEL-1(50-58) exhibited a marked response in ELISPOT assays after in vitro sensitization of CD8 T cells from HLA-A *0201(+) melanoma patients. In vitro digestion assays using purified proteasomes provided evidence of natural processing of RAB38/NY-MEL-1(50-58) peptide. Accordingly, monoclonal RAB38/NY-MEL-1(50-58)-specific T cell populations were capable of specifically recognizing HLA-A2(+) melanoma cell lines expressing RAB38/NY-MEL-1. Applying fluorescent HLA-A2/RAB38/NY-MEL-1(50-58) multimeric constructs, we were able to document a spontaneously developed memory/effector CD8 T cell response against this peptide in a melanoma patient. To elucidate the Ag-processing pathway, we demonstrate that RAB38/NY-MEL-1(50-58) is produced efficiently by the standard proteasome and the immunoproteasome. In addition to the identification of a RAB38/NY-MEL-1-derived immunogenic CD8 T cell epitope, this study is instrumental for both the onset and monitoring of future RAB38/NY-MEL-1-based vaccination trials.

  10. Rabs on the fly

    DEFF Research Database (Denmark)

    Caviglia, Sara; Flores-Benitez, David; Lattner, Johanna

    2017-01-01

    The organisation of intracellular transport processes is adapted specifically to different cell types, developmental stages, and physiological requirements. Some protein traffic routes are universal to all cells and constitutively active, while other routes are cell-type specific, transient...

  11. An overexpression screen of Toxoplasma gondii Rab-GTPases reveals distinct transport routes to the micronemes.

    Directory of Open Access Journals (Sweden)

    Katrin Kremer

    2013-03-01

    Full Text Available The basic organisation of the endomembrane system is conserved in all eukaryotes and comparative genome analyses provides compelling evidence that the endomembrane system of the last common eukaryotic ancestor (LCEA is complex with many genes required for regulated traffic being present. Although apicomplexan parasites, causative agents of severe human and animal diseases, appear to have only a basic set of trafficking factors such as Rab-GTPases, they evolved unique secretory organelles (micronemes, rhoptries and dense granules that are sequentially secreted during invasion of the host cell. In order to define the secretory pathway of apicomplexans, we performed an overexpression screen of Rabs in Toxoplasma gondii and identified Rab5A and Rab5C as important regulators of traffic to micronemes and rhoptries. Intriguingly, we found that not all microneme proteins traffic depends on functional Rab5A and Rab5C, indicating the existence of redundant microneme targeting pathways. Using two-colour super-resolution stimulated emission depletion (STED we verified distinct localisations of independent microneme proteins and demonstrate that micronemal organelles are organised in distinct subsets or subcompartments. Our results suggest that apicomplexan parasites modify classical regulators of the endocytic system to carryout essential parasite-specific roles in the biogenesis of their unique secretory organelles.

  12. Activation-Inactivation Cycling of Rab35 and ARF6 Is Required for Phagocytosis of Zymosan in RAW264 Macrophages

    Directory of Open Access Journals (Sweden)

    Youhei Egami

    2015-01-01

    Full Text Available Phagocytosis of zymosan by phagocytes is a widely used model of microbial recognition by the innate immune system. Live-cell imaging showed that fluorescent protein-fused Rab35 accumulated in the membranes of phagocytic cups and then dissociated from the membranes of newly formed phagosomes. By our novel pull-down assay for Rab35 activity, we found that Rab35 is deactivated immediately after zymosan internalization into the cells. Phagosome formation was inhibited in cells expressing the GDP- or GTP-locked Rab35 mutant. Moreover, the simultaneous expression of ACAP2—a Rab35 effector protein—with GTP-locked Rab35 or the expression of plasma membrane-targeted ACAP2 showed a marked inhibitory effect on phagocytosis through ARF6 inactivation by the GAP activity of ACAP2. ARF6, a substrate for ACAP2, was also localized on the phagocytic cups and dissociated from the membranes of internalized phagosomes. In support of the microscopic observations, ARF6-GTP pull-down experiments showed that ARF6 is transiently activated during phagosome formation. Furthermore, the expression of GDP- or GTP-locked ARF6 mutants also suppresses the uptake of zymosan. These data suggest that the activation-inactivation cycles of Rab35 and ARF6 are required for the uptake of zymosan and that ACAP2 is an important component that links Rab35/ARF6 signaling during phagocytosis of zymosan.

  13. Rab21, a Novel PS1 Interactor, Regulates γ-Secretase Activity via PS1 Subcellular Distribution.

    Science.gov (United States)

    Sun, Zhenzhen; Xie, Yujie; Chen, Yintong; Yang, Qinghu; Quan, Zhenzhen; Dai, Rongji; Qing, Hong

    2018-05-01

    γ-Secretase has been a therapeutical target for its key role in cleaving APP to generate β-amyloid (Aβ), the primary constituents of senile plaques and a hallmark of Alzheimer's disease (AD) pathology. Recently, γ-secretase-associating proteins showed promising role in specifically modulating APP processing while sparing Notch signaling; however, the underlying mechanism is still unclear. A co-immunoprecipitation (Co-IP) coupled with mass spectrometry proteomic assay for Presenilin1 (PS1, the catalytic subunit of γ-secretase) was firstly conducted to find more γ-secretase-associating proteins. Gene ontology analysis of these results identified Rab21 as a potential PS1 interacting protein, and the interaction between them was validated by reciprocal Co-IP and immunofluorescence assay. Then, molecular and biochemical methods were used to investigate the effect of Rab21 on APP processing. Results showed that overexpression of Rab21 enhanced Aβ generation, while silencing of Rab21 reduced the accumulation of Aβ, which resulted due to change in γ-secretase activity rather than α- or β-secretase. Finally, we demonstrated that Rab21 had no effect on γ-secretase complex synthesis or metabolism but enhanced PS1 endocytosis and translocation to late endosome/lysosome. In conclusion, we identified a novel γ-secretase-associating protein Rab21 and illustrate that Rab21 promotes γ-secretase internalization and translocation to late endosome/lysosome. Moreover, silencing of Rab21 decreases the γ-secretase activity in APP processing thus production of Aβ. All these results open new gateways towards the understanding of γ-secretase-associating proteins in APP processing and make inhibition of Rab21 a promising strategy for AD therapy.

  14. 32 CFR 202.9 - Conducting RAB meetings.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Conducting RAB meetings. 202.9 Section 202.9... members. Group consensus is not a prerequisite for RAB input. Each member of the RAB may provide advice as an individual; however, when a RAB decides to vote or poll for consensus, only community members...

  15. Wnt6 maintains anterior escort cells as an integral component of the germline stem cell niche.

    Science.gov (United States)

    Wang, Xiaoxi; Page-McCaw, Andrea

    2018-02-07

    Stem cells reside in a niche, a local environment whose cellular and molecular complexity is still being elucidated. In Drosophila ovaries, germline stem cells depend on cap cells for self-renewing signals and physical attachment. Germline stem cells also contact the anterior escort cells, and here we report that anterior escort cells are absolutely required for germline stem cell maintenance. When escort cells die from impaired Wnt signaling or hid expression, the loss of anterior escort cells causes loss of germline stem cells. Anterior escort cells function as an integral niche component by promoting DE-cadherin anchorage and by transiently expressing the Dpp ligand to promote full-strength BMP signaling in germline stem cells. Anterior escort cells are maintained by Wnt6 ligands produced by cap cells; without Wnt6 signaling, anterior escort cells die leaving vacancies in the niche, leading to loss of germline stem cells. Our data identify anterior escort cells as constituents of the germline stem cell niche, maintained by a cap cell-produced Wnt6 survival signal. © 2018. Published by The Company of Biologists Ltd.

  16. The glyceraldehyde-3-phosphate dehydrogenase and the small GTPase Rab 2 are crucial for Brucella replication.

    Directory of Open Access Journals (Sweden)

    Emilie Fugier

    2009-06-01

    Full Text Available The intracellular pathogen Brucella abortus survives and replicates inside host cells within an endoplasmic reticulum (ER-derived replicative organelle named the "Brucella-containing vacuole" (BCV. Here, we developed a subcellular fractionation method to isolate BCVs and characterize for the first time the protein composition of its replicative niche. After identification of BCV membrane proteins by 2 dimensional (2D gel electrophoresis and mass spectrometry, we focused on two eukaryotic proteins: the glyceraldehyde-3-phosphate dehydrogenase (GAPDH and the small GTPase Rab 2 recruited to the vacuolar membrane of Brucella. These proteins were previously described to localize on vesicular and tubular clusters (VTC and to regulate the VTC membrane traffic between the endoplasmic reticulum (ER and the Golgi. Inhibition of either GAPDH or Rab 2 expression by small interfering RNA strongly inhibited B. abortus replication. Consistent with this result, inhibition of other partners of GAPDH and Rab 2, such as COPI and PKC iota, reduced B. abortus replication. Furthermore, blockage of Rab 2 GTPase in a GDP-locked form also inhibited B. abortus replication. Bacteria did not fuse with the ER and instead remained in lysosomal-associated membrane vacuoles. These results reveal an essential role for GAPDH and the small GTPase Rab 2 in B. abortus virulence within host cells.

  17. The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

    Science.gov (United States)

    Harris, Janelle L; Dave, Keyur; Gorman, Jeffrey; Khanna, Kum Kum

    2018-06-01

    5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics

  18. Expression of Rab25 in non-small cell lung cancer and its clinical significance

    Directory of Open Access Journals (Sweden)

    Pu ZHOU

    2014-03-01

    Full Text Available Objective To assess the expression of Rab25 protein in non-small cell lung cancer (NSCLC, and explore the correlation of its expression with tumor proliferation and metastasis. Methods Sixty-one cases of NSCLC specimens (31 cases of squamous cell carcinoma, 26 cases of adenocarcinoma, and 4 cases of adenosquamous carcinoma undergone surgical treatment, and 40 specimens of adjacent normal lung tissues were obtained from Jan. 2009 to Jun. 2010 at Xingqiao Hospital of Third Military Medical University. Immunochemistry method of MaxVision was used to detect the expression of Rab25 in the specimens, and then the correlation of the expression with the clinicopathological parameters (patients' sex, age, smoking history, tumor type, differentiation, volume, TNM stage, lymph metastasis, etc. was analyzed using statistical software SPSS 21.0. Results  Rab25 protein was mainly expressed in cytoplasm and cell membrane. The positive rate of Rab25 in NSCLC was 93.4%, which was significantly higher than that in adjacent normal tissues (27.5%, P<0.01. The expression of Rab25 protein was significantly associated with the TNM stage and tumor size (P<0.05. Conclusions The expression of Rab25 is obviously higher in NSCLC than in the adjacent normal tissues, and the expression is associated with TNM stage and tumor size. Moreover, the later of the NSCLC stage, the larger of tumor size, and the higher of Rab25 expression will be in the NSCLC tissue. DOI: 10.11855/j.issn.0577-7402.2014.02.16

  19. Rab3a is critical for trapping alpha-MSH granules in the high Ca²⁺-affinity pool by preventing constitutive exocytosis.

    Directory of Open Access Journals (Sweden)

    Simon Sedej

    Full Text Available Rab3a is a small GTPase of the Rab3 subfamily that acts during late stages of Ca²⁺-regulated exocytosis. Previous functional analysis in pituitary melanotrophs described Rab3a as a positive regulator of Ca²⁺-dependent exocytosis. However, the precise role of the Rab3a isoform on the kinetics and intracellular [Ca²⁺] sensitivity of regulated exocytosis, which may affect the availability of two major peptide hormones, α-melanocyte stimulating hormone (α-MSH and β-endorphin in plasma, remain elusive. We employed Rab3a knock-out mice (Rab3a KO to explore the secretory phenotype in melanotrophs from fresh pituitary tissue slices. High resolution capacitance measurements showed that Rab3a KO melanotrophs possessed impaired Ca²⁺-triggered secretory activity as compared to wild-type cells. The hampered secretion was associated with the absence of cAMP-guanine exchange factor II/ Epac2-dependent secretory component. This component has been attributed to high Ca²⁺-sensitive release-ready vesicles as determined by slow photo-release of caged Ca²⁺. Radioimmunoassay revealed that α-MSH, but not β-endorphin, was elevated in the plasma of Rab3a KO mice, indicating increased constitutive exocytosis of α-MSH. Increased constitutive secretion of α-MSH from incubated tissue slices was associated with reduced α-MSH cellular content in Rab3a-deficient pituitary cells. Viral re-expression of the Rab3a protein in vitro rescued the secretory phenotype of melanotrophs from Rab3a KO mice. In conclusion, we suggest that Rab3a deficiency promotes constitutive secretion and underlies selective impairment of Ca²⁺-dependent release of α-MSH.

  20. Folliculin directs the formation of a Rab34-RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.

    Science.gov (United States)

    Starling, Georgina P; Yip, Yan Y; Sanger, Anneri; Morton, Penny E; Eden, Emily R; Dodding, Mark P

    2016-06-01

    The spatial distribution of lysosomes is important for their function and is, in part, controlled by cellular nutrient status. Here, we show that the lysosome associated Birt-Hoge-Dubé (BHD) syndrome renal tumour suppressor folliculin (FLCN) regulates this process. FLCN promotes the peri-nuclear clustering of lysosomes following serum and amino acid withdrawal and is supported by the predominantly Golgi-associated small GTPase Rab34. Rab34-positive peri-nuclear membranes contact lysosomes and cause a reduction in lysosome motility and knockdown of FLCN inhibits Rab34-induced peri-nuclear lysosome clustering. FLCN interacts directly via its C-terminal DENN domain with the Rab34 effector RILP Using purified recombinant proteins, we show that the FLCN-DENN domain does not act as a GEF for Rab34, but rather, loads active Rab34 onto RILP We propose a model whereby starvation-induced FLCN association with lysosomes drives the formation of contact sites between lysosomes and Rab34-positive peri-nuclear membranes that restrict lysosome motility and thus promote their retention in this region of the cell. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  1. Crystallization and preliminary X-ray crystallographic studies of Rab6A′(Q72L): a GTP-locked form

    International Nuclear Information System (INIS)

    Shin, Young-Cheul; Jang, Tae-Ho; Yoon, Jong Hwan; Jeon, Ju-Hong; Park, Hyun Ho

    2012-01-01

    Rab6A′(Q72L), a constitutively active GTP-binding form of Rab6A, was purified and crystallized. The crystals were found to belong to space group P22 1 2 1 , with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The crystals were obtained at 293 K and diffracted to a resolution of 1.9 Å. Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A′. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A′ and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A′(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A′(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22 1 2 1 with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules

  2. Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway

    DEFF Research Database (Denmark)

    Madsen, Kenneth Lindegaard; Thorsen, Thor Seneca; Rahbek-Clemmensen, Troels

    2012-01-01

    The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing...... functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which...

  3. Agent-Based Simulation of Children’s School Travel Mode with Parental Escort Decisions

    Directory of Open Access Journals (Sweden)

    Peng Jing

    2018-02-01

    Full Text Available In the last few years, the number of private cars has expanding quickly in China, more and more parents use cars to escort their children to school, thus cause serious traffic congestions near school in many cities. In this paper, we developed an agent-based model (ABM of the parents’ choice of escort mode. The core of this model is an escort mode choice motivation adjustment function that combines distance, traffic safety and social influence. We also used ABM to exhibit the emergent decoy effect phenomenon, which is a dynamic phenomenon that the introduction of a decoy to the choice-set could increase the share of other alternatives. The model reveals the parents’ inner psychological mechanism when facing competing escort mode choice in transportation system. The simulation results show that the proportion of parents to choose bus escort was 62.45% without the decoy effect was introduced, while the proportion of parents to choose bus escort increased to 74.29% with the decoy effect was entry. The use of the ABM method gives the potential to cope with the dynamic changes in studying parent escort mode choice behavior.

  4. Rab GTPases in Immunity and Inflammation

    Directory of Open Access Journals (Sweden)

    Akriti Prashar

    2017-09-01

    Full Text Available Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

  5. Rab GTPases in Immunity and Inflammation.

    Science.gov (United States)

    Prashar, Akriti; Schnettger, Laura; Bernard, Elliott M; Gutierrez, Maximiliano G

    2017-01-01

    Strict spatiotemporal control of trafficking events between organelles is critical for maintaining homeostasis and directing cellular responses. This regulation is particularly important in immune cells for mounting specialized immune defenses. By controlling the formation, transport and fusion of intracellular organelles, Rab GTPases serve as master regulators of membrane trafficking. In this review, we discuss the cellular and molecular mechanisms by which Rab GTPases regulate immunity and inflammation.

  6. α-MSH stimulates glucose uptake in mouse muscle and phosphorylates Rab-GTPase-activating protein TBC1D1 independently of AMPK

    DEFF Research Database (Denmark)

    Møller, Cathrine Laustrup; Kjøbsted, Rasmus; Enriori, Pablo J

    2016-01-01

    The melanocortin system includes five G-protein coupled receptors (family A) defined as MC1R-MC5R, which are stimulated by endogenous agonists derived from proopiomelanocortin (POMC). The melanocortin system has been intensely studied for its central actions in body weight and energy expenditure...... pathway involved in α-MSH-stimulated glucose uptake in differentiated L6 myotubes and mouse muscle explants. In order to examine the involvement of AMPK, we investigate -MSH stimulation in both wild type and AMPK deficient mice. We found that -MSH significantly induces phosphorylation of TBC1 domain (TBC1...

  7. Strain-Specific Altered Regulatory Response of Rab7a and Tau in Creutzfeldt-Jakob Disease and Alzheimer's Disease.

    Science.gov (United States)

    Zafar, Saima; Younas, Neelam; Correia, Susana; Shafiq, Mohsin; Tahir, Waqas; Schmitz, Matthias; Ferrer, Isidre; Andréoletti, Olivier; Zerr, Inga

    2017-01-01

    There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the disease, we checked the regulatory interactive response of Rab7a, Rab9, and known biomarkers PrP C and tau forms in frontal cortex at pre-symptomatic stage of the disease in tg340 mice expressing about fourfold of human PrP-M129 with PrP-null background that had been inoculated with human sCJD MM1 brain tissue homogenates (sCJD MM1 mice). In addition, we analyzed 5XFAD mice, exhibiting five mutations in the APP and presenilin genes related to familial Alzheimer's disease (FAD), to validate specific regulatory response of Rab7a, Rab9, tau, and phosphorylated form of tau by immunostaining 5XFAD mice in comparison with the wild-type age-matched mice brain. The cortical region of 5XFAD mice brain showed accumulated form of Rab7a in puncta that co-label for p-Tau, indicating colocalization by using confocal laser-scanning microscopy and was confirmed by using reverse co-immunoprecipitation. Furthermore, synthetic RNA (siRNA) against the Rab7a gene decreased expression of Rab7a protein, in cortical

  8. Anterograde trafficking of KCa3.1 in polarized epithelia is Rab1- and Rab8-dependent and recycling endosome-independent.

    Directory of Open Access Journals (Sweden)

    Claudia A Bertuccio

    Full Text Available The intermediate conductance, Ca2+-activated K+ channel (KCa3.1 targets to the basolateral (BL membrane in polarized epithelia where it plays a key role in transepithelial ion transport. However, there are no studies defining the anterograde and retrograde trafficking of KCa3.1 in polarized epithelia. Herein, we utilize Biotin Ligase Acceptor Peptide (BLAP-tagged KCa3.1 to address these trafficking steps in polarized epithelia, using MDCK, Caco-2 and FRT cells. We demonstrate that KCa3.1 is exclusively targeted to the BL membrane in these cells when grown on filter supports. Following endocytosis, KCa3.1 degradation is prevented by inhibition of lysosomal/proteosomal pathways. Further, the ubiquitylation of KCa3.1 is increased following endocytosis from the BL membrane and PR-619, a deubiquitylase inhibitor, prevents degradation, indicating KCa3.1 is targeted for degradation by ubiquitylation. We demonstrate that KCa3.1 is targeted to the BL membrane in polarized LLC-PK1 cells which lack the μ1B subunit of the AP-1 complex, indicating BL targeting of KCa3.1 is independent of μ1B. As Rabs 1, 2, 6 and 8 play roles in ER/Golgi exit and trafficking of proteins to the BL membrane, we evaluated the role of these Rabs in the trafficking of KCa3.1. In the presence of dominant negative Rab1 or Rab8, KCa3.1 cell surface expression was significantly reduced, whereas Rabs 2 and 6 had no effect. We also co-immunoprecipitated KCa3.1 with both Rab1 and Rab8. These results suggest these Rabs are necessary for the anterograde trafficking of KCa3.1. Finally, we determined whether KCa3.1 traffics directly to the BL membrane or through recycling endosomes in MDCK cells. For these studies, we used either recycling endosome ablation or dominant negative RME-1 constructs and determined that KCa3.1 is trafficked directly to the BL membrane rather than via recycling endosomes. These results are the first to describe the anterograde and retrograde trafficking of KCa3

  9. 78 FR 261 - Moving Security Zone Around Escorted Vessels on the Lower Mississippi River

    Science.gov (United States)

    2013-01-03

    ... provides for enhanced protection of escorted vessels while causing little if any disruption to other routine navigation since most vessels will be allowed to transit within the outer 250 yards of the...

  10. RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

    International Nuclear Information System (INIS)

    Bello, Oscar D.; Zanetti, M. Natalia; Mayorga, Luis S.; Michaut, Marcela A.

    2012-01-01

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: ► RIM and Munc13 are present in human sperm and localize to the acrosomal region. ► RIM and Munc13 are necessary for acrosomal exocytosis. ► RIM and Munc13 participate before the acrosomal calcium efflux. ► RIM, Munc13 and Rab3A interplay in human sperm acrosomal exocytosis. ► RIM and Rab3A

  11. RIM, Munc13, and Rab3A interplay in acrosomal exocytosis

    Energy Technology Data Exchange (ETDEWEB)

    Bello, Oscar D.; Zanetti, M. Natalia [Laboratorio de Biologia Celular y Molecular, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Mayorga, Luis S. [Laboratorio de Biologia Celular y Molecular, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Michaut, Marcela A., E-mail: mmichaut@fcm.uncu.edu.ar [Laboratorio de Biologia Reproductiva, Instituto de Histologia y Embriologia, IHEM (CONICET-UNCuyo), Facultad de Ciencias Medicas (Argentina); Instituto de Ciencias Basicas, Universidad Nacional de Cuyo, Mendoza (5500) (Argentina)

    2012-03-10

    Exocytosis is a highly regulated, multistage process consisting of multiple functionally definable stages, including recruitment, targeting, tethering, priming, and docking of secretory vesicles with the plasma membrane, followed by calcium-triggered membrane fusion. The acrosome reaction of spermatozoa is a complex, calcium-dependent regulated exocytosis. Fusion at multiple sites between the outer acrosomal membrane and the cell membrane causes the release of the acrosomal contents and the loss of the membranes surrounding the acrosome. Not much is known about the molecules that mediate membrane docking in this particular fusion model. In neurons, the formation of the ternary RIM/Munc13/Rab3A complex has been suggested as a critical component of synaptic vesicles docking. Previously, we demonstrated that Rab3A localizes to the acrosomal region in human sperm, stimulates acrosomal exocytosis, and participates in an early stage during membrane fusion. Here, we report that RIM and Munc13 are also present in human sperm and localize to the acrosomal region. Like Rab3A, RIM and Munc13 participate in a prefusion step before the efflux of intra-acrosomal calcium. By means of a functional assay using antibodies and recombinant proteins, we show that RIM, Munc13 and Rab3A interplay during acrosomal exocytosis. Finally, we report by electron transmission microscopy that sequestering RIM and Rab3A alters the docking of the acrosomal membrane to the plasma membrane during calcium-activated acrosomal exocytosis. Our results suggest that the RIM/Munc13/Rab3 A complex participates in acrosomal exocytosis and that RIM and Rab3A have central roles in membrane docking. -- Highlights: Black-Right-Pointing-Pointer RIM and Munc13 are present in human sperm and localize to the acrosomal region. Black-Right-Pointing-Pointer RIM and Munc13 are necessary for acrosomal exocytosis. Black-Right-Pointing-Pointer RIM and Munc13 participate before the acrosomal calcium efflux. Black

  12. Patterns of client behavior with their most recent male escort: an application of latent class analysis.

    Science.gov (United States)

    Grov, Christian; Starks, Tyrel J; Wolff, Margaret; Smith, Michael D; Koken, Juline A; Parsons, Jeffrey T

    2015-05-01

    Research examining interactions between male escorts and clients has relied heavily on data from escorts, men working on the street, and behavioral data aggregated over time. In the current study, 495 clients of male escorts answered questions about sexual behavior with their last hire. Latent class analysis identified four client sets based on these variables. The largest (n = 200, 40.4 %, labeled Typical Escort Encounter) included men endorsing behavior prior research found typical of paid encounters (e.g., oral sex and kissing). The second largest class (n = 157, 31.7 %, Typical Escort Encounter + Erotic Touching) included men reporting similar behaviors, but with greater variety along a spectrum of touching (e.g., mutual masturbation and body worship). Those classed BD/SM and Kink (n = 76, 15.4 %) reported activity along the kink spectrum (BD/SM and role play). Finally, men classed Erotic Massage Encounters (n = 58, 11.7 %) primarily engaged in erotic touch. Clients reporting condomless anal sex were in the minority (12.2 % overall). Escorts who engage in anal sex with clients might be appropriate to train in HIV prevention and other harm reduction practices-adopting the perspective of "sex workers as sex educators."

  13. Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues

    Directory of Open Access Journals (Sweden)

    E Wyroba

    2009-08-01

    Full Text Available Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein and LAMP-2 (lysosomal membrane protein 2 as well as a7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2 in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100 000 x g of equal load were quantified by immunoblotting. LAMP-2 crossreacting polypeptide of ~106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The a7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILPrelated polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.

  14. Intracellular Transport of Vaccinia Virus in HeLa Cells Requires WASH-VPEF/FAM21-Retromer Complexes and Recycling Molecules Rab11 and Rab22

    Science.gov (United States)

    Hsiao, Jye-Chian; Chu, Li-Wei; Lo, Yung-Tsun; Lee, Sue-Ping; Chen, Tzu-Jung; Huang, Cheng-Yen

    2015-01-01

    ABSTRACT Vaccinia virus, the prototype of the Orthopoxvirus genus in the family Poxviridae, infects a wide range of cell lines and animals. Vaccinia mature virus particles of the WR strain reportedly enter HeLa cells through fluid-phase endocytosis. However, the intracellular trafficking process of the vaccinia mature virus between cellular uptake and membrane fusion remains unknown. We used live imaging of single virus particles with a combination of various cellular vesicle markers, to track fluorescent vaccinia mature virus particle movement in cells. Furthermore, we performed functional interference assays to perturb distinct vesicle trafficking processes in order to delineate the specific route undertaken by vaccinia mature virus prior to membrane fusion and virus core uncoating in cells. Our results showed that vaccinia virus traffics to early endosomes, where recycling endosome markers Rab11 and Rab22 are recruited to participate in subsequent virus trafficking prior to virus core uncoating in the cytoplasm. Furthermore, we identified WASH-VPEF/FAM21-retromer complexes that mediate endosome fission and sorting of virus-containing vesicles prior to virus core uncoating in the cytoplasm. IMPORTANCE Vaccinia mature virions of the WR strain enter HeLa cells through fluid phase endocytosis. We previously demonstrated that virus-containing vesicles are internalized into phosphatidylinositol 3-phosphate positive macropinosomes, which are then fused with Rab5-positive early endosomes. However, the subsequent process of sorting the virion-containing vesicles prior to membrane fusion remains unclear. We dissected the intracellular trafficking pathway of vaccinia mature virions in cells up to virus core uncoating in cytoplasm. We show that vaccinia mature virions first travel to early endosomes. Subsequent trafficking events require the important endosome-tethered protein VPEF/FAM21, which recruits WASH and retromer protein complexes to the endosome. There, the complex

  15. Sheep internal parasites on Rab and Pag

    Directory of Open Access Journals (Sweden)

    Relja Beck

    2010-01-01

    Full Text Available The purpose of our research was to determine which groups and species of internal parasites endanger the health of sheep on the islands of Rab and Pag. The research was carried out in 10 flocks on both islands taking the fresh dung out of 30% of the total number of sheep in each flock. It was ascertained that the gastrointestinal parasites and protozoa of Eimeria genus are present in most flocks on both islands. The presence of the fluke Dicrocoelium dendriticum was ascertained in considerably larger number of flocks on the island of Rab than on the island of Pag. On the other hand, the presence of parasites of Moniezia and Nematodirus genus was ascertained in larger number of flocks on the island of Pag. In two flocks on Rab parasites of Protostrongylus genus were ascertained while on the island of Pag they were not found in any flock.

  16. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Kagiwada, Satoshi; Shimazu, Sayuri; Takegawa, Kaoru; Noguchi, Tetsuko; Miyamoto, Masaaki

    2015-01-01

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed

  17. Coordinated regulation by two VPS9 domain-containing guanine nucleotide exchange factors in small GTPase Rab5 signaling pathways in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Kagiwada, Satoshi [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Shimazu, Sayuri [Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Takegawa, Kaoru [Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581 (Japan); Noguchi, Tetsuko [Department of Biological Sciences, Faculty of Science, Nara Women' s University, Kitauoyanishi-machi, Nara 630-8506 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho, Nada, Kobe 657-8501 (Japan)

    2015-03-20

    The small GTPase Rab5 is reported to regulate various cellular functions, such as vesicular transport and endocytosis. VPS9 domain-containing proteins are thought to activate Rab5(s) by their guanine-nucleotide exchange activities. Numerous VPS9 proteins have been identified and are structurally conserved from yeast to mammalian cells. However, the functional relationships among VPS9 proteins in cells remain unclear. Only one Rab5 and two VPS9 proteins were identified in the Schizosaccharomyces pombe genome. Here, we examined the cellular function of two VPS9 proteins and the relationship between these proteins in cellular functions. Vps901-GFP and Vps902-GFP exhibited dotted signals in vegetative and differentiated cells. vps901 deletion mutant (Δvps901) cells exhibited a phenotype deficient in the mating process and responses to high concentrations of ions, such as calcium and metals, and Δvps901Δvps902 double mutant cells exhibited round cell shapes similar to ypt5-909 (Rab5 mutant allele) cells. Deletion of both vps901 and vps902 genes completely abolished the mating process and responses to various stresses. A lack of vacuole formation and aberrant inner cell membrane structures were also observed in Δvps901Δvps902 cells by electron microscopy. These data strongly suggest that Vps901 and Vps902 are cooperatively involved in the regulation of cellular functions, such as cell morphology, sexual development, response to ion stresses, and vacuole formation, via Rab5 signaling pathways in fission yeast cells. - Highlights: • Roles of Rab5 activator VPS9 proteins in cellular functions. • Cooperation between VPS9 proteins in Rab5 signaling pathway. • Roles of each VPS9 protein in Rab5 signaling pathway are discussed.

  18. Regulatory elements in vivo in the promoter of the abscisic acid responsive gene rab17 from maize.

    Science.gov (United States)

    Busk, P K; Jensen, A B; Pagès, M

    1997-06-01

    The rab17 gene from maize is transcribed in late embryonic development and is responsive to abscisic acid and water stress in embryo and vegetative tissues. In vivo footprinting and transient transformation of rab17 were performed in embryos and vegetative tissues to characterize the cis-elements involved in regulation of the gene. By in vivo footprinting, protein binding was observed to nine elements in the promoter, which correspond to five putative ABREs (abscisic acid responsive elements) and four other sequences. The footprints indicated that distinct proteins interact with these elements in the two developmental stages. In transient transformation, six of the elements were important for high level expression of the rab17 promoter in embryos, whereas only three elements were important in leaves. The cis-acting sequences can be divided in embryo-specific, ABA-specific and leaf-specific elements on the basis of protein binding and the ability to confer expression of rab17. We found one positive, new element, called GRA, with the sequence CACTGGCCGCCC. This element was important for transcription in leaves but not in embryos. Two other non-ABRE elements that stimulated transcription from the rab17 promoter resemble previously described abscisic acid and drought-inducible elements. There were differences in protein binding and function of the five ABREs in the rab17 promoter. The possible reasons for these differences are discussed. The in vivo data obtained suggest that an embryo-specific pathway regulates transcription of the rab genes during development, whereas another pathway is responsible for induction in response to ABA and drought in vegetative tissues.

  19. Structural basis for Rab1 de-AMPylation by the Legionella pneumophila effector SidD.

    Directory of Open Access Journals (Sweden)

    Yang Chen

    Full Text Available The covalent attachment of adenosine monophosphate (AMP to proteins, a process called AMPylation (adenylylation, has recently emerged as a novel theme in microbial pathogenesis. Although several AMPylating enzymes have been characterized, the only known virulence protein with de-AMPylation activity is SidD from the human pathogen Legionella pneumophila. SidD de-AMPylates mammalian Rab1, a small GTPase involved in secretory vesicle transport, thereby targeting the host protein for inactivation. The molecular mechanisms underlying Rab1 recognition and de-AMPylation by SidD are unclear. Here, we report the crystal structure of the catalytic region of SidD at 1.6 Å resolution. The structure reveals a phosphatase-like fold with additional structural elements not present in generic PP2C-type phosphatases. The catalytic pocket contains a binuclear metal-binding site characteristic of hydrolytic metalloenzymes, with strong dependency on magnesium ions. Subsequent docking and molecular dynamics simulations between SidD and Rab1 revealed the interface contacts and the energetic contribution of key residues to the interaction. In conjunction with an extensive structure-based mutational analysis, we provide in vivo and in vitro evidence for a remarkable adaptation of SidD to its host cell target Rab1 which explains how this effector confers specificity to the reaction it catalyses.

  20. Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae.

    Science.gov (United States)

    Chen, Ming-Chyuan; Cheng, Ying-Min; Hong, Min-Chang; Fang, Lee-Shing

    2004-11-19

    The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts.

  1. Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae

    International Nuclear Information System (INIS)

    Chen, M.-C.; Cheng, Y.-M; Hong, M.-C.; Fang, L.-S.

    2004-01-01

    The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts

  2. Rab9-dependent retrograde transport and endosomal sorting of the endopeptidase furin

    Science.gov (United States)

    Chia, Pei Zhi Cheryl; Gasnereau, Isabelle; Lieu, Zi Zhao; Gleeson, Paul A.

    2011-01-01

    The endopeptidase furin and the trans-Golgi network protein TGN38 are membrane proteins that recycle between the TGN and plasma membrane. TGN38 is transported by a retromer-dependent pathway from early endosomes to the TGN, whereas the intracellular transport of furin is poorly defined. Here we have identified the itinerary and transport requirements of furin. Using internalisation assays, we show that furin transits the early and late endosomes en route to the TGN. The GTPase Rab9 and the TGN golgin GCC185, components of the late endosome-to-TGN pathway, were required for efficient TGN retrieval of furin. By contrast, TGN38 trafficking was independent of Rab9 and GCC185. To identify the sorting signals for the early endosome-to-TGN pathway, the trafficking of furin–TGN38 chimeras was investigated. The diversion of furin from the Rab9-dependent late-endosome-to-TGN pathway to the retromer-dependent early-endosome-to-TGN pathway required both the transmembrane domain and cytoplasmic tail of TGN38. We present evidence to suggest that the length of the transmembrane domain is a contributing factor in endosomal sorting. Overall, these data show that furin uses the Rab9-dependent pathway from late endosomes and that retrograde transport directly from early endosomes is dependent on both the transmembrane domain and the cytoplasmic tail. PMID:21693586

  3. Genetics Home Reference: choroideremia

    Science.gov (United States)

    ... out its protein escort function. This lack of functional REP-1 prevents Rab proteins from reaching and ... the cells of the retina causes the progressive vision loss characteristic of choroideremia . Learn more about the ...

  4. Charcot Marie Tooth 2B Peripheral Sensory Neuropathy: How Rab7 Mutations Impact NGF Signaling?

    Directory of Open Access Journals (Sweden)

    Harry Liu

    2017-02-01

    Full Text Available Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A “gain of toxicity” model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s causes a “loss of function”, resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons of CMT2B are needed to precisely define the disease mechanisms.

  5. Charcot Marie Tooth 2B Peripheral Sensory Neuropathy: How Rab7 Mutations Impact NGF Signaling?

    Science.gov (United States)

    Liu, Harry; Wu, Chengbiao

    2017-02-04

    Charcot-Marie-Tooth 2B peripheral sensory neuropathy (CMT2B) is a debilitating autosomal dominant hereditary sensory neuropathy. Patients with this disease lose pain sensation and frequently need amputation. Axonal dysfunction and degeneration of peripheral sensory neurons is a major clinical manifestation of CMT2B. However, the cellular and molecular pathogenic mechanisms remain undefined. CMT2B is caused by missense point mutations (L129F, K157N, N161T/I, V162M) in Rab7 GTPase. Strong evidence suggests that the Rab7 mutation(s) enhances the cellular levels of activated Rab7 proteins, thus resulting in increased lysosomal activity and autophagy. As a consequence, trafficking and signaling of neurotrophic factors such as nerve growth factor (NGF) in the long axons of peripheral sensory neurons are particularly vulnerable to premature degradation. A "gain of toxicity" model has, thus, been proposed based on these observations. However, studies of fly photo-sensory neurons indicate that the Rab7 mutation(s) causes a "loss of function", resulting in haploinsufficiency. In the review, we summarize experimental evidence for both hypotheses. We argue that better models (rodent animals and human neurons) of CMT2B are needed to precisely define the disease mechanisms.

  6. CED-10/Rac1 regulates endocytic recycling through the RAB-5 GAP TBC-2.

    Directory of Open Access Journals (Sweden)

    Lin Sun

    Full Text Available Rac1 is a founding member of the Rho-GTPase family and a key regulator of membrane remodeling. In the context of apoptotic cell corpse engulfment, CED-10/Rac1 acts with its bipartite guanine nucleotide exchange factor, CED-5/Dock180-CED-12/ELMO, in an evolutionarily conserved pathway to promote phagocytosis. Here we show that in the context of the Caenorhabditis elegans intestinal epithelium CED-10/Rac1, CED-5/Dock180, and CED-12/ELMO promote basolateral recycling. Furthermore, we show that CED-10 binds to the RAB-5 GTPase activating protein TBC-2, that CED-10 contributes to recruitment of TBC-2 to endosomes, and that recycling cargo is trapped in recycling endosomes in ced-12, ced-10, and tbc-2 mutants. Expression of GTPase defective RAB-5(Q78L also traps recycling cargo. Our results indicate that down-regulation of early endosome regulator RAB-5/Rab5 by a CED-5, CED-12, CED-10, TBC-2 cascade is an important step in the transport of cargo through the basolateral recycling endosome for delivery to the plasma membrane.

  7. RINL, guanine nucleotide exchange factor Rab5-subfamily, is involved in the EphA8-degradation pathway with odin.

    Directory of Open Access Journals (Sweden)

    Hiroaki Kajiho

    Full Text Available The Rab family of small guanosine triphosphatases (GTPases plays a vital role in membrane trafficking. Its active GTP-bound state is driven by guanine nucleotide-exchange factors (GEFs. Ras and Rab interactor (or Ras interaction/interference-like (RINL, which contains a conserved VPS9 domain critical for GEF action, was recently identified as a new Rab5 subfamily GEF in vitro. However, its detailed function and interacting molecules have not yet been fully elucidated. Here we found that RINL has GEF activity for the Rab5 subfamily proteins by measuring their GTP-bound forms in cultured cells. We also found that RINL interacts with odin, a member of the ankyrin-repeat and sterile-alpha motif (SAM domain-containing (Anks protein family. In addition, the Eph tyrosine kinase receptor EphA8 formed a ternary complex with both RINL and odin. Interestingly, RINL expression in cultured cells reduced EphA8 levels in a manner dependent on both its GEF activity and interaction with odin. In addition, knockdown of RINL increased EphA8 level in HeLa cells. Our findings suggest that RINL, as a GEF for Rab5 subfamily, is implicated in the EphA8-degradation pathway via its interaction with odin.

  8. 阴道毛滴虫Rab11鸟苷三磷酸酶cDNA克隆和序列分析%Molecular Cloning and Sequence Analysis of Rab11 GTPase in Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    张仁利; 许铭炎; 许锦阶; 高世同; 黄达娜; 耿艺介; 傅玉才

    2006-01-01

    Objective Rab11 GTPases play an essential role in regulating membrane trafficking pathways in eukaryotic cells. Nonetheless, there has been little work done on characterizing the transport machinery of Trichomonas. The aim of this study is to clone and characterize a Rab11 gene of Trichomonas vaginalis.Methods A cDNA expression library was constructed with T. vaginalis total RNA. A cDNA clone, which showed a high degree of homology with Rab proteins of different species, was isolated and sequenced. Sequence analysis was performed using BLASTP, RPS-BLAST and ClustalW programs. The genomic DNA corresponding to the cDNA sequence was amplified using PCR techniques and following by sequencing. Results cDNA with a length of 710 base pairs and an open reading frame of 636 bp was obtained. The deduced amino acid sequence from the open reading frame was found to possess 211 residuals. Sequence analysis demonstrated that this cDNA clone was homologous to the Rab11 subfamily of different species (60% identity and 79% similarity with Arabidopsis thaliana Rab11c, 58% identity and 78% similarity with human Rab11b), and that the amino acid sequence contains all the well known conserved sequence elements of Rab family. Specific Rab motifs were also detected in the deduced amino acid sequence. Phylogenetic analysis showed that its closest homologues are Rab11 proteins from other species. Sequencing of the PCR product of genomic DNA revealed that the genomic DNA sequence encompassing the putative 5'-ATG and 3'-stop codon is identical to the cDNA sequence.Conclusion A cDNA clone corresponding to the T. vaginalis Rab11 gene was obtained.The function of this gene in regulating membrane trafficking pathways of the parasitic protist is still under investigation.%目的近年研究表明Rab11鸟苷三磷酸酶在调节各种真核细胞的膜转运通道中发挥着重要作用.但是,对于原生动物毛滴虫膜转运系统的研究仍甚少.本研究旨在克隆和分析阴道毛滴虫Rab

  9. [School escorts in inclusive education- the current situation in the state of Baden-Wurttemberg].

    Science.gov (United States)

    Henn, Katharina; Thurn, Leonore; Besier, Tanja; Künster, Anne K; Fegert, Jörg M; Ziegenhain, Ute

    2014-11-01

    In the context of inclusive education, so-called «Schulbegleiter» (school escorts) have been installed to assist children with special needs. However, adequate conceptions of their role are lacking, and no provisions exist describing the exact tasks and duties of these school escorts. This article presents data on a statewide empirical study and provides information on the professional background, qualifications, and assignments of such escorts as well as the nature of the assisted children in the German state of Baden-Württemberg. The data were collected in nearly 50 % of all schools providing general education in Baden-Württemberg, and a total of 526 school escorts completed a questionnaire on their work. Children with special needs who received such assistance were aged 6 to 19 years (M = 10.4) and had a mental disorder in nearly three-fourths of all cases, with autism spectrum disorders being most common (59.4 %). The most frequent assignments of the escorts were providing practical assistance during classes and mobilizing the child as well as providing direct support in emotionally challenging situations. Such escorts can make a significant contribution to the inclusive education of disabled children. They do, however, need an adequate qualification and practical competences to meet the diverse challenges of children with special needs. Child and adolescent psychiatrists are supposed to stay involved in the process, not only by planning interventions, but also during the course of inclusive education itself. Thus, the emotional situation and adjustment of included children with mental disorders over time must be monitored adequately.

  10. Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

    Science.gov (United States)

    Lamers, Ideke J C; Reijnders, Margot R F; Venselaar, Hanka; Kraus, Alison; Jansen, Sandra; de Vries, Bert B A; Houge, Gunnar; Gradek, Gyri Aasland; Seo, Jieun; Choi, Murim; Chae, Jong-Hee; van der Burgt, Ineke; Pfundt, Rolph; Letteboer, Stef J F; van Beersum, Sylvia E C; Dusseljee, Simone; Brunner, Han G; Doherty, Dan; Kleefstra, Tjitske; Roepman, Ronald

    2017-11-02

    The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization. Copyright © 2017 American Society of Human Genetics. All rights reserved.

  11. Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

    2010-09-01

    The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

  12. Social-emotional aspects of male escorting: experiences of men working for an agency.

    Science.gov (United States)

    Smith, Michael D; Grov, Christian; Seal, David W; Bernhardt, Nicholas; McCall, Peter

    2015-05-01

    Social situations and emotional correlates associated with male sex work have not been well documented. Most of the research in this area focuses on sexual activity with little mention of other aspects of the job. Yet, research with female sex workers finds significant social and emotional components to sex work. The current study focused on how male sex workers (MSWs) perceived and adapted to the social-emotional aspects of their job. As part of a larger project examining MSWs working for a single escort agency, 40 men (M age, 22.3 years, 75 % Caucasian) located in the mid-Atlantic U.S. participated in semi-structured interviews. The agency owner was also interviewed. Participants reported a range of social and emotional factors regarding sex work and employed a variety of strategies to provide good customer service and adapt to negative experiences. For most, social support was inhibited due to fear of stigmatization that might result if participants disclosed sex work to significant others outside the agency. Instead, interactions within the agency provided core work-related social support for most MSWs. Emotional and relational tasks inherent to escort work grew easier with experience and negativity about the job declined. Our data suggested that socially connected individuals seemed to be more satisfied with sex work. Social and emotional requirements represented a significant but unanticipated component of male sex work to which escorts actively adapted. Escorting may be similar to other service occupations in terms of the social-emotional situations and skills involved.

  13. 33 CFR 165.114 - Safety and Security Zones: Escorted Vessels-Boston Harbor, Massachusetts.

    Science.gov (United States)

    2010-07-01

    ... 33 Navigation and Navigable Waters 2 2010-07-01 2010-07-01 false Safety and Security Zones... COAST GUARD, DEPARTMENT OF HOMELAND SECURITY (CONTINUED) PORTS AND WATERWAYS SAFETY REGULATED NAVIGATION... Guard District § 165.114 Safety and Security Zones: Escorted Vessels—Boston Harbor, Massachusetts. (a...

  14. A study at Manchester Dental Hospital, of the compliance of IV sedation patients and their escorts.

    Science.gov (United States)

    Lightfoot, Kate

    2014-01-01

    Oral surgery, under intravenous sedation, is carried out on a daily basis at Manchester Dental Hospital. This essay discusses the background of conscious sedation in the dental setting and looks at the compliance of patients (and escorts) when having intravenous sedation. The patient journey, from pre-operative assessment through to treatment is considered. Hospital instructions indicate an obligation for patients to attend with a responsible adult (eighteen or over) who remains with them throughout their treatment, escorts them home,and arranges their care for the next twenty-four hours. By following these instructions the patient should receive optimal care. Two questionnaires were devised and given to patients/escorts to assess their compliance with Manchester Dental Hospital postoperative protocols. 100% compliance was the set standard, with results presented as pie charts. This work began during the undergraduate summer vacation of 2012 and continued over a five-month period. Initial results suggested that compliance was generally good but certain improvements could be made. Suggestions for future development are presented. These aim to give clarity to all patients/escorts and their specific roles in the anticipated treatment.

  15. Rab11-FIP3 Regulation of Lck Endosomal Traffic Controls TCR Signal Transduction.

    Science.gov (United States)

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Vázquez-Chávez, Elena; Lasserre, Rémi; Agüera-González, Sonia; Cuche, Céline; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2017-04-01

    The role of endosomes in receptor signal transduction is a long-standing question, which remains largely unanswered. The T cell Ag receptor and various components of its proximal signaling machinery are associated with distinct endosomal compartments, but how endosomal traffic affects T cell signaling remains ill-defined. In this article, we demonstrate in human T cells that the subcellular localization and function of the protein tyrosine kinase Lck depends on the Rab11 effector FIP3 (Rab11 family interacting protein-3). FIP3 overexpression or silencing and its ability to interact with Rab11 modify Lck subcellular localization and its delivery to the immunological synapse. Importantly, FIP3-dependent Lck localization controls early TCR signaling events, such as tyrosine phosphorylation of TCRζ, ZAP70, and LAT and intracellular calcium concentration, as well as IL-2 gene expression. Interestingly, FIP3 controls both steady-state and poststimulation phosphotyrosine and calcium levels. Finally, our findings indicate that FIP3 modulates TCR-CD3 cell surface expression via the regulation of steady-state Lck-mediated TCRζ phosphorylation, which in turn controls TCRζ protein levels. This may influence long-term T cell activation in response to TCR-CD3 stimulation. Therefore, our data underscore the importance of finely regulated endosomal traffic in TCR signal transduction and T cell activation leading to IL-2 production. Copyright © 2017 by The American Association of Immunologists, Inc.

  16. Site-directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia.

    Science.gov (United States)

    Wyroba, E; Kwaśniak, P; Miller, K; Kobyłecki, K; Osińska, M

    2016-04-11

    Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post-translational modifications (PTM) in proper targeting of Paramecium Rab7b paralogue - distinct from that of Rab7a directly involved in phagocytosis - was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14-]UDP-glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non-mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non- mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC-MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14-]UDP- glucose, the suggested composition of the adduct attached to Thr200 might be (Hex)1(HexNAc)1(Phos)3 or (HexNAc)1 (Deoxyhexose)1 (Phos)1 (HexA)1. These data indicate that PTM of Thr200 located in the hypervariable C-region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.

  17. Site‐directed mutagenesis, in vivo electroporation and mass spectrometry in search for determinants of the subcellular targeting of Rab7b paralogue in the model eukaryote Paramecium octaurelia

    Directory of Open Access Journals (Sweden)

    E. Wyroba

    2016-04-01

    Full Text Available Protein products of the paralogous genes resulting from the whole genome duplication may acquire new function. The role of post‐translational modifications (PTM in proper targeting of Paramecium Rab7b paralogue – distinct from that of Rab7a directly involved in phagocytosis ‐ was studied using point mutagenesis, proteomic analysis and double immunofluorescence after in vivo electroporation of the mutagenized protein. Here we show that substitution of Thr200 by Ala200 resulted in diminished incorporation of [P32] by 37.4% and of 32 [C14–]UDP‐glucose by 24%, respectively, into recombinant Rab7b_200 in comparison to the non‐mutagenized control. Double confocal imaging revealed that Rab7b_200 was mistargeted upon electroporation into living cells contrary to non‐ mutagenized recombinant Rab7b correctly incorporated in the cytostome area. We identified the peptide ion at m/z=677.63+ characteristic for the glycan group attached to Thr200 in Rab7b using nano LC‐MS/MS and comparing the peptide map of this protein with that after deglycosylation with the mixture of five enzymes of different specificity. Based on the mass of this peptide ion and quantitative radioactive assays with [P32]and  [C14‐]UDP‐ glucose, the suggested composition of the adduct attached to Thr200 might be (Hex1(HexNAc1(Phos3 or (HexNAc1 (Deoxyhexose1 (Phos1 (HexA1. These data indicate that PTM of Thr200 located in the hypervariable C‐region of Rab7b in Paramecium is crucial for the proper localization/function of this protein. Moreover, these proteins differ also in other PTM: the number of phosphorylated amino acids in Rab7b is much higher than in Rab7a.   

  18. Venture from the Interior-Herpesvirus pUL31 Escorts Capsids from Nucleoplasmic Replication Compartments to Sites of Primary Envelopment at the Inner Nuclear Membrane.

    Science.gov (United States)

    Bailer, Susanne M.

    2017-11-25

    Herpesviral capsid assembly is initiated in the nucleoplasm of the infected cell. Size constraints require that newly formed viral nucleocapsids leave the nucleus by an evolutionarily conserved vescular transport mechanism called nuclear egress. Mature capsids released from the nucleoplasm are engaged in a membrane-mediated budding process, composed of primary envelopment at the inner nuclear membrane and de-envelopment at the outer nuclear membrane. Once in the cytoplasm, the capsids receive their secondary envelope for maturation into infectious virions. Two viral proteins conserved throughout the herpesvirus family, the integral membrane protein pUL34 and the phosphoprotein pUL31, form the nuclear egress complex required for capsid transport from the infected nucleus to the cytoplasm. Formation of the nuclear egress complex results in budding of membrane vesicles revealing its function as minimal virus-encoded membrane budding and scission machinery. The recent structural analysis unraveled details of the heterodimeric nuclear egress complex and the hexagonal coat it forms at the inside of budding vesicles to drive primary envelopment. With this review, I would like to present the capsid-escort-model where pUL31 associates with capsids in nucleoplasmic replication compartments for escort to sites of primary envelopment thereby coupling capsid maturation and nuclear egress.

  19. A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton

    Directory of Open Access Journals (Sweden)

    Sarah M. Carpanini

    2014-06-01

    Full Text Available Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM. Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic

  20. A novel mouse model of Warburg Micro syndrome reveals roles for RAB18 in eye development and organisation of the neuronal cytoskeleton.

    Science.gov (United States)

    Carpanini, Sarah M; McKie, Lisa; Thomson, Derek; Wright, Ann K; Gordon, Sarah L; Roche, Sarah L; Handley, Mark T; Morrison, Harris; Brownstein, David; Wishart, Thomas M; Cousin, Michael A; Gillingwater, Thomas H; Aligianis, Irene A; Jackson, Ian J

    2014-06-01

    Mutations in RAB18 have been shown to cause the heterogeneous autosomal recessive disorder Warburg Micro syndrome (WARBM). Individuals with WARBM present with a range of clinical symptoms, including ocular and neurological abnormalities. However, the underlying cellular and molecular pathogenesis of the disorder remains unclear, largely owing to the lack of any robust animal models that phenocopy both the ocular and neurological features of the disease. We report here the generation and characterisation of a novel Rab18-mutant mouse model of WARBM. Rab18-mutant mice are viable and fertile. They present with congenital nuclear cataracts and atonic pupils, recapitulating the characteristic ocular features that are associated with WARBM. Additionally, Rab18-mutant cells exhibit an increase in lipid droplet size following treatment with oleic acid. Lipid droplet abnormalities are a characteristic feature of cells taken from WARBM individuals, as well as cells taken from individuals with other neurodegenerative conditions. Neurological dysfunction is also apparent in Rab18-mutant mice, including progressive weakness of the hind limbs. We show that the neurological defects are, most likely, not caused by gross perturbations in synaptic vesicle recycling in the central or peripheral nervous system. Rather, loss of Rab18 is associated with widespread disruption of the neuronal cytoskeleton, including abnormal accumulations of neurofilament and microtubule proteins in synaptic terminals, and gross disorganisation of the cytoskeleton in peripheral nerves. Global proteomic profiling of peripheral nerves in Rab18-mutant mice reveals significant alterations in several core molecular pathways that regulate cytoskeletal dynamics in neurons. The apparent similarities between the WARBM phenotype and the phenotype that we describe here indicate that the Rab18-mutant mouse provides an important platform for investigation of the disease pathogenesis and therapeutic interventions.

  1. Enrichment of Phosphatidylethanolamine in Viral Replication Compartments via Co-opting the Endosomal Rab5 Small GTPase by a Positive-Strand RNA Virus.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    2016-10-01

    Full Text Available Positive-strand RNA viruses build extensive membranous replication compartments to support replication and protect the virus from antiviral responses by the host. These viruses require host factors and various lipids to form viral replication complexes (VRCs. The VRCs built by Tomato bushy stunt virus (TBSV are enriched with phosphatidylethanolamine (PE through a previously unknown pathway. To unravel the mechanism of PE enrichment within the TBSV replication compartment, in this paper, the authors demonstrate that TBSV co-opts the guanosine triphosphate (GTP-bound active form of the endosomal Rab5 small GTPase via direct interaction with the viral replication protein. Deletion of Rab5 orthologs in a yeast model host or expression of dominant negative mutants of plant Rab5 greatly decreases TBSV replication and prevents the redistribution of PE to the sites of viral replication. We also show that enrichment of PE in the viral replication compartment is assisted by actin filaments. Interestingly, the closely related Carnation Italian ringspot virus, which replicates on the boundary membrane of mitochondria, uses a similar strategy to the peroxisomal TBSV to hijack the Rab5-positive endosomes into the viral replication compartments. Altogether, usurping the GTP-Rab5-positive endosomes allows TBSV to build a PE-enriched viral replication compartment, which is needed to support peak-level replication. Thus, the Rab family of small GTPases includes critical host factors assisting VRC assembly and genesis of the viral replication compartment.

  2. A Rab-centric perspective of bacterial pathogen-occupied vacuoles.

    Science.gov (United States)

    Sherwood, Racquel Kim; Roy, Craig R

    2013-09-11

    The ability to create and maintain a specialized organelle that supports bacterial replication is an important virulence property for many intracellular pathogens. Living in a membrane-bound vacuole presents inherent challenges, including the need to remodel a plasma membrane-derived organelle into a novel structure that will expand and provide essential nutrients to support replication, while also having the vacuole avoid membrane transport pathways that target bacteria for destruction in lysosomes. It is clear that pathogenic bacteria use different strategies to accomplish these tasks. The dynamics by which host Rab GTPases associate with pathogen-occupied vacuoles provide insight into the mechanisms used by different bacteria to manipulate host membrane transport. In this review we highlight some of the strategies bacteria use to maintain a pathogen-occupied vacuole by focusing on the Rab proteins involved in biogenesis and maintenance of these novel organelles. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Arf6, Rab11 and transferrin receptor define distinct populations of recycling endosomes.

    Science.gov (United States)

    Kobayashi, Hotaka; Fukuda, Mitsunori

    2013-09-01

    Recycling endosomes are key platforms for endocytic recycling that return internalized molecules back to the plasma membrane. To determine how recycling endosomes perform their functions, searching for proteins and lipids that specifically localized at recycling endosomes has often been performed by colocalization analyses between candidate molecules and conventional recycling endosome markers. However, it remains unclear whether all the conventional markers have identical localizations. Here we report finding that three well-known recycling endosome markers, i.e., Arf6, Rab11 and transferrin receptor (TfR), have different intracellular localizations in PC12 cells. The results of immunofluorescence analyses showed that the signals of endogenous Arf6, Rab11 and TfR in nerve growth factor-stimulated PC12 cells generally differed, although there was some overlapping. Our findings provide new information about recycling endosome markers, and they highlight the heterogeneity of recycling endosomes.

  4. Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

    Science.gov (United States)

    Lin, Mingqun; Liu, Hongyan; Xiong, Qingming; Niu, Hua; Cheng, Zhihui; Yamamoto, Akitsugu; Rikihisa, Yasuko

    2016-11-01

    Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

  5. Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Jing-Fang, E-mail: jfwang@gordonlifescience.org [Key Laboratory of Systems Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shanghai Center for Bioinformation Technology, 100 Qinzhou Road, Shanghai 200235 (China); Gordon Life Science Institute, 13784 Torrey Del Mar Drive, San Diego, CA 92130 (United States); Chou, Kuo-Chen [Gordon Life Science Institute, 13784 Torrey Del Mar Drive, San Diego, CA 92130 (United States)

    2009-12-18

    Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.

  6. Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania*

    Science.gov (United States)

    Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

    2015-01-01

    Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. PMID:26499792

  7. Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania.

    Science.gov (United States)

    Bahl, Surbhi; Parashar, Smriti; Malhotra, Himanshu; Raje, Manoj; Mukhopadhyay, Amitabha

    2015-12-11

    Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Poly(ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair.

    Science.gov (United States)

    Robu, Mihaela; Shah, Rashmi G; Purohit, Nupur K; Zhou, Pengbo; Naegeli, Hanspeter; Shah, Girish M

    2017-08-15

    Xeroderma pigmentosum C (XPC) protein initiates the global genomic subpathway of nucleotide excision repair (GG-NER) for removal of UV-induced direct photolesions from genomic DNA. The XPC has an inherent capacity to identify and stabilize at the DNA lesion sites, and this function is facilitated in the genomic context by UV-damaged DNA-binding protein 2 (DDB2), which is part of a multiprotein UV-DDB ubiquitin ligase complex. The nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) has been shown to facilitate the lesion recognition step of GG-NER via its interaction with DDB2 at the lesion site. Here, we show that PARP1 plays an additional DDB2-independent direct role in recruitment and stabilization of XPC at the UV-induced DNA lesions to promote GG-NER. It forms a stable complex with XPC in the nucleoplasm under steady-state conditions before irradiation and rapidly escorts it to the damaged DNA after UV irradiation in a DDB2-independent manner. The catalytic activity of PARP1 is not required for the initial complex formation with XPC in the nucleoplasm but it enhances the recruitment of XPC to the DNA lesion site after irradiation. Using purified proteins, we also show that the PARP1-XPC complex facilitates the handover of XPC to the UV-lesion site in the presence of the UV-DDB ligase complex. Thus, the lesion search function of XPC in the genomic context is controlled by XPC itself, DDB2, and PARP1. Our results reveal a paradigm that the known interaction of many proteins with PARP1 under steady-state conditions could have functional significance for these proteins.

  9. 32 CFR 202.4 - Composition of a RAB.

    Science.gov (United States)

    2010-07-01

    ... appropriate. At closing installations where BRAC Cleanup Teams (BCT) exist, representatives of the BCT may... installation and the other the community. Co-chairs shall be responsible for directing and managing the RAB...

  10. The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

    Directory of Open Access Journals (Sweden)

    Sanda Ljubicic

    Full Text Available Rab37 belongs to a subclass of Rab GTPases regulating exocytosis, including also Rab3a and Rab27a. Proteomic studies indicate that Rab37 is associated with insulin-containing large dense core granules of pancreatic β-cells. In agreement with these observations, we detected Rab37 in extracts of β-cell lines and human pancreatic islets and confirmed by confocal microscopy the localization of the GTPase on insulin-containing secretory granules. We found that, as is the case for Rab3a and Rab27a, reduction of Rab37 levels by RNA interference leads to impairment in glucose-induced insulin secretion and to a decrease in the number of granules in close apposition to the plasma membrane. Pull-down experiments revealed that, despite similar functional effects, Rab37 does not interact with known Rab3a or Rab27a effectors and is likely to operate through a different mechanism. Exposure of insulin-secreting cells to proinflammatory cytokines, fatty acids or oxidized low-density lipoproteins, mimicking physiopathological conditions that favor the development of diabetes, resulted in a decrease in Rab37 expression. Our data identify Rab37 as an additional component of the machinery governing exocytosis of β-cells and suggest that impaired expression of this GTPase may contribute to defective insulin release in pre-diabetic and diabetic conditions.

  11. Male clients' behaviours with and perspectives about their last male escort encounter: comparing repeat versus first-time hires.

    Science.gov (United States)

    Wolff, Margaret M; Grov, Christian; Smith, Michael D; Koken, Juline A; Parsons, Jeffrey T

    2014-01-01

    Research on men who have sex with men suggests that condomless anal intercourse occurs more frequently in established sexual relationships. While comparable data regarding male-for-male escorting is unavailable, research implies that many clients seek emotional as well as physical connections with the men they hire. In 2012, 495 male clients, recruited via daddysreviews.com completed an online survey about their last hiring experience. Most participants were from the USA (85.7%), the UK and Canada (3.2% each). In total, 75% of encounters involved an escort hired for the first time; 25% were with a previously hired escort ('repeat encounter'). The client's age, lifetime number of escorts hired and number hired in the past year were positively associated with the last encounter being a repeat encounter. Cuddling, sharing a meal, drinking alcohol, taking a walk, watching a show and shopping were also positively associated with repeat encounters. Conversely, none of the sexual behaviours were significantly associated with repeat encounters. Repeat encounters were significantly more likely to include non-sexual behaviours alongside sexual activities, but no more likely to involve condomless anal intercourse. Moreover, clients' knowledge of escorts' HIV status was not significantly associated with engaging in condomless anal intercourse with repeat encounters.

  12. Mutations in the small GTPase gene RAB39B are responsible for X-linked mental retardation associated with autism, epilepsy, and macrocephaly.

    Science.gov (United States)

    Giannandrea, Maila; Bianchi, Veronica; Mignogna, Maria Lidia; Sirri, Alessandra; Carrabino, Salvatore; D'Elia, Errico; Vecellio, Matteo; Russo, Silvia; Cogliati, Francesca; Larizza, Lidia; Ropers, Hans-Hilger; Tzschach, Andreas; Kalscheuer, Vera; Oehl-Jaschkowitz, Barbara; Skinner, Cindy; Schwartz, Charles E; Gecz, Jozef; Van Esch, Hilde; Raynaud, Martine; Chelly, Jamel; de Brouwer, Arjan P M; Toniolo, Daniela; D'Adamo, Patrizia

    2010-02-12

    Human Mental Retardation (MR) is a common and highly heterogeneous pediatric disorder affecting around 3% of the general population; at least 215 X-linked MR (XLMR) conditions have been described, and mutations have been identified in 83 different genes, encoding proteins with a variety of function, such as chromatin remodeling, synaptic function, and intracellular trafficking. The small GTPases of the RAB family, which play an essential role in intracellular vesicular trafficking, have been shown to be involved in MR. We report here the identification of mutations in the small GTPase RAB39B gene in two male patients. One mutation in family X (D-23) introduced a stop codon seven amino acids after the start codon (c.21C > A; p.Y7X). A second mutation, in the MRX72 family, altered the 5' splice site (c.215+1G > A) and normal splicing. Neither instance produced a protein. Mutations segregate with the disease in the families, and in some family members intellectual disabilities were associated with autism spectrum disorder, epileptic seizures, and macrocephaly. We show that RAB39B, a novel RAB GTPase of unknown function, is a neuronal-specific protein that is localized to the Golgi compartment. Its downregulation leads to an alteration in the number and morphology of neurite growth cones and a significant reduction in presynaptic buttons, suggesting that RAB39B is required for synapse formation and maintenance. Our results demonstrate developmental and functional neuronal alteration as a consequence of downregulation of RAB39B and emphasize the critical role of vesicular trafficking in the development of neurons and human intellectual abilities. Copyright (c) 2010 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

  13. On the laws of thermodynamics from the escort average and on the uniqueness of statistical factors

    International Nuclear Information System (INIS)

    Yamano, Takuya

    2003-01-01

    We consider the relation between the statistical weight and the laws of thermodynamics. Our path bases on the infinitesimal perturbation from outside of the thermodynamical system. It is fair to say that the form of the first laws of thermodynamics and the Clausius' definition of thermodynamic entropy are commensurately altered once we employ the escort average of statistical weight but preserves the forms in the limit of the usual weight. We also see an example for the unique determination of the statistical factor (so-called the Gibbs theorem) in addition to the Boltzmann and the Tsallis ones

  14. 41 CFR 303-70.600 - How many persons may we authorize travel expenses for to escort the remains of a deceased employee?

    Science.gov (United States)

    2010-07-01

    ... 41 Public Contracts and Property Management 4 2010-07-01 2010-07-01 false How many persons may we authorize travel expenses for to escort the remains of a deceased employee? 303-70.600 Section 303-70.600... DEATH OF CERTAIN EMPLOYEES Escort of Remains § 303-70.600 How many persons may we authorize travel...

  15. Plasma membrane cholesterol level and agonist-induced internalization of delta-opioid receptors; colocalization study with intracellular membrane markers of Rab family\

    Czech Academy of Sciences Publication Activity Database

    Brejchová, Jana; Vošahlíková, Miroslava; Roubalová, Lenka; Parenti, M.; Mauri, M.; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-01-01

    Roč. 48, č. 4 (2016), s. 375-396 ISSN 0145-479X R&D Projects: GA ČR(CZ) GAP207/12/0919 Institutional support: RVO:67985823 Keywords : cholesterol * plasma membrane * delta-opioid receptor * internalization * Rab proteins Subject RIV: CE - Biochemistry Impact factor: 2.576, year: 2016

  16. Porn star/stripper/escort: economic and sexual dynamics in a sex work career.

    Science.gov (United States)

    Escoffier, Jeffrey

    2007-01-01

    This article explores the career dynamics of performers in the gay male pornography industry, by focusing on a common career path- from porn star to stripper to escort. Between 1995 and 2005, most men performing in gay porn films, unlike contract actresses in the straight porn industry, have been unable to earn enough income to work exclusively as performers in front of the camera. The industry's constant search for new faces and fresh performers creates what sociologist Paul Cressey has called "the retrogressive dynamic": The longer a person works in a sexual occupation, the less one is paid, and the lower the status of the work venue. In the porn industry, one aspect of this process is referred to as "overexposure," during which the performer experiences a diminishing "fantasy potential" as fans lose erotic interest in the porn star who has appeared too frequently in too many movies. Performers attempt to confront the retrogressive dynamic by limiting the number of adult films in which they appear in a year, diversifying their sexual repertoire, or shifting into other roles within the industry (behind the camera, marketing, production, etc.). One common option is to pursue work in economically complementary forms of sex work such as stripping and escorting.

  17. The N-Myc down regulated Gene1 (NDRG1) Is a Rab4a effector involved in vesicular recycling of E-cadherin.

    Science.gov (United States)

    Kachhap, Sushant K; Faith, Dennis; Qian, David Z; Shabbeer, Shabana; Galloway, Nathan L; Pili, Roberto; Denmeade, Samuel R; DeMarzo, Angelo M; Carducci, Michael A

    2007-09-05

    Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1) increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

  18. The N-Myc down regulated Gene1 (NDRG1 Is a Rab4a effector involved in vesicular recycling of E-cadherin.

    Directory of Open Access Journals (Sweden)

    Sushant K Kachhap

    2007-09-01

    Full Text Available Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1 increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

  19. Two Rab5 Homologs Are Essential for the Development and Pathogenicity of the Rice Blast Fungus Magnaporthe oryzae

    Directory of Open Access Journals (Sweden)

    Cheng D. Yang

    2017-05-01

    Full Text Available The rice blast fungus, Magnaporthe oryzae, infects many economically important cereal crops, particularly rice. It has emerged as an important model organism for studying the growth, development, and pathogenesis of filamentous fungi. RabGTPases are important molecular switches in regulation of intracellular membrane trafficking in all eukaryotes. MoRab5A and MoRab5B are Rab5 homologs in M. oryzae, but their functions in the fungal development and pathogenicity are unknown. In this study, we have employed a genetic approach and demonstrated that both MoRab5A and MoRab5B are crucial for vegetative growth and development, conidiogenesis, melanin synthesis, vacuole fusion, endocytosis, sexual reproduction, and plant pathogenesis in M. oryzae. Moreover, both MoRab5A and MoRab5B show similar localization in hyphae and conidia. To further investigate possible functional redundancy between MoRab5A and MoRab5B, we overexpressed MoRAB5A and MoRAB5B, respectively, in MoRab5B:RNAi and MoRab5A:RNAi strains, but neither could rescue each other’s defects caused by the RNAi. Taken together, we conclude that both MoRab5A and MoRab5B are necessary for the development and pathogenesis of the rice blast fungus, while they may function independently.

  20. Rab11b mediates melanin transfer between donor melanocytes and acceptor keratinocytes via coupled exo/endocytosis.

    Science.gov (United States)

    Tarafder, Abul K; Bolasco, Giulia; Correia, Maria S; Pereira, Francisco J C; Iannone, Lucio; Hume, Alistair N; Kirkpatrick, Niall; Picardo, Mauro; Torrisi, Maria R; Rodrigues, Inês P; Ramalho, José S; Futter, Clare E; Barral, Duarte C; Seabra, Miguel C

    2014-04-01

    The transfer of melanin from melanocytes to keratinocytes is a crucial process underlying maintenance of skin pigmentation and photoprotection against UV damage. Here, we present evidence supporting coupled exocytosis of the melanin core, or melanocore, by melanocytes and subsequent endocytosis by keratinocytes as a predominant mechanism of melanin transfer. Electron microscopy analysis of human skin samples revealed three lines of evidence supporting this: (1) the presence of melanocores in the extracellular space; (2) within keratinocytes, melanin was surrounded by a single membrane; and (3) this membrane lacked the melanosomal membrane protein tyrosinase-related protein 1 (TYRP1). Moreover, co-culture of melanocytes and keratinocytes suggests that melanin exocytosis is specifically induced by keratinocytes. Furthermore, depletion of Rab11b, but not Rab27a, caused a marked decrease in both keratinocyte-stimulated melanin exocytosis and transfer to keratinocytes. Thus, we propose that the predominant mechanism of melanin transfer is keratinocyte-induced exocytosis, mediated by Rab11b through remodeling of the melanosome membrane, followed by subsequent endocytosis by keratinocytes.

  1. Early stages of functional diversification in the Rab GTPase gene family revealed by genomic and localization studies in Paramecium species.

    Science.gov (United States)

    Bright, Lydia J; Gout, Jean-Francois; Lynch, Michael

    2017-04-15

    New gene functions arise within existing gene families as a result of gene duplication and subsequent diversification. To gain insight into the steps that led to the functional diversification of paralogues, we tracked duplicate retention patterns, expression-level divergence, and subcellular markers of functional diversification in the Rab GTPase gene family in three Paramecium aurelia species. After whole-genome duplication, Rab GTPase duplicates are more highly retained than other genes in the genome but appear to be diverging more rapidly in expression levels, consistent with early steps in functional diversification. However, by localizing specific Rab proteins in Paramecium cells, we found that paralogues from the two most recent whole-genome duplications had virtually identical localization patterns, and that less closely related paralogues showed evidence of both conservation and diversification. The functionally conserved paralogues appear to target to compartments associated with both endocytic and phagocytic recycling functions, confirming evolutionary and functional links between the two pathways in a divergent eukaryotic lineage. Because the functionally diversifying paralogues are still closely related to and derived from a clade of functionally conserved Rab11 genes, we were able to pinpoint three specific amino acid residues that may be driving the change in the localization and thus the function in these proteins. © 2017 Bright et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  2. Male Escorts' and Male Clients' Sexual Behavior During Their Last Commercial Sexual Encounter: Comparing and Contrasting Findings from Two Online Studies.

    Science.gov (United States)

    Grov, Christian; Rodríguez-Díaz, Carlos E; Jovet-Toledo, Gerardo G

    2016-05-01

    Much of what is known about commercial sexual encounters between men is based on data gathered from escorts. With few exceptions, studies have not compared male clients' reports of behavior during commercial sexual encounters with male escorts'. The present study draws from two datasets, a 2012 survey of clients (n = 495) and a 2013 survey of escorts (n = 387)--both used virtually identical measures of sexual behavior during the most recent commercial sexual encounter. For clients and escorts, the majority eschewed having sex without a condom, and kissing and oral sex were among the most common behaviors reported. Using logistic regression, both samples were compared across 15 sexual behaviors, finding significant differences in six--the escort sample had greater odds of reporting their last commercial sexual encounter involved watching the client masturbate, viewing porn, role play (dad/son, dominant/submissive), and having prior sexual experience with their commercial partner. The escort sample had lower odds of reporting that the client watched the escort masturbate, and being told partner's HIV status. In multivariable modeling, both samples did not significantly differ in reports of condomless anal sex. Male-male commercial sexual encounters appear to be involved in a wide range of sexual behaviors, many of which convey low-to-no risk of HIV transmission.

  3. CSFV proliferation is associated with GBF1 and Rab2

    Indian Academy of Sciences (India)

    2016-12-29

    Dec 29, 2016 ... 2.4 GBF1and Rab2 shRNA vector construction and cell ... CSFV RNA in ST cells at the earliest time point were selected as ... Real-time PCR for detection of GBF1 ..... pathways are also regulated by GBF1 and are involved in.

  4. Characterization of the human RAB38 and RAB7 genes: exclusion of new major pathological loci for Japanese OCA.

    Science.gov (United States)

    Suzuki, Tamio; Miyamura, Yoshinori; Inagaki, Katsuhiko; Tomita, Yasushi

    2003-08-01

    Oculocutaneous albinisms (OCAs) are due to various gene mutations that cause a disruption of melanogenesis in the melanocyte. Four different genes associated with human OCA have been reported, however, not all of OCA patients can be classified according to these four genes. We have sought to find a new major locus for Japanese OCA. Recently two genes, RAB38 and RAB7, were reported to play an important role in melanogenesis in the melanocyte, suggesting that these two genes could be good candidates for new OCA loci. To determine the structures of the human RAB38 and RAB7 genes, and examine if the two genes are new major loci for Japanese OCA. We screened mutations in these genes of 25 Japanese OCA patients who lacked mutations in the OCA1 and OCA2 genes with SSCP/heteroduplexes method. We determined the both genes, and their genomic organizations to design the primers for SSCP/heteroduplexes method. And then we screened mutations, but no mutation was detected. Neither of the genes is a new major locus for Japanese OCA.

  5. Molecular characterization and expression of Rab7 from Clonorchis sinensis and its potential role in autophagy.

    Science.gov (United States)

    Jia, Feifei; Li, Ye; Huang, Yan; Chen, Tingjin; Li, Shan; Xu, Yanquan; Wu, Zhongdao; Li, Xuerong; Yu, Xinbing

    2013-07-01

    Accumulating evidences suggest that Rab7 GTPase is important for the normal progression of autophagy. However, the role of Rab7 GTPase in regulation of autophagy in Clonorchis sinensis is not known. In this study, a gene encoding Rab7 was isolated from C. sinensis adult cDNA. Recombinant CsRab7 was expressed and purified from Escherichia coli. CsRab7 transcripts were detected in the cDNA of adult worm, metacercaria, cercaria, and egg of C. sinensis, and were highly expressed in the metacercaria. Immunohistochemical localization results revealed that CsRab7 was specifically deposited on the vitellarium and eggs of adult worm. Furthermore, EGFP signal of CsRab7WT and the active mutant CsRab7Q67L were associated with autophagic vesicles in transiently transfected 293T cells. It is concluded from the present study that CsRab7 GTPase possibly contributes to the development of C. sinensis and that the autophagy pathway could be an important site of action with respect to the developmental role of CsRab7 in C. sinensis.

  6. Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

    Directory of Open Access Journals (Sweden)

    Michael G Connor

    2015-10-01

    Full Text Available Yersinia pestis is a facultative intracellular pathogen that causes the disease known as plague. During infection of macrophages Y. pestis actively evades the normal phagosomal maturation pathway to establish a replicative niche within the cell. However, the mechanisms used by Y. pestis to subvert killing by the macrophage are unknown. Host Rab GTPases are central mediators of vesicular trafficking and are commonly targeted by bacterial pathogens to alter phagosome maturation and killing by macrophages. Here we demonstrate for the first time that host Rab1b is required for Y. pestis to effectively evade killing by macrophages. We also show that Rab1b is specifically recruited to the Yersinia containing vacuole (YCV and that Y. pestis is unable to subvert YCV acidification when Rab1b expression is knocked down in macrophages. Furthermore, Rab1b knockdown also altered the frequency of association between the YCV with the lysosomal marker Lamp1, suggesting that Rab1b recruitment to the YCV directly inhibits phagosome maturation. Finally, we show that Rab1b knockdown also impacts the pH of the Legionella pneumophila containing vacuole, another pathogen that recruits Rab1b to its vacuole. Together these data identify a novel role for Rab1b in the subversion of phagosome maturation by intracellular pathogens and suggest that recruitment of Rab1b to the pathogen containing vacuole may be a conserved mechanism to control vacuole pH.

  7. Integration of two RAB5 groups during endosomal transport in plants

    Science.gov (United States)

    Ebine, Kazuo; Choi, Seung-won; Ichinose, Sakura; Uemura, Tomohiro; Nakano, Akihiko

    2018-01-01

    RAB5 is a key regulator of endosomal functions in eukaryotic cells. Plants possess two different RAB5 groups, canonical and plant-unique types, which act via unknown counteracting mechanisms. Here, we identified an effector molecule of the plant-unique RAB5 in Arabidopsis thaliana, ARA6, which we designated PLANT-UNIQUE RAB5 EFFECTOR 2 (PUF2). Preferential colocalization with canonical RAB5 on endosomes and genetic interaction analysis indicated that PUF2 coordinates vacuolar transport with canonical RAB5, although PUF2 was identified as an effector of ARA6. Competitive binding of PUF2 with GTP-bound ARA6 and GDP-bound canonical RAB5, together interacting with the shared activating factor VPS9a, showed that ARA6 negatively regulates canonical RAB5-mediated vacuolar transport by titrating PUF2 and VPS9a. These results suggest a unique and unprecedented function for a RAB effector involving the integration of two RAB groups to orchestrate endosomal trafficking in plant cells. PMID:29749929

  8. Rab18 dynamics in adipocytes in relation to lipogenesis, lipolysis and obesity.

    Directory of Open Access Journals (Sweden)

    Marina R Pulido

    Full Text Available Lipid droplets (LDs are organelles that coordinate lipid storage and mobilization, both processes being especially important in cells specialized in managing fat, the adipocytes. Proteomic analyses of LDs have consistently identified the small GTPase Rab18 as a component of the LD coat. However, the specific contribution of Rab18 to adipocyte function remains to be elucidated. Herein, we have analyzed Rab18 expression, intracellular localization and function in relation to the metabolic status of adipocytes. We show that Rab18 production increases during adipogenic differentiation of 3T3-L1 cells. In addition, our data show that insulin induces, via phosphatidylinositol 3-kinase (PI3K, the recruitment of Rab18 to the surface of LDs. Furthermore, Rab18 overexpression increased basal lipogenesis and Rab18 silencing impaired the lipogenic response to insulin, thereby suggesting that this GTPase promotes fat accumulation in adipocytes. On the other hand, studies of the β-adrenergic receptor agonist isoproterenol confirmed and extended previous evidence for the participation of Rab18 in lipolysis. Together, our data support the view that Rab18 is a common mediator of lipolysis and lipogenesis and suggests that the endoplasmic reticulum (ER is the link that enables Rab18 action on these two processes. Finally, we describe, for the first time, the presence of Rab18 in human adipose tissue, wherein the expression of this GTPase exhibits sex- and depot-specific differences and is correlated to obesity. Taken together, these findings indicate that Rab18 is involved in insulin-mediated lipogenesis, as well as in β-adrenergic-induced lipolysis, likely facilitating interaction of LDs with ER membranes and the exchange of lipids between these compartments. A role for Rab18 in the regulation of adipocyte biology under both normal and pathological conditions is proposed.

  9. Molecular Analysis of Gli3, Ihh, Rab23, and Jag1 in a Rabbit Model of Craniosynostosis: Likely Exclusion as the Loci of Origin.

    Science.gov (United States)

    Gilbert, James R; Taylor, Gwen M; Losee, Joseph E; Mooney, Mark P; Cooper, Gregory M

    2018-03-01

    Craniosynostosis (CS) involves the premature fusion of one or more cranial sutures. The etiology of CS is complex and mutations in more than 50 distinct genes have been causally linked to the disorder. Many of the genes that have been associated with CS in humans play an essential role in tissue patterning and early craniofacial development. Among these genes are members of the Hedgehog (HH) and Notch signal transduction pathways, including the GLI family member Gli3, Indian Hedgehog ( Ihh), the RAS oncogene family member Rab23, and the Notch ligand JAGGED1 ( Jag1). We have previously described a colony of rabbits with a heritable pattern of coronal suture synostosis, although the genetic basis for synostosis within this model remains unknown. The present study was performed to determine if coding errors in Gli3, Ihh, Rab23, or Jag1 could be causally linked to craniosynostosis in this unique animal model. Sequencing of cDNA templates was performed using samples obtained from wild-type and craniosynostotic rabbits. Several nucleotide polymorphisms were identified in Gli3, Ihh, and Rab23, although these variants failed to segregate by phenotype. No nucleotide polymorphisms were identified in Jag1. These data indicate that the causal locus for heritable craniosynostosis in this rabbit model is not located within the protein coding regions of Gli3, Ihh, Rab23, or Jag1.

  10. The RabGAP TBC1D1 plays a central role in exercise-regulated glucose metabolism in skeletal muscle

    DEFF Research Database (Denmark)

    Stöckli, Jacqueline; Meoli, Christopher C; Hoffman, Nolan J

    2015-01-01

    Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated...... weight, insulin action, and exercise. TBC1D1(-/-) mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1(-/-) muscle, and TBC1D1(-/-) mice showed impaired exercise endurance...... together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers....

  11. miR-184 and miR-150 promote renal glomerular mesangial cell aging by targeting Rab1a and Rab31.

    Science.gov (United States)

    Liu, Xiujuan; Fu, Bo; Chen, Dapeng; Hong, Quan; Cui, Jing; Li, Jin; Bai, Xueyuan; Chen, Xiangmei

    2015-08-15

    The molecular mechanism of kidney aging is not well understood, but the abnormal expression of miRNAs with aging is considered to be an important contributor. miR-184 and miR-150 were screened using a miRNA microarray and qRT-PCR and found to be significantly upregulated in 24-month-old rats. Rat renal primary glomerular mesangial cells (GMCs) were isolated from 3-month and 24-month-old rats for the in vitro analysis of the roles of miR-184 and miR-150 in kidney aging. Bioinformatics analyses suggested that Rab1a and Rab31, which are associated with cell autophagy, were targeted by both miR-184 and miR-150. miR-184 and miR-150 were increased significantly in aging GMCs versus young cells, while Rab1a and Rab31 were significantly lower in aging cells. Furthermore, dual luciferase reporter assays revealed that miR-184 and miR-150 bound to the 3'-UTR of Rab1a and Rab31 mRNAs. Transfection of miR-184 and miR-150 mimics into young GMCs suppressed the expression of Rab1a and Rab31. Transfected cells showed lower autophagy activities and higher levels of cellular oxidative products, leading to the aging of young GMCs. However, miR-184 and miR-150 inhibitors promoted autophagy and reduced oxidative damage by upregulating Rab1a and Rab31 in old GMCs. In conclusion, miR-184 and miR-150 inhibited autophagy, promoting GMC aging. Copyright © 2015 Elsevier Inc. All rights reserved.

  12. Steady-state Manoeuvring of a Generic ASD Tug in Escort Pull and Bow-rope Aided Push Operation

    Directory of Open Access Journals (Sweden)

    Jaroslaw Artyszuk

    2014-09-01

    Full Text Available This paper is devoted to expand the very promising research undertaken in the author's previous work, basically done on simplified modelling the escort push operation. Now, the other two modes of a tug's employment, as stated in the title, are covered. The special focus is again set on the indirect towing in that the towline force is much higher than the thruster force. The ratio of these two forces, referred to as the relative towing force (or amplification ratio is evaluated together with the hull drift angle and the thruster(-s angle for a given escort speed. This mutual relationship is known as the tug performance diagram. Although rather generic (container-type formulas are derived, they are supplied for exemplification purposes with simple, analytically given hull hydrodynamic forces. The aim is also here to provide a basis for further sensitivity analysis of the model and possible improvement/optimisation to the tug design. The obtained charts also could serve as rough and clear guidance for towmasters while escorting.

  13. Rab from the white shrimp Litopenaeus vannamei: characterization and its regulation upon environmental stress.

    Science.gov (United States)

    Wang, Lei; Wang, Xiao-Rong; Liu, Jin; Chen, Chu-Xian; Liu, Yuan; Wang, Wei-Na

    2015-10-01

    With the destruction of the ecological environment, shrimp cultivation in China has been seriously affected by outbreaks of infectious diseases. Rab, which belong to small GTPase Ras superfamily, can regulate multiple steps in eukaryotic vesicle trafficking including vesicle budding, vesicle tethering, and membrane fusion. Knowledge of Rab in shrimp is essential to understanding regulation and detoxification mechanisms of environmental stress. In this study, we analyzed the functions of Rab from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNA of Rab was obtained, which was 751 bp long, with open reading frame encoding 206 amino acids. In this study, for the first time, the gene expression of Rab of L. vannamei was analyzed by quantitative real-time PCR after exposure to five kinds of environmental stresses (bacteria, pH, Cd, salinity and low temperature). The results demonstrate that Rab is sensitive and involved in bacteria, pH, and Cd stress responses and Rab is more sensitive to bacteria than other stresses. Therefore we infer that Rab may have relationship with the anti-stress mechanism induced by environment stress in shrimp and Rab could be used as critical biomarkers for environmental quality assessment.

  14. Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin.

    Science.gov (United States)

    Stokes, Leanne

    2013-03-01

    The P2X4 receptor is an ATP-gated ion channel expressed in neurons, endothelia and immune cells. Plasma membrane expression of P2X4 is regulated by dynamin-dependent endocytosis, and this study identifies a Rab5-dependent pathway of receptor internalisation. Expression of Rab5 constructs altered the distribution of P2X4 in HEK-293 cells, and both constitutive internalisation and agonist-induced desensitisation of P2X4 were increased by co-expression of wild-type Rab5 or constitutively active Rab5 (Q79L). Expression of inactive dynamin K44A and Rab5 S34N constructs abolished agonist-induced desensitisation, suggesting internalisation as the underlying mechanism. Blocking P2X4 internalisation in this way also abolished potentiation of ATP-induced currents by the allosteric modulator ivermectin. This suggests that the dynamin-Rab5 internalisation pathway is essential for the ivermectin potentiation effect. In agreement with this hypothesis, the co-expression of wild-type dynamin, wild-type Rab5 or active Rab5 (Q79L) could increase the potentiation of the ATP-induced P2X4 response by ivermectin. These findings highlight Rab5 GTPase as a key regulator of P2X4 receptor cell surface expression and internalisation.

  15. The late endocytic Rab39a GTPase regulates the interaction between multivesicular bodies and chlamydial inclusions.

    Science.gov (United States)

    Gambarte Tudela, Julian; Capmany, Anahi; Romao, Maryse; Quintero, Cristian; Miserey-Lenkei, Stephanie; Raposo, Graca; Goud, Bruno; Damiani, Maria Teresa

    2015-08-15

    Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development. © 2015. Published by The Company of Biologists Ltd.

  16. Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana

    OpenAIRE

    Diana Castaño; Mauricio Rojas

    2010-01-01

    En el fagosoma, Mycobacterium spp. altera la activación y reclutamiento de diferentes proteínas "del gen Ras de cerebro de rata", comúnmente conocidas como Rab. En este manuscrito se revisa una serie de reportes que han demostrado que los fagosomas que contienen micobacterias tienen una expresión mayor y sostenida de Rab5, Rab11, Rab14 y Rab22a, y menor o ninguna expresión de Rab7, Rab9 y Rab6. Esto se correlaciona con aumento de la fusión de estos fagosomas con endosomas tempranos y de recic...

  17. Plasma membrane cholesterol level and agonist-induced internalization of δ-opioid receptors; colocalization study with intracellular membrane markers of Rab family.

    Science.gov (United States)

    Brejchova, Jana; Vosahlikova, Miroslava; Roubalova, Lenka; Parenti, Marco; Mauri, Mario; Chernyavskiy, Oleksandr; Svoboda, Petr

    2016-08-01

    Decrease of cholesterol level in plasma membrane of living HEK293 cells transiently expressing FLAG-δ-OR by β-cyclodextrin (β-CDX) resulted in a slight internalization of δ-OR. Massive internalization of δ-OR induced by specific agonist DADLE was diminished in cholesterol-depleted cells. These results suggest that agonist-induced internalization of δ-OR, which has been traditionally attributed exclusively to clathrin-mediated pathway, proceeds at least partially via membrane domains. Identification of internalized pools of FLAG-δ-OR by colocalization studies with proteins of Rab family indicated the decreased presence of receptors in early endosomes (Rab5), late endosomes and lysosomes (Rab7) and fast recycling vesicles (Rab4). Slow type of recycling (Rab11) was unchanged by cholesterol depletion. As expected, agonist-induced internalization of oxytocin receptors was totally suppressed in β-CDX-treated cells. Determination of average fluorescence lifetime of TMA-DPH, the polar derivative of hydrophobic membrane probe diphenylhexatriene, in live cells by FLIM indicated a significant alteration of the overall PM structure which may be interpreted as an increased "water-accessible space" within PM area. Data obtained by studies of HEK293 cells transiently expressing FLAG-δ-OR by "antibody feeding" method were extended by analysis of the effect of cholesterol depletion on distribution of FLAG-δ-OR in sucrose density gradients prepared from HEK293 cells stably expressing FLAG-δ-OR. Major part of FLAG-δ-OR was co-localized with plasma membrane marker Na,K-ATPase and β-CDX treatment resulted in shift of PM fragments containing both FLAG-δ-OR and Na,K-ATPase to higher density. Thus, the decrease in content of the major lipid constituent of PM resulted in increased density of resulting PM fragments.

  18. Rab14 and its exchange factor FAM116 link endocytic recycling and adherens junction stability in migrating cells.

    Science.gov (United States)

    Linford, Andrea; Yoshimura, Shin-ichiro; Nunes Bastos, Ricardo; Langemeyer, Lars; Gerondopoulos, Andreas; Rigden, Daniel J; Barr, Francis A

    2012-05-15

    Rab GTPases define the vesicle trafficking pathways underpinning cell polarization and migration. Here, we find that Rab4, Rab11, and Rab14 and the candidate Rab GDP-GTP exchange factors (GEFs) FAM116A and AVL9 are required for cell migration. Rab14 and its GEF FAM116A localize to and act on an intermediate compartment of the transferrin-recycling pathway prior to Rab11 and after Rab5 and Rab4. This Rab14 intermediate recycling compartment has specific functions in migrating cells discrete from early and recycling endosomes. Rab14-depleted cells show increased N-cadherin levels at junctional complexes and cannot resolve cell-cell junctions. This is due to decreased shedding of cell-surface N-cadherin by the ADAM family protease ADAM10/Kuzbanian. In FAM116A- and Rab14-depleted cells, ADAM10 accumulates in a transferrin-positive endocytic compartment, and the cell-surface level of ADAM10 is correspondingly reduced. FAM116 and Rab14 therefore define an endocytic recycling pathway needed for ADAM protease trafficking and regulation of cell-cell junctions. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Rab11-dependent Recycling of the Human Ether-a-go-go-related Gene (hERG) Channel*

    Science.gov (United States)

    Chen, Jeffery; Guo, Jun; Yang, Tonghua; Li, Wentao; Lamothe, Shawn M.; Kang, Yudi; Szendrey, John A.; Zhang, Shetuan

    2015-01-01

    The human ether-a-go-go-related gene (hERG) encodes the pore-forming subunit of the rapidly activating delayed rectifier potassium channel (IKr). A reduction in the hERG current causes long QT syndrome, which predisposes affected individuals to ventricular arrhythmias and sudden death. We reported previously that hERG channels in the plasma membrane undergo vigorous internalization under low K+ conditions. In the present study, we addressed whether hERG internalization occurs under normal K+ conditions and whether/how internalized channels are recycled back to the plasma membrane. Using patch clamp, Western blot, and confocal imaging analyses, we demonstrated that internalized hERG channels can effectively recycle back to the plasma membrane. Low K+-enhanced hERG internalization is accompanied by an increased rate of hERG recovery in the plasma membrane upon reculture following proteinase K-mediated clearance of cell-surface proteins. The increased recovery rate is not due to enhanced protein synthesis, as hERG mRNA expression was not altered by low K+ exposure, and the increased recovery was observed in the presence of the protein biosynthesis inhibitor cycloheximide. GTPase Rab11, but not Rab4, is involved in the recycling of hERG channels. Interfering with Rab11 function not only delayed hERG recovery in cells after exposure to low K+ medium but also decreased hERG expression and function in cells under normal culture conditions. We concluded that the recycling pathway plays an important role in the homeostasis of plasma membrane-bound hERG channels. PMID:26152716

  20. Homologous Recombination as a Replication Fork Escort: Fork-Protection and Recovery

    Directory of Open Access Journals (Sweden)

    Audrey Costes

    2012-12-01

    Full Text Available Homologous recombination is a universal mechanism that allows DNA repair and ensures the efficiency of DNA replication. The substrate initiating the process of homologous recombination is a single-stranded DNA that promotes a strand exchange reaction resulting in a genetic exchange that promotes genetic diversity and DNA repair. The molecular mechanisms by which homologous recombination repairs a double-strand break have been extensively studied and are now well characterized. However, the mechanisms by which homologous recombination contribute to DNA replication in eukaryotes remains poorly understood. Studies in bacteria have identified multiple roles for the machinery of homologous recombination at replication forks. Here, we review our understanding of the molecular pathways involving the homologous recombination machinery to support the robustness of DNA replication. In addition to its role in fork-recovery and in rebuilding a functional replication fork apparatus, homologous recombination may also act as a fork-protection mechanism. We discuss that some of the fork-escort functions of homologous recombination might be achieved by loading of the recombination machinery at inactivated forks without a need for a strand exchange step; as well as the consequence of such a model for the stability of eukaryotic genomes.

  1. 阴道毛滴虫TvRab11C基因的克隆及原核表达%Cloning and prokaryotic expression of TvRab11C gene of Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    刘畅; 丁鹤; 宫鹏涛; 李建华; 李淑红; 李赫; 张国才; 张西臣

    2012-01-01

    目的 克隆并原核表达阴道毛滴虫TvRab11C(G3 Ras-related protein Rab11C)基因.方法 利用PCR技术扩增阴道毛滴虫TvRab1 1C基因,与原核表达载体pET-28a连接,构建重组原核表达质粒pET-28a-TvRab1 1C,转化大肠杆菌BL21(DE3),IPTG诱导表达,SDS-PAGE分析表达产物的可溶性,Western blot分析表达产物的反应原性.结果 重组原核表达质粒pET-28a-TvRab11C经双酶切及测序证明构建正确;表达的重组蛋白相对分子质量约为30000,主要以包涵体形式存在,可被抗阴道毛滴虫多克隆抗体识别.结论 成功克隆了阴道毛滴虫TvRab11C基因,并在E.coli BL21 (DE3)中获得了表达,为进一步研究TvRas基因和G蛋白与阴道毛滴虫寄生能力和致病性的关系奠定了基础.%Objective To clone the TvRabllC gene of Trichomonas vaginalis and express in prokaryotic cells. Methods The TvRabllC gene was amplified by PCR from T. vaginalis and inserted into prokaryotic expression vector pET-28a. The constructed recombinant plasmid pET-28a-TvRabllC was transformed to E. coli BL21 (DE3) and induced by IPTG. The expressed product was analyzed for solubility by SDS-PAGE and for reactogenicity by Western blot. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-28a-TvRabllC was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 30 000,mainly existed in a form of inclusion body,and was recognized by polyclonal antibody against T. vaginalis. Conclusion The TvRas gene of T. vaginalis was successfully cloned and expressed in E. coli BL21 (DE3),which laid a foundation of further study on relationship of TvRas gene and protein to the parasitic ability and pathogenicity of T. vaginalis.

  2. Rab5 induces Rac-independent lamellipodia formation and cell migration

    NARCIS (Netherlands)

    Spaargaren, M.; Bos, J. L.

    1999-01-01

    Rab5 is a regulatory GTPase of vesicle docking and fusion that is involved in receptor-mediated endocytosis and pinocytosis. Introduction of active Rab5 in cells stimulates the rate of endocytosis and vesicle fusion, resulting in the formation of large endocytic vesicles, whereas dominant negative

  3. Clinical presentation of Griscelli syndrome type 2 and spectrum of RAB27A mutations

    DEFF Research Database (Denmark)

    Meeths, Marie; Bryceson, Yenan T; Rudd, Eva

    2010-01-01

    Griscelli syndrome type 2 (GS2) is an autosomal-recessive immunodeficiency caused by mutations in RAB27A, clinically characterized by partial albinism and haemophagocytic lymphohistocytosis (HLH). We evaluated the frequency of RAB27A mutations in 21 unrelated patients with haemophagocytic syndromes...

  4. Deficient Rab11 activity underlies glucose hypometabolism in primary neurons of Huntington’s disease mice

    International Nuclear Information System (INIS)

    Li, Xueyi; Valencia, Antonio; McClory, Hollis; Sapp, Ellen; Kegel, Kimberly B.; DiFiglia, Marian

    2012-01-01

    Highlights: ► Primary Huntington’s disease neurons are impaired in taking up glucose. ► Rab11 modulates glucose uptake in neurons. ► Increasing Rab11 activity attenuates the glucose uptake defect in disease neurons. ► We provide a novel mechanism for glucose hypometabolism in Huntington’s disease. -- Abstract: Huntington’s disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Positron emission tomography studies have revealed a decline in glucose metabolism in the brain of patients with HD by a mechanism that has not been established. We examined glucose utilization in embryonic primary cortical neurons of wild-type (WT) and HD knock-in mice, which have 140 CAG repeats inserted in the endogenous mouse huntingtin gene (HD 140Q/140Q ). Primary HD 140Q/140Q cortical neurons took up significantly less glucose than did WT neurons. Expression of permanently inactive and permanently active forms of Rab11 correspondingly altered glucose uptake in WT neurons, suggesting that normal activity of Rab11 is needed for neuronal uptake of glucose. It is known that Rab11 activity is diminished in HD 140Q/140Q neurons. Expression of dominant active Rab11 to enhance the activity of Rab11 normalized glucose uptake in HD 140Q/140Q neurons. These results suggest that deficient activity of Rab11 is a novel mechanism for glucose hypometabolism in HD.

  5. The Rab2A GTPase Promotes Breast Cancer Stem Cells and Tumorigenesis via Erk Signaling Activation

    Directory of Open Access Journals (Sweden)

    Man-Li Luo

    2015-04-01

    Full Text Available Proline-directed phosphorylation is regulated by the prolyl isomerase Pin1, which plays a fundamental role in driving breast cancer stem-like cells (BCSCs. Rab2A is a small GTPase critical for vesicle trafficking. Here, we show that Pin1 increases Rab2A transcription to promote BCSC expansion and tumorigenesis in vitro and in vivo. Mechanistically, Rab2A directly interacts with and prevents dephosphorylation/inactivation of Erk1/2 by the MKP3 phosphatase, resulting in Zeb1 upregulation and β-catenin nuclear translocation. In cancer cells, Rab2A is activated via gene amplification, mutation or Pin1 overexpression. Rab2A overexpression or mutation endows BCSC traits to primary normal human breast epithelial cells, whereas silencing Rab2A potently inhibits the expansion and tumorigenesis of freshly isolated BCSCs. Finally, Rab2A overexpression correlates with poor clinical outcome in breast cancer patients. Thus, Pin1/Rab2A/Erk drives BCSC expansion and tumorigenicity, suggesting potential drug targets.

  6. A Rab11A/myosin Vb/Rab11-FIP2 complex frames two late recycling steps of langerin from the ERC to the plasma membrane.

    Science.gov (United States)

    Gidon, Alexandre; Bardin, Sabine; Cinquin, Bertrand; Boulanger, Jerome; Waharte, François; Heliot, Laurent; de la Salle, Henri; Hanau, Daniel; Kervrann, Charles; Goud, Bruno; Salamero, Jean

    2012-06-01

    A large body of knowledge relating to the constitution of Rab GTPase/Rab effector complexes and their impact on both membrane domain organization and overall membrane trafficking has been built up in recent years. However in the context of the live cell there are still many questions that remain to be answered, such as where and when these complexes assemble and where they perform their primary function(s). We describe here the dynamic processes that take place in the final steps of the Rab11A dependent recycling pathway, in the context of the membrane platform constituted by Myosin Vb, Rab11A, and Rab11-FIP2. We first confirm that a series of previously reported observations obtained during the study of a number of trafficking cargoes also apply to langerin. Langerin is a cargo molecule that traffics through Rab11A-positive membrane domains of the endosomal recycling pathway. In order to explore the relative dynamics of this set of partners, we make extensive use of a combinatory approach of Live-FRET, fast FRAP video, fast confocal and TIRF microscopy modalities. Our data show that the Myosin Vb/Rab11A/Rab11-FIP2 platform is spatially involved in the regulation of langerin trafficking at two distinct sites within live cells, first at the sorting site in the endosomal recycling compartment (ERC) where transport vesicles are formed, and subsequently, in a strict time-defined order, at the very late stage of docking/tethering and fusion of these langerin recycling vesicles to the plasma membrane. © 2012 John Wiley & Sons A/S.

  7. The Rab GTPase Rab8 as a shared regulator of ciliogenesis and immune synapse assembly: From a conserved pathway to diverse cellular structures.

    Science.gov (United States)

    Patrussi, Laura; Baldari, Cosima T

    2016-01-01

    Rab GTPases, which form the largest branch of the Ras GTPase superfamily, regulate almost every step of vesicle-mediated trafficking. Among them, Rab8 is an essential participant in primary cilium formation. In a report recently published in the Journal of Cell Science, Finetti and colleagues identify Rab8 as a novel player in vesicular traffic in the non-ciliated T lymphocytes, which contributes to the assembly of the specialized signaling platform known as the immune synapse. By interacting with the v-SNARE VAMP-3, Rab8 is indeed responsible for the final docking/fusion step in T cell receptor (TCR) recycling to the immune synapse. A second important take-home message which comes to light from this work is that VAMP-3 also interacts with Rab8 at the base of the cilium in NIH-3T3 cells, where it regulates ciliary growth and targeting of Smoothened at the plasma membrane. Hence the data presented in this report, in addition to identifying Rab8 as a novel player in vesicular traffic to the immune synapse, reveal how both ciliated and non-ciliated cells take advantage of a conserved pathway to build highly specific cellular structures.

  8. Les clients des services d'escorte tels que perçus par des femmes offrant ces services

    Directory of Open Access Journals (Sweden)

    Jacqueline

    2015-07-01

    Full Text Available La discussion de projets de loi criminalisant l'achat des services sexuels constitue un sujet brûlant d'actualité dans plusieurs pays, dont le Canada. Pourtant, bon nombre d'études indiquent que la plupart des clients agissent de manière civilisée envers la personne travailleuse du sexe, alors que seule une infime minorité de clients présentent des comportements violents. Interviewant 16 femmes offrant des services d'escorte, quant à leur expérience de sexualité dans le cadre du travail, celles-ci ont beaucoup discuté des besoins qu'elles percevaient chez leurs clients, de même que des attitudes de ces derniers envers elles. Les participantes décrivent ceux-ci comme étant le plus souvent respectueux, la plupart recherchant un contact chaleureux avec elle et agissant en conséquence. En outre, ceux des clients qui ne recherchent qu'une expérience sexuelle tendent à être également respectueux envers l'escorte. Par contre, certains clients présentent une attitude ou un comportement désagréable. Quelques participantes ont également eu affaire à un client violent au cours de leur parcours comme escorte. La plupart des participantes étaient en mesure de choisir leurs clients, ce qui leur a permis de surtout rencontrer des clients aimables et qui, à l'occasion, leur procure également du plaisir sexuel. Par contre, les participantes plus vulnérables et moins capables de choisir et de maintenir leurs limites ont rencontré plus de clients désagréables ou agressifs.

  9. Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase

    Directory of Open Access Journals (Sweden)

    Isabelle Bouchez

    2015-07-01

    Full Text Available It has now been clearly shown that lipid droplets (LDs play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+ ATPase (V-ATPase. Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

  10. The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28.

    Science.gov (United States)

    Pla, M; Vilardell, J; Guiltinan, M J; Marcotte, W R; Niogret, M F; Quatrano, R S; Pagès, M

    1993-01-01

    The maize gene rab28 has been identified as ABA-inducible in embryos and vegetative tissues. It is also induced by water stress in young leaves. The proximal promoter region contains the conserved cis-acting element CCACGTGG (ABRE) reported for ABA induction in other plant genes. Transient expression assays in rice protoplasts indicate that a 134 bp fragment (-194 to -60 containing the ABRE) fused to a truncated cauliflower mosaic virus promoter (35S) is sufficient to confer ABA-responsiveness upon the GUS reporter gene. Gel retardation experiments indicate that nuclear proteins from tissues in which the rab28 gene is expressed can interact specifically with this 134 bp DNA fragment. Nuclear protein extracts from embryo and water-stressed leaves generate specific complexes of different electrophoretic mobility which are stable in the presence of detergent and high salt. However, by DMS footprinting the same guanine-specific contacts with the ABRE in both the embryo and leaf binding activities were detected. These results indicate that the rab28 promoter sequence CCACGTGG is a functional ABA-responsive element, and suggest that distinct regulatory factors with apparent similar affinity for the ABRE sequence may be involved in the hormone action during embryo development and in vegetative tissues subjected to osmotic stress.

  11. RosettaAntibodyDesign (RAbD): A general framework for computational antibody design.

    Science.gov (United States)

    Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Weitzner, Brian D; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R; Dunbrack, Roland L

    2018-04-01

    A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228-256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody-antigen complexes, using two design strategies-optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the native

  12. RosettaAntibodyDesign (RAbD): A general framework for computational antibody design

    Science.gov (United States)

    Adolf-Bryfogle, Jared; Kalyuzhniy, Oleks; Kubitz, Michael; Hu, Xiaozhen; Adachi, Yumiko; Schief, William R.

    2018-01-01

    A structural-bioinformatics-based computational methodology and framework have been developed for the design of antibodies to targets of interest. RosettaAntibodyDesign (RAbD) samples the diverse sequence, structure, and binding space of an antibody to an antigen in highly customizable protocols for the design of antibodies in a broad range of applications. The program samples antibody sequences and structures by grafting structures from a widely accepted set of the canonical clusters of CDRs (North et al., J. Mol. Biol., 406:228–256, 2011). It then performs sequence design according to amino acid sequence profiles of each cluster, and samples CDR backbones using a flexible-backbone design protocol incorporating cluster-based CDR constraints. Starting from an existing experimental or computationally modeled antigen-antibody structure, RAbD can be used to redesign a single CDR or multiple CDRs with loops of different length, conformation, and sequence. We rigorously benchmarked RAbD on a set of 60 diverse antibody–antigen complexes, using two design strategies—optimizing total Rosetta energy and optimizing interface energy alone. We utilized two novel metrics for measuring success in computational protein design. The design risk ratio (DRR) is equal to the frequency of recovery of native CDR lengths and clusters divided by the frequency of sampling of those features during the Monte Carlo design procedure. Ratios greater than 1.0 indicate that the design process is picking out the native more frequently than expected from their sampled rate. We achieved DRRs for the non-H3 CDRs of between 2.4 and 4.0. The antigen risk ratio (ARR) is the ratio of frequencies of the native amino acid types, CDR lengths, and clusters in the output decoys for simulations performed in the presence and absence of the antigen. For CDRs, we achieved cluster ARRs as high as 2.5 for L1 and 1.5 for H2. For sequence design simulations without CDR grafting, the overall recovery for the

  13. Rab GTPases and the Autophagy Pathway: Bacterial Targets for a Suitable Biogenesis and Trafficking of Their Own Vacuoles

    Directory of Open Access Journals (Sweden)

    María Milagros López de Armentia

    2016-03-01

    Full Text Available Autophagy is an intracellular process that comprises degradation of damaged organelles, protein aggregates and intracellular pathogens, having an important role in controlling the fate of invading microorganisms. Intracellular pathogens are internalized by professional and non-professional phagocytes, localizing in compartments called phagosomes. To degrade the internalized microorganism, the microbial phagosome matures by fusion events with early and late endosomal compartments and lysosomes, a process that is regulated by Rab GTPases. Interestingly, in order to survive and replicate in the phagosome, some pathogens employ different strategies to manipulate vesicular traffic, inhibiting phagolysosomal biogenesis (e.g., Staphylococcus aureus and Mycobacterium tuberculosis or surviving in acidic compartments and forming replicative vacuoles (e.g., Coxiella burnetti and Legionella pneumophila. The bacteria described in this review often use secretion systems to control the host’s response and thus disseminate. To date, eight types of secretion systems (Type I to Type VIII are known. Some of these systems are used by bacteria to translocate pathogenic proteins into the host cell and regulate replicative vacuole formation, apoptosis, cytokine responses, and autophagy. Herein, we have focused on how bacteria manipulate small Rab GTPases to control many of these processes. The growing knowledge in this field may facilitate the development of new treatments or contribute to the prevention of these types of bacterial infections.

  14. Isolation and functional characterisation of two new bZIP maize regulators of the ABA responsive gene rab28.

    Science.gov (United States)

    Nieva, Claudia; Busk, Peter K; Domínguez-Puigjaner, Eva; Lumbreras, Victoria; Testillano, Pilar S; Risueño, Maria-Carmen; Pagès, Montserrat

    2005-08-01

    The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

  15. A Bacterial Pathogen Targets a Host Rab-Family GTPase Defense Pathway with a GAP.

    Science.gov (United States)

    Spanò, Stefania; Gao, Xiang; Hannemann, Sebastian; Lara-Tejero, María; Galán, Jorge E

    2016-02-10

    Cell-autonomous defense mechanisms are potent strategies that protect individual cells against intracellular pathogens. The Rab-family GTPase Rab32 was previously shown to restrict the intracellular human pathogen Salmonella Typhi, but its potential broader role in antimicrobial defense remains unknown. We show that Rab32 represents a general cell-autonomous, antimicrobial defense that is counteracted by two Salmonella effectors. Mice lacking Rab-32 or its nucleotide exchange factor BLOC-3 are permissive to S. Typhi infection and exhibit increased susceptibility to S. Typhimurium. S. Typhimurium counters this defense pathway by delivering two type III secretion effectors, SopD2, a Rab32 GAP, and GtgE, a specific Rab32 protease. An S. Typhimurium mutant strain lacking these two effectors exhibits markedly reduced virulence, which is fully restored in BLOC-3-deficient mice. These results demonstrate that a cell-autonomous, Rab32-dependent host defense pathway plays a central role in the defense against vacuolar pathogens and describe a mechanism evolved by a bacterial pathogen to counter it. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. A comparison of male sex workers in Prague: Internet escorts versus men who work in specialized bars and clubs.

    Science.gov (United States)

    Bar-Johnson, Michael David; Weiss, Petr

    2015-01-01

    Prague, the Czech Republic, is a popular sex tourism destination where sex work is decriminalized and young men offer sexual services at low prices relative to countries in Western Europe. This quantitative survey aimed to identify some of the demographic characteristics of these young men and their experiences in the sex industry. Internet escorts (N = 20) and sex workers in bars and clubs (N = 20) completed the survey anonymously in spring 2011. The results showed that sex workers in clubs often had troubled pasts and were forced into sex work to survive. They also reported incidents of violence, serious alcohol and drug use, as well as frequent gambling. The larger group of sex workers in Prague is made up of Internet escorts who have backgrounds that are not atypical for the average Czech youth. They had fewer problems with drugs and alcohol but were twice as likely as sex workers in bars and clubs to be victims of violent crime. Plans for interventions to help those who would change their line of work, as well as the importance of sociocultural context in understanding sex workers, are discussed.

  17. Rab9-dependent autophagy is required for the IGF-IIR triggering mitophagy to eliminate damaged mitochondria.

    Science.gov (United States)

    Huang, Chih-Yang; Kuo, Wei-Wen; Ho, Tsung-Jung; Chiang, Shu-Fen; Pai, Pei-Ying; Lin, Jing-Ying; Lin, Ding-Yu; Kuo, Chia-Hua; Huang, Chih-Yang

    2018-03-25

    Mitochondria dysfunction is the major characteristic of mitophagy, which is essential in mitochondrial quality control. However, excessive mitophagy contributes to cell death in a number of diseases, including ischemic stroke and hepatotoxicity. Insulin-like growth factor II (IGF-II) and its receptor (IGF-IIR) play vital roles in the development of heart failure during hypertension. We found that IGF-II triggers IGF-IIR receptor activation, causing mitochondria dysfunction, resulting in mitophagy, and cardiomyocyte cell death. These results indicated that IGF-IIR activation triggers mitochondria fragmentation, leading to autophagosome formation, and loss of mitochondria content. These results are associated with Parkin-dependent mitophagy. Additionally, autophagic proteins Atg5, and Atg7 deficiency did not suppress IGF-IIR-induced mitophagy. However, Rab9 knockdown reduced mitophagy and maintained mitochondrial function. These constitutive mitophagies through IGF-IIR activation trigger mitochondria loss and mitochondrial ROS accumulation for cardiomyocyte viability decrease. Together, our results indicate that IGF-IIR predominantly induces mitophagy through the Rab9-dependent alternative autophagy. © 2018 Wiley Periodicals, Inc.

  18. C9orf72’s interaction with Rab GTPases - modulation of membrane traffic and autophagy

    Directory of Open Access Journals (Sweden)

    Bor Luen Tang

    2016-10-01

    Full Text Available Hexanucleotide repeat expansion in an intron of Chromosome 9 open reading frame 72 (C9orf72 is the most common genetic cause of Amyotrophic Lateral Sclerosis (ALS and Frontotemporal Dementia (FTD. While functional haploinsufficiency of C9orf72 resulting from the mutation may play a role in ALS/FTD, the actual cellular role of the protein has been unclear. Recent findings have now shown that C9orf72 physically and functionally interacts with multiple members of the Rab small GTPases family, consequently exerting important influences on cellular membrane traffic and the process of autophagy. Loss of C9orf72 impairs endocytosis in neuronal cell lines, and attenuated autophagosome formation. Interestingly, C9orf72 could influence autophagy both as part of a Guanine nucleotide exchange factor (GEF complex, or as a Rab effector that facilitates transport of the Unc-51-like Autophagy Activating Kinase 1 (Ulk1 autophagy initiation complex. The cellular function of C9orf72 is discussed in the light of these recent findings

  19. 78 FR 19103 - Safety Zone; Spanish Navy School Ship San Sebastian El Cano Escort; Bahia de San Juan; San Juan, PR

    Science.gov (United States)

    2013-03-29

    ...-AA00 Safety Zone; Spanish Navy School Ship San Sebastian El Cano Escort; Bahia de San Juan; San Juan... temporary moving safety zone on the waters of Bahia de San Juan during the transit of the Spanish Navy... Channel entrance, and to protect the high ranking officials on board the Spanish Navy School Ship San...

  20. Ectopic expression of a vesicle trafficking gene, OsRab7, from Oryza ...

    African Journals Online (AJOL)

    Jane

    2011-07-18

    Jul 18, 2011 ... OsRab7, from Oryza sativa, confers tolerance to several ... Key Laboratory of Molecular Biology and Gene Engineering, College of Life Science, Nanchang University, ... The reverse transcription was performed using MMLV.

  1. Salmonella Disrupts Host Endocytic Trafficking by SopD2-Mediated Inhibition of Rab7

    Directory of Open Access Journals (Sweden)

    Vanessa M. D’Costa

    2015-09-01

    Full Text Available Intracellular bacterial pathogens of a diverse nature share the ability to evade host immunity by impairing trafficking of endocytic cargo to lysosomes for degradation, a process that is poorly understood. Here, we show that the Salmonella enterica type 3 secreted effector SopD2 mediates this process by binding the host regulatory GTPase Rab7 and inhibiting its nucleotide exchange. Consequently, this limits Rab7 interaction with its dynein- and kinesin-binding effectors RILP and FYCO1 and thereby disrupts host-driven regulation of microtubule motors. Our study identifies a bacterial effector capable of directly binding and thereby modulating Rab7 activity and a mechanism of endocytic trafficking disruption that may provide insight into the pathogenesis of other bacteria. Additionally, we provide a powerful tool for the study of Rab7 function, and a potential therapeutic target.

  2. Smart I’rab: Smart Aplicasion for Arabic Grammar Learning

    Directory of Open Access Journals (Sweden)

    Syd. Ali Zein Farmadi

    2013-12-01

    Full Text Available Arabic grammar, known as nahwu, is necessary to comprehend the Holy Qur’an that is completely written in Arabic. However, many people get trouble to study this skill because there are various kinds of word formation and sentences that may be created from a single verb, noun, adjective, subject, predicate, object, adverb or another formation. This research proposes a new approach to identify the position and word function in Arabic sentence. The approach creates smart process that employs Natural Language Processing (NLP and expert system with modeling based on knowledge and inference engine in determining the word position. The knowledge base determines the part of speech while the inference engine shows the word function in the sentence. On processing, the system uses 82 templates consisting of 34 verb templates, 34 subject pronouns, 14 pronouns for object or possessive word. All the templates are in the form of char array for harakat (vowel and letters which become the comparators for determining the part of speech from input word sentence. Output from the system is an i’rab (the explanation of word function in sentence written in Arabic. The system has been tested for 159 times to examine word and sentence. The examination for word that is done 117 times has not made any error except for the word that is really like another word. While the detection for word function in sentence that is done 42 times experiment, there is no error too. An error happens when the part of speech from the word being examined is not included in the system yet, influencing the following word function detection. Keywords: I’rab, Arabic grammar, NLP, expert system, knowledge base, inference engine

  3. 阴道毛滴虫Rab1a重组蛋白的表达和细胞内定位%Expression of Recombinant TvRab1a and Intracellular Location of TvRab1a in Trichomonas vaginalis

    Institute of Scientific and Technical Information of China (English)

    徐晓园; 傅玉才; 许铭炎; 许锦阶; 张仁利

    2006-01-01

    目的 制备阴道毛滴虫(Trichomonas vaginalis,Tv)Rab1a重组蛋白及其多克隆抗体,并对TvRab1a蛋白进行阴道毛滴虫的细胞内定位.方法 将我们已构建的pQE80L/TvRab1a重组表达质粒转入大肠埃希菌E.coli M15,在IPTG诱导下表达重组蛋白,经Ni-NTA亲和柱层析后获得纯度较高的TvRab1a的重组蛋白;用经复性处理的重组蛋白免疫动物,获得TvRab1a重组蛋白抗血清,免疫印迹Western Blot鉴定抗血清.采用荧光免疫细胞化学对TvRab1a蛋白进行细胞内定位.结果 Western Blot分析显示,TvRab1a重组蛋白可与豚鼠的抗TvRab1a血清反应,同时该抗血清在滴虫提取蛋白中检测到与预测TvRab1a分子量一致的条带;免疫荧光化学检测发现TvRab1a分布于细胞核周围的高尔基复合体与内质网.结论 获得的抗TvRab1a蛋白的多抗血清可用于TvRab1a基因功能的研究;TvRab1a功能场所位于高尔基复合体与内质网.

  4. BLOC-3 mutated in Hermansky-Pudlak syndrome is a Rab32/38 guanine nucleotide exchange factor.

    Science.gov (United States)

    Gerondopoulos, Andreas; Langemeyer, Lars; Liang, Jin-Rui; Linford, Andrea; Barr, Francis A

    2012-11-20

    Hermansky-Pudlak syndrome (HPS) is a human disease characterized by partial loss of pigmentation and impaired blood clotting. These symptoms are caused by defects in the biogenesis of melanosomes and platelet dense granules, often referred to as lysosome-related organelles. Genes mutated in HPS encode subunits of the biogenesis of lysosome-related organelles complexes (BLOCs). BLOC-1 and BLOC-2, together with the AP-3 clathrin adaptor complex, act at early endosomes to sort components required for melanin formation and melanosome biogenesis away from the degradative lysosomal pathway toward early stage melanosomes. However the molecular functions of the Hps1-Hps4 complex BLOC-3 remain mysterious. Like other trafficking pathways, melanosome biogenesis and transport of enzymes involved in pigmentation involves specific Rab GTPases, in this instance Rab32 and Rab38. We now demonstrate that BLOC-3 is a Rab32 and Rab38 guanine nucleotide exchange factor (GEF). Silencing of the BLOC-3 subunits Hps1 and Hps4 results in the mislocalization of Rab32 and Rab38 and reduction in pigmentation. In addition, we show that BLOC-3 can promote specific membrane recruitment of Rab32/38. BLOC-3 therefore defines a novel Rab GEF family with a specific function in the biogenesis of lysosome-related organelles. Copyright © 2012 Elsevier Ltd. All rights reserved.

  5. Rab3A Inhibition of Ca2+ -Dependent Dopamine Release From PC12 Cells Involves Interaction With Synaptotagmin I.

    Science.gov (United States)

    Dai, Zhipan; Tang, Xia; Chen, Jia; Tang, Xiaochao; Wang, Xianchun

    2017-11-01

    Rab3 and synaptotagmin have been suggested to play important roles in the regulation of neurotransmitter release and, however, the molecular mechanism has not been completely clear. Here, we studied the effects of Rab3A and synaptotagmin I (Syt I) on dopamine release using PC12 cells as a model system. Rab3A was demonstrated to have effects on both Ca 2+ -independent and Ca 2+ -dependent dopamine releases from the PC12 cells. Application of Rab3A (up to 2500 nM) gradually decreased the amount of Ca 2+ -dependently released dopamine, indicating that Rab3A is a negative modulator that was further supported by the increase in dopamine release caused by Rab3A knockdown. Syt I knockdown weakened the Ca 2+ -dependent dopamine release, suggesting that Syt I plays a positive regulatory role in the cellular process. Treatment of the Syt I-knocked down PC12 cells with Rab3A further decreased Ca 2+ -dependent dopamine release and, however, the decrease magnitude was significantly reduced compared with that before Syt I knockdown, thus for the first time demonstrating that the inhibitory effect of Rab3A on Ca 2+ -dependent dopamine release involves the interaction with Syt I. This work has shed new light on the molecular mechanism for Rab3 and synaptotamin regulation of neurotransmitter release. J. Cell. Biochem. 118: 3696-3705, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  6. 46-kDa protein located in the flagellar pocket of Leishmania ...

    Indian Academy of Sciences (India)

    NII

    Cloning and expression of endocytic Rab GTPases from Leishmania. Fractionation of early compartment from. Leishmania containing endocytic probes. Rab7:WT. Rab5: WT. Localization of Rab5 and Rab7 in Leishmania. Phase. LTR. Merge. Rab7-GFP. Rab5-GFP. LTR. Phase. Merge. Rab5-GFP. Rab7-GFP. Lysosomes.

  7. Retinal gene therapy in patients with choroideremia: initial findings from a phase 1/2 clinical trial

    NARCIS (Netherlands)

    Maclaren, R.E.; Groppe, M.; Barnard, A.R.; Cottriall, C.L.; Tolmachova, T.; Seymour, L.; Clark, K.; During, M.J.; Cremers, F.P.M.; Black, G.C.M.; Lotery, A.J.; Downes, S.M.; Webster, A.R.; Seabra, M.C.

    2014-01-01

    BACKGROUND: Choroideremia is an X-linked recessive disease that leads to blindness due to mutations in the CHM gene, which encodes the Rab escort protein 1 (REP1). We assessed the effects of retinal gene therapy with an adeno-associated viral (AAV) vector encoding REP1 (AAV.REP1) in patients with

  8. Evaluating algal growth performance and water use efficiency of pilot-scale revolving algal biofilm (RAB) culture systems.

    Science.gov (United States)

    Gross, Martin; Mascarenhas, Vernon; Wen, Zhiyou

    2015-10-01

    A Revolving Algal Biofilm (RAB) growth system in which algal cells are attached to a flexible material rotating between liquid and gas phases has been developed. In this work, different configurations of RAB systems were developed at pilot-scale by retrofitting the attachment materials to a raceway pond (2000-L with 8.5 m(2) footprint area) and a trough reservoir (150 L with 3.5 m(2) footprint area). The algal growth performance and chemical composition, as well as the water evaporative loss and specific water consumption were evaluated over a period of nine months in a greenhouse environment near Boone, Iowa USA. Additionally a raceway pond was run in parallel, which served as a control. On average the raceway-based RAB and the trough-based RAB outperformed the control pond by 309% and 697%, respectively. A maximum productivity of 46.8 g m(-2) day(-1) was achieved on the trough-based RAB system. The evaporative water loss of the RAB system was modeled based on an energy balance analysis and was experimentally validated. While the RAB system, particularly the trough-based RAB, had higher water evaporative loss, the specific water consumption per unit of biomass produced was only 26% (raceway-based RAB) and 7% (trough-based RAB) of that of the control pond. Collectively, this research shows that the RAB system is an efficient algal culture system and has great potential to commercially produce microalgae with high productivity and efficient water use. © 2015 Wiley Periodicals, Inc.

  9. PKN1 Directs Polarized RAB21 Vesicle Trafficking via RPH3A and Is Important for Neutrophil Adhesion and Ischemia-Reperfusion Injury

    Directory of Open Access Journals (Sweden)

    Qianying Yuan

    2017-06-01

    Full Text Available Polarized vesicle transport plays an important role in cell polarization, but the mechanisms underlying this process and its role in innate immune responses are not well understood. Here, we describe a phosphorylation-regulated polarization mechanism that is important for neutrophil adhesion to endothelial cells during inflammatory responses. We show that the protein kinase PKN1 phosphorylates RPH3A, which enhances binding of RPH3A to guanosine triphosphate (GTP-bound RAB21. These interactions are important for polarized localization of RAB21 and RPH3A in neutrophils, which leads to PIP5K1C90 polarization. Consistent with the roles of PIP5K1C90 polarization, the lack of PKN1 or RPH3A impairs neutrophil integrin activation, adhesion to endothelial cells, and infiltration in inflammatory models. Furthermore, myeloid-specific loss of PKN1 decreases tissue injury in a renal ischemia-reperfusion model. Thus, this study characterizes a mechanism for protein polarization in neutrophils and identifies a potential protein kinase target for therapeutic intervention in reperfusion-related tissue injury.

  10. Conservation of the TRAPPII-specific subunits of a Ypt/Rab exchanger complex

    Directory of Open Access Journals (Sweden)

    Yoo Eunice

    2007-02-01

    Full Text Available Abstract Background Ypt/Rab GTPases and their GEF activators regulate intra-cellular trafficking in all eukaryotic cells. In S. cerivisiae, the modular TRAPP complex acts as a GEF for the Golgi gatekeepers: Ypt1 and the functional pair Ypt31/32. While TRAPPI, which acts in early Golgi, is conserved from fungi to animals, not much is known about TRAPPII, which acts in late Golgi and consists of TRAPPI plus three additional subunits. Results Here, we show a phylogenetic analysis of the three TRAPPII-specific subunits. One copy of each of the two essential subunits, Trs120 and Trs130, is present in almost every fully sequenced eukaryotic genome. Moreover, the primary, as well as the predicted secondary, structure of the Trs120- and Trs130-related sequences are conserved from fungi to animals. The mammalian orthologs of Trs120 and Trs130, NIBP and TMEM1, respectively, are candidates for human disorders. Currently, NIBP is implicated in signaling, and TMEM1 is suggested to have trans-membrane domains (TMDs and to function as a membrane channel. However, we show here that the yeast Trs130 does not function as a trans-membrane protein, and the human TMEM1 does not contain putative TMDs. The non-essential subunit, Trs65, is conserved only among many fungi and some unicellular eukaryotes. Multiple alignment analysis of each TRAPPII-specific subunit revealed conserved domains that include highly conserved amino acids. Conclusion We suggest that the function of both NIBP and TMEM1 in the regulation of intra-cellular trafficking is conserved from yeast to man. The conserved domains and amino acids discovered here can be used for functional analysis that should help to resolve the differences in the assigned functions of these proteins in fungi and animals.

  11. Rab39a interacts with phosphatidylinositol 3-kinase and negatively regulates autophagy induced by lipopolysaccharide stimulation in macrophages.

    Directory of Open Access Journals (Sweden)

    Shintaro Seto

    Full Text Available Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K. Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34(th to 41(st in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.

  12. Cloning and Expression of A Rab1a Gene from Trichomonas vaginalis%阴道毛滴虫Rab1a基因的cDNA克隆及重组表达

    Institute of Scientific and Technical Information of China (English)

    徐晓园; 傅玉才; 许铭炎; 史咏梅; 刘红

    2006-01-01

    目的克隆和分析阴道毛滴虫(Trichomonas vaginalis,Tv)Rab1a基因,构建Rab1a基因表达重组载体并表达其融合蛋白,以进一步探讨其功能.方法提取阴道毛滴虫基因组DNA为模板,扩增TvRab1a基因,用pQE80L载体与TvRab1a cDNA克隆构建原核表达重组体并表达融合蛋白,纯化表达产物并由SDS-PAGE鉴定.结果在阴道毛滴虫cDNA文库克隆中发现了Rab1a基因,序列分析显示Rab1a基因的基因组DNA序列含有一个25 bp大小的内含子.成功构建了pQE/Rab1a原核表达重组体,并表达出预期大小的重组蛋白质.结论分析表明TvRab1a基因是阴道毛滴虫Rab1鸟苷三磷酸酶同源基因,它含有一个25 bp的内含子.获得了该基因的重组蛋白,将对TvRab1a基因的功能进行进一步研究.

  13. Rab23 is overexpressed in human astrocytoma and promotes cell migration and invasion through regulation of Rac1.

    Science.gov (United States)

    Wang, Minghao; Dong, Qianze; Wang, Yunjie

    2016-08-01

    Rab23 overexpression has been implicated in several human cancers. However, its biological roles and molecular mechanism in astrocytoma have not been elucidated. The aim of this study is to explore clinical significance and biological roles of Rab23 in astrocytoma. We observed negative Rab23 staining in normal astrocytes and positive staining in 39 out of 86 (45 %) astrocytoma specimens using immunohistochemistry. The positive rate of Rab23 was higher in grades III and IV (56.5 %, 26/46) than grades I + II astrocytomas (32.5 %, 13/40, p Rac1 activity. Treatment of transfected cells with a Rac1 inhibitor decreased Rac1 activity and invasion. In conclusion, Rab23 serves as an important oncoprotein in human astrocytoma by regulating cell invasion and migration through Rac1 activity.

  14. Control of MarRAB Operon in Escherichia coli via Autoactivation and Autorepression

    Science.gov (United States)

    Prajapat, Mahendra Kumar; Jain, Kirti; Saini, Supreet

    2015-01-01

    Choice of network topology for gene regulation has been a question of interest for a long time. How do simple and more complex topologies arise? In this work, we analyze the topology of the marRAB operon in Escherichia coli, which is associated with control of expression of genes associated with conferring resistance to low-level antibiotics to the bacterium. Among the 2102 promoters in E. coli, the marRAB promoter is the only one that encodes for an autoactivator and an autorepressor. What advantages does this topology confer to the bacterium? In this work, we demonstrate that, compared to control by a single regulator, the marRAB regulatory arrangement has the least control cost associated with modulating gene expression in response to environmental stimuli. In addition, the presence of dual regulators allows the regulon to exhibit a diverse range of dynamics, a feature that is not observed in genes controlled by a single regulator. PMID:26445450

  15. Golgi-associated Rab14, a new regulator for Chlamydia trachomatis infection outcome.

    Science.gov (United States)

    Capmany, Anahí; Leiva, Natalia; Damiani, María Teresa

    2011-09-01

    Chlamydia trachomatis is the causing agent of the most frequent bacterial sexually-transmitted diseases worldwide and is an underlying cause of chronic pelvic inflammatory diseases and cervical cancer. It is an obligate intracellular bacterium that establishes a close relationship with the Golgi complex and parasites the biosynthetic machinery of host cells. In a recent study, we have demonstrated that Rab14, a newly-described Golgi-associated Rab, is involved in the delivery of sphingolipids to the growing bacteria-containing vacuole. The interference with Rab14-controlled trafficking pathways delays chlamydial inclusion enlargement, decreases bacterial lipid uptake, negatively impact on bacterial differentiation, and reduces bacterial progeny and infectivity. C. trachomatis manipulation of host trafficking pathways for the acquisition of endogenously-biosynthesized nutrients arises as one of the characteristics of this highly evolved pathogen. The development of therapeutic strategies targeted to interfere with bacterium-host cell interaction is a new challenge for pharmacological approaches to control chlamydial infections.

  16. LMTK1 regulates dendritic formation by regulating movement of Rab11A-positive endosomes.

    Science.gov (United States)

    Takano, Tetsuya; Urushibara, Tomoki; Yoshioka, Nozomu; Saito, Taro; Fukuda, Mitsunori; Tomomura, Mineko; Hisanaga, Shin-Ichi

    2014-06-01

    Neurons extend two types of neurites-axons and dendrites-that differ in structure and function. Although it is well understood that the cytoskeleton plays a pivotal role in neurite differentiation and extension, the mechanisms by which membrane components are supplied to growing axons or dendrites is largely unknown. We previously reported that the membrane supply to axons is regulated by lemur kinase 1 (LMTK1) through Rab11A-positive endosomes. Here we investigate the role of LMTK1 in dendrite formation. Down-regulation of LMTK1 increases dendrite growth and branching of cerebral cortical neurons in vitro and in vivo. LMTK1 knockout significantly enhances the prevalence, velocity, and run length of anterograde movement of Rab11A-positive endosomes to levels similar to those expressing constitutively active Rab11A-Q70L. Rab11A-positive endosome dynamics also increases in the cell body and growth cone of LMTK1-deficient neurons. Moreover, a nonphosphorylatable LMTK1 mutant (Ser34Ala, a Cdk5 phosphorylation site) dramatically promotes dendrite growth. Thus LMTK1 negatively controls dendritic formation by regulating Rab11A-positive endosomal trafficking in a Cdk5-dependent manner, indicating the Cdk5-LMTK1-Rab11A pathway as a regulatory mechanism of dendrite development as well as axon outgrowth. © 2014 Takano et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  17. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Tsukamoto, Yuta; Katayama, Chisako [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Shinohara, Miki; Shinohara, Akira [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Maekawa, Shohei [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Miyamoto, Masaaki, E-mail: miya@kobe-u.ac.jp [Graduate School of Science, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan); Center for Supports to Research and Education Activities, Kobe University, 1-1 Rokkodai-cho Nada, Kobe 657-8501 (Japan)

    2013-11-29

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions.

  18. The small GTPase Rab5 homologue Ypt5 regulates cell morphology, sexual development, ion-stress response and vacuolar formation in fission yeast

    International Nuclear Information System (INIS)

    Tsukamoto, Yuta; Katayama, Chisako; Shinohara, Miki; Shinohara, Akira; Maekawa, Shohei; Miyamoto, Masaaki

    2013-01-01

    Highlights: •Multiple functions of Rab5 GTPase in fission yeast were found. •Roles of Rab5 in fission yeast were discussed. •Relation between Rab5 and actin cytoskeleton were discussed. -- Abstract: Inner-membrane transport is critical to cell function. Rab family GTPases play an important role in vesicle transport. In mammalian cells, Rab5 is reported to be involved in the regulation of endosome formation, phagocytosis and chromosome alignment. Here, we examined the role of the fission yeast Rab5 homologue Ypt5 using a point mutant allele. Mutant cells displayed abnormal cell morphology, mating, sporulation, endocytosis, vacuole fusion and responses to ion stress. Our data strongly suggest that fission yeast Rab5 is involved in the regulation of various types of cellular functions

  19. A novel marRAB operon contributes to the rifampicin resistance in Mycobacterium smegmatis.

    Science.gov (United States)

    Zhang, Haiwei; Gao, Long; Zhang, Jiaoling; Li, Weihui; Yang, Min; Zhang, Hua; Gao, Chunhui; He, Zheng-Guo

    2014-01-01

    The multiple-antibiotic resistance regulator (MarR) plays an important role in modulating bacterial antibiotic resistance. However, the regulatory model of the marRAB operon in mycobacteria remains to be characterized. Here we report that a MarR, encoded by Ms6508, and its marRAB operon specifically contribute to rifampicin (RIF) resistance in Mycobacterium smegmatis. We show that the MarR recognizes a conserved 21-bp palindromic motif and negatively regulates the expression of two ABC transporters in the operon, encoded by Ms6509-6510. Unlike other known drug efflux pumps, overexpression of these two ABC transporters unexpectedly increased RIF sensitivity and deletion of these two genes increased mycobacterial resistance to the antibiotic. No change can be detected for the sensitivity of recombinant mycobacterial strains to three other anti-TB drugs. Furthermore, HPLC experiments suggested that Ms6509-Ms6510 could pump RIF into the mycobacterial cells. These findings indicated that the mycobacterial MarR functions as a repressor and constitutively inhibits the expression of the marRAB operon, which specifically contributes to RIF resistance in M. smegmatis. Therefore, our data suggest a new regulatory mechanism of RIF resistance and also provide the new insight into the regulatory model of a marRAB operon in mycobacteria.

  20. Roles of the Drosophila LRRK2 homolog in Rab7-dependent lysosomal positioning.

    Science.gov (United States)

    Dodson, Mark W; Zhang, Ting; Jiang, Changan; Chen, Shengdi; Guo, Ming

    2012-03-15

    LRRK2 (PARK8) is the most common genetic determinant of Parkinson's disease (PD), with dominant mutations in LRRK2 causing inherited PD and sequence variation at the LRRK2 locus associated with increased risk for sporadic PD. Although LRRK2 has been implicated in diverse cellular processes encompassing almost all cellular compartments, the precise functions of LRRK2 remain unclear. Here, we show that the Drosophila homolog of LRRK2 (Lrrk) localizes to the membranes of late endosomes and lysosomes, physically interacts with the crucial mediator of late endosomal transport Rab7 and negatively regulates rab7-dependent perinuclear localization of lysosomes. We also show that a mutant form of lrrk analogous to the pathogenic LRRK2(G2019S) allele behaves oppositely to wild-type lrrk in that it promotes rather than inhibits rab7-dependent perinuclear lysosome clustering, with these effects of mutant lrrk on lysosome position requiring both microtubules and dynein. These data suggest that LRRK2 normally functions in Rab7-dependent lysosomal positioning, and that this function is disrupted by the most common PD-causing LRRK2 mutation, linking endolysosomal dysfunction to the pathogenesis of LRRK2-mediated PD.

  1. RAB-like 2 has an essential role in male fertility, sperm intra-flagellar transport, and tail assembly.

    Directory of Open Access Journals (Sweden)

    Jennifer C Y Lo

    Full Text Available A significant percentage of young men are infertile and, for the majority, the underlying cause remains unknown. Male infertility is, however, frequently associated with defective sperm motility, wherein the sperm tail is a modified flagella/cilia. Conversely, a greater understanding of essential mechanisms involved in tail formation may offer contraceptive opportunities, or more broadly, therapeutic strategies for global cilia defects. Here we have identified Rab-like 2 (RABL2 as an essential requirement for sperm tail assembly and function. RABL2 is a member of a poorly characterized clade of the RAS GTPase superfamily. RABL2 is highly enriched within developing male germ cells, where it localizes to the mid-piece of the sperm tail. Lesser amounts of Rabl2 mRNA were observed in other tissues containing motile cilia. Using a co-immunoprecipitation approach and RABL2 affinity columns followed by immunochemistry, we demonstrated that within developing haploid germ cells RABL2 interacts with intra-flagella transport (IFT proteins and delivers a specific set of effector (cargo proteins, including key members of the glycolytic pathway, to the sperm tail. RABL2 binding to effector proteins is regulated by GTP. Perturbed RABL2 function, as exemplified by the Mot mouse line that contains a mutation in a critical protein-protein interaction domain, results in male sterility characterized by reduced sperm output, and sperm with aberrant motility and short tails. Our data demonstrate a novel function for the RABL protein family, an essential role for RABL2 in male fertility and a previously uncharacterised mechanism for protein delivery to the flagellum.

  2. PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic.

    Science.gov (United States)

    Deng, Bangli; Zhu, Xiaocui; Zhao, Yihe; Zhang, Da; Pannu, Alisha; Chen, Liming; Niu, Wenyan

    2018-01-08

    Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca 2+ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca 2+ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs.

    Science.gov (United States)

    Levine, Timothy P; Daniels, Rachel D; Gatta, Alberto T; Wong, Louise H; Hayes, Matthew J

    2013-02-15

    Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS, also called motor neuron disease, MND) are severe neurodegenerative diseases that show considerable overlap at the clinical and cellular level. The most common single mutation in families with FTD or ALS has recently been mapped to a non-coding repeat expansion in the uncharacterized gene C9ORF72. Although a plausible mechanism for disease is that aberrant C9ORF72 mRNA poisons splicing, it is important to determine the cellular function of C9ORF72, about which nothing is known. Sensitive homology searches showed that C9ORF72 is a full-length distant homologue of proteins related to Differentially Expressed in Normal and Neoplasia (DENN), which is a GDP/GTP exchange factor (GEF) that activates Rab-GTPases. Our results suggest that C9ORF72 is likely to regulate membrane traffic in conjunction with Rab-GTPase switches, and we propose to name the gene and its product DENN-like 72 (DENNL72).

  4. Understanding the drive to escort: a cross-sectional analysis examining parental attitudes towards children’s school travel and independent mobility

    Directory of Open Access Journals (Sweden)

    Mammen George

    2012-10-01

    Full Text Available Abstract Background The declining prevalence of Active School Transportation (AST has been accompanied by a decrease in independent mobility internationally. The objective of this study was to compare family demographics and AST related perceptions of parents who let their children walk unescorted to/from school to those parents who escort (walk and drive their children to/from school. By comparing these groups, insight was gained into how we may encourage greater AST and independent mobility in youth living in the Greater Toronto and Hamilton Area, Canada. Methods This study involved a cross-sectional design, using data from a self-reported questionnaire (n =1,016 that examined parental perceptions and attitudes regarding AST. A multinomial logistic regression analysis was used to explore the differences between households where children travelled independently to school or were escorted. Results Findings revealed that unescorted children were: significantly older, the families spoke predominantly English at home, more likely to live within one kilometer from school, and their parents agreed to a greater extent that they chose to reside in the current neighborhood in order for their child to walk to/from school. The parents of the escorted children worried significantly more about strangers and bullies approaching their child as well as the traffic volume around school. Conclusions From both a policy and research perspective, this study highlights the value of distinguishing between mode (i.e., walking or driving and travel independence. For policy, our findings highlight the need for planning decisions about the siting of elementary schools to include considerations of the impact of catchment size on how children get to/from school. Given the importance of age, distance, and safety issues as significant correlates of independent mobility, research and practice should focus on the development and sustainability of non-infrastructure programs

  5. Rac1-Rab11-FIP3 regulatory hub coordinates vesicle traffic with actin remodeling and T-cell activation.

    Science.gov (United States)

    Bouchet, Jérôme; Del Río-Iñiguez, Iratxe; Lasserre, Rémi; Agüera-Gonzalez, Sonia; Cuche, Céline; Danckaert, Anne; McCaffrey, Mary W; Di Bartolo, Vincenzo; Alcover, Andrés

    2016-06-01

    The immunological synapse generation and function is the result of a T-cell polarization process that depends on the orchestrated action of the actin and microtubule cytoskeleton and of intracellular vesicle traffic. However, how these events are coordinated is ill defined. Since Rab and Rho families of GTPases control intracellular vesicle traffic and cytoskeleton reorganization, respectively, we investigated their possible interplay. We show here that a significant fraction of Rac1 is associated with Rab11-positive recycling endosomes. Moreover, the Rab11 effector FIP3 controls Rac1 intracellular localization and Rac1 targeting to the immunological synapse. FIP3 regulates, in a Rac1-dependent manner, key morphological events, like T-cell spreading and synapse symmetry. Finally, Rab11-/FIP3-mediated regulation is necessary for T-cell activation leading to cytokine production. Therefore, Rac1 endosomal traffic is key to regulate T-cell activation. © 2016 The Authors.

  6. Deficient Rab11 activity underlies glucose hypometabolism in primary neurons of Huntington's disease mice

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xueyi, E-mail: xli12@partners.org [Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 (United States); Valencia, Antonio; McClory, Hollis; Sapp, Ellen; Kegel, Kimberly B. [Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 (United States); DiFiglia, Marian, E-mail: difiglia@helix.mgh.harvard.edu [Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129 (United States)

    2012-05-18

    Highlights: Black-Right-Pointing-Pointer Primary Huntington's disease neurons are impaired in taking up glucose. Black-Right-Pointing-Pointer Rab11 modulates glucose uptake in neurons. Black-Right-Pointing-Pointer Increasing Rab11 activity attenuates the glucose uptake defect in disease neurons. Black-Right-Pointing-Pointer We provide a novel mechanism for glucose hypometabolism in Huntington's disease. -- Abstract: Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a CAG repeat expansion in the huntingtin gene. Positron emission tomography studies have revealed a decline in glucose metabolism in the brain of patients with HD by a mechanism that has not been established. We examined glucose utilization in embryonic primary cortical neurons of wild-type (WT) and HD knock-in mice, which have 140 CAG repeats inserted in the endogenous mouse huntingtin gene (HD{sup 140Q/140Q}). Primary HD{sup 140Q/140Q} cortical neurons took up significantly less glucose than did WT neurons. Expression of permanently inactive and permanently active forms of Rab11 correspondingly altered glucose uptake in WT neurons, suggesting that normal activity of Rab11 is needed for neuronal uptake of glucose. It is known that Rab11 activity is diminished in HD{sup 140Q/140Q} neurons. Expression of dominant active Rab11 to enhance the activity of Rab11 normalized glucose uptake in HD{sup 140Q/140Q} neurons. These results suggest that deficient activity of Rab11 is a novel mechanism for glucose hypometabolism in HD.

  7. Essential role of RAB27A in determining constitutive human skin color.

    Directory of Open Access Journals (Sweden)

    Yasuko Yoshida-Amano

    Full Text Available Human skin color is predominantly determined by melanin produced in melanosomes within melanocytes and subsequently distributed to keratinocytes. There are many studies that have proposed mechanisms underlying ethnic skin color variations, whereas the processes involved from melanin synthesis in melanocytes to the transfer of melanosomes to keratinocytes are common among humans. Apart from the activities in the melanogenic rate-limiting enzyme, tyrosinase, in melanocytes and the amounts and distribution patterns of melanosomes in keratinocytes, the abilities of the actin-associated factors in charge of melanosome transport within melanocytes also regulate pigmentation. Mutations in genes encoding melanosome transport-related molecules, such as MYO5A, RAB27A and SLAC-2A, have been reported to cause a human pigmentary disease known as Griscelli syndrome, which is associated with diluted skin and hair color. Thus we hypothesized that process might play a role in modulating skin color variations. To address that hypothesis, the correlations of expression of RAB27A and its specific effector, SLAC2-A, to melanogenic ability were evaluated in comparison with tyrosinase, using human melanocytes derived from 19 individuals of varying skin types. Following the finding of the highest correlation in RAB27A expression to the melanogenic ability, darkly-pigmented melanocytes with significantly higher RAB27A expression were found to transfer significantly more melanosomes to keratinocytes than lightly-pigmented melanocytes in co-culture and in human skin substitutes (HSSs in vivo, resulting in darker skin color in concert with the difference observed in African-descent and Caucasian skins. Additionally, RAB27A knockdown by a lentivirus-derived shRNA in melanocytes concomitantly demonstrated a significantly reduced number of transferred melanosomes to keratinocytes in co-culture and a significantly diminished epidermal melanin content skin color intensity (

  8. Improving feedback from outpatient medical appointments attended by escorted psychiatric patients in the North London Forensic Service.

    Science.gov (United States)

    McCurdy, Kathleen; Croxford, Anna

    2015-01-01

    It is well established that patients with mental illness are known to have a high level of morbidity and mortality compared to the general population. This is particularly prominent in long-stay psychiatric patients, such as those in secure settings. The Royal College of Psychiatrists recommends that psychiatrists should promote the physical health of their patients and liaise with other specialties. However, there is evidence that communication between psychiatry and other specialties is poor. A survey was carried out at the North London Forensic Service in June 2014. This looked at the views of clinical staff about the frequency and quality of feedback obtained when inpatients attend outpatient hospital appointments at local general hospitals. This survey highlighted the general perception among staff that feedback is poor, with 68.43% of respondents saying that they were "very unsatisfied" or "unsatisfied" with the level and quality of feedback. Clinical staff felt that many patients who attended hospital outpatient appointments, even when escorted by staff, returned with little or no feedback. This was confirmed by a baseline audit across 3 wards showing that details of the appointment (date, time, hospital, and specialty) were only documented in 54.5% of cases and the content of the appointment documented in even fewer cases. A form was designed by junior doctors that provided a simple framework of 6 questions to be answered at the outpatient clinic about the problem, diagnosis, and further actions needed. This was introduced and its impact assessed with a 3-month and 6-month audit of electronic notes, as well as a follow-up survey after 6 months. The audit showed significant improvement in the quality of feedback about the appointment at both the 3-month and 6-month point. The follow-up survey showed that 70% of respondents were aware of the form and 100% of those who were aware of the form had used it at least once and found it helpful. The general

  9. Characterisation of SalRAB a salicylic acid inducible positively regulated efflux system of Rhizobium leguminosarum bv viciae 3841.

    Directory of Open Access Journals (Sweden)

    Adrian J Tett

    Full Text Available Salicylic acid is an important signalling molecule in plant-microbe defence and symbiosis. We analysed the transcriptional responses of the nitrogen fixing plant symbiont, Rhizobium leguminosarum bv viciae 3841 to salicylic acid. Two MFS-type multicomponent efflux systems were induced in response to salicylic acid, rmrAB and the hitherto undescribed system salRAB. Based on sequence similarity salA and salB encode a membrane fusion and inner membrane protein respectively. salAB are positively regulated by the LysR regulator SalR. Disruption of salA significantly increased the sensitivity of the mutant to salicylic acid, while disruption of rmrA did not. A salA/rmrA double mutation did not have increased sensitivity relative to the salA mutant. Pea plants nodulated by salA or rmrA strains did not have altered nodule number or nitrogen fixation rates, consistent with weak expression of salA in the rhizosphere and in nodule bacteria. However, BLAST analysis revealed seventeen putative efflux systems in Rlv3841 and several of these were highly differentially expressed during rhizosphere colonisation, host infection and bacteroid differentiation. This suggests they have an integral role in symbiosis with host plants.

  10. Rab27A mediated by NF-κB promotes the stemness of colon cancer cells via up-regulation of cytokine secretion.

    Science.gov (United States)

    Feng, Feixue; Jiang, Yinghao; Lu, Huanyu; Lu, Xiaozhao; Wang, Shan; Wang, Lifeng; Wei, Mengying; Lu, Wei; Du, Zhichao; Ye, Zichen; Yang, Guodong; Yuan, Fang; Ma, Yanxia; Lei, Xiaoying; Lu, Zifan

    2016-09-27

    Recent evidences have unveiled critical roles of cancer stem cells (CSCs) in tumorigenicity, but how interactions between CSC and tumor environments help maintain CSC initiation remains obscure. The small GTPases Rab27A regulates autocrine and paracrine cytokines by monitoring exocytosis of extracellular vesicles, and is reported to promote certain tumor progression. We observe that overexpression of Rab27A increased sphere formation efficiency (SFE) by increasing the proportion of CD44+ and PKH26high cells in HT29 cell lines, and accelerating the growth of colosphere with higher percentage of cells at S phase. Mechanism study revealed that the supernatant derived from HT29 sphere after Rab27A overexpression was able to expand sphere numbers with elevated secretion of VEGF and TGF-β. In tumor implanting nude mice model, tumor initiation rates and tumor sizes were enhanced by Rab27A with obvious angiogenesis. As a contrast, knocking down Rab27A impaired the above effects. More importantly, the correlation between higher p65 level and Rab27A in colon sphere was detected, p65 was sufficient to induce up-regulation of Rab27A and a functional NF-κB binding site in the Rab27A promoter was demonstrated. Altogether, our findings reveal a unique mechanism that tumor environment related NF-κB signaling promotes various colon cancer stem cells (cCSCs) properties via an amplified paracrine mechanism regulated by higher Rab27A level.

  11. MUC1 intra-cellular trafficking is clathrin, dynamin, and rab5 dependent

    International Nuclear Information System (INIS)

    Liu Xiaolong; Yuan Zhenglong; Chung, Maureen

    2008-01-01

    MUC1, a transmembrane glycoprotein, is abnormally over-expressed in most human adenocarcinomas. MUC1 association with cytoplasmic cell signal regulators and nuclear accumulation are important for its tumor related activities. Little is known about how MUC1 translocates from the cell membrane to the cytoplasm. In this study, live cell imaging was used to study MUC1 intracellular trafficking. The interaction between EGFR and MUC1 was mapped by FRET analysis and EGF stimulated MUC1 endocytosis was observed directly through live cell imaging. MUC1-CT endocytosis was clathrin and dynamin dependent. Rab5 over-expression resulted in decreased cell membrane localization of MUC1, with accumulation of MUC1 endocytic vesicles in the peri-nuclear region. Conversely, over-expression of a Rab5 dominant negative mutant (S34N) resulted in redistribution of MUC1 from the peri-nuclear region to the cytoplasm. Collectively, these results indicated that MUC1 intra-cellular trafficking occurs through a regulated process that was stimulated by direct EGFR and MUC1 interaction, mediated by clathrin coated pits that were dynamin dependent and regulated by Rab5

  12. The Rab7 effector PLEKHM1 binds Arl8b to promote cargo traffic to lysosomes.

    Science.gov (United States)

    Marwaha, Rituraj; Arya, Subhash B; Jagga, Divya; Kaur, Harmeet; Tuli, Amit; Sharma, Mahak

    2017-04-03

    Endocytic, autophagic, and phagocytic vesicles move on microtubule tracks to fuse with lysosomes. Small GTPases, such as Rab7 and Arl8b, recruit their downstream effectors to mediate this transport and fusion. However, the potential cross talk between these two GTPases is unclear. Here, we show that the Rab7 effector PLEKHM1 simultaneously binds Rab7 and Arl8b, bringing about clustering and fusion of late endosomes and lysosomes. We show that the N-terminal RUN domain of PLEKHM1 is necessary and sufficient for interaction with Arl8b and its subsequent localization to lysosomes. Notably, we also demonstrate that Arl8b mediates recruitment of HOPS complex to PLEKHM1-positive vesicle contact sites. Consequently, Arl8b binding to PLEKHM1 is required for its function in delivery and, therefore, degradation of endocytic and autophagic cargo in lysosomes. Finally, we also show that PLEKHM1 competes with SKIP for Arl8b binding, which dictates lysosome positioning. These findings suggest that Arl8b, along with its effectors, orchestrates lysosomal transport and fusion. © 2017 Marwaha et al.

  13. Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

    International Nuclear Information System (INIS)

    Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon; Nguyen, Hong-Hoa; Yang, Jun-Mo; Kang, Jong-Sun; Hahn, Myong-Joon

    2011-01-01

    Highlights: ► APPL1 regulates the protein level of EGFR in response to EGF stimulation. ► Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. ► Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation. Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.

  14. Over-expression of a Rab family GTPase from phreatophyte Prosopis juliflora confers tolerance to salt stress on transgenic tobacco.

    Science.gov (United States)

    George, Suja; Parida, Ajay

    2011-03-01

    Plant growth and productivity are adversely affected by various abiotic and biotic stress factors. In our previous study, we used Prosopis juliflora, an abiotic stress tolerant tree species of Fabaceae, as a model plant system for isolating genes functioning in abiotic stress tolerance. Here we report the isolation and characterization of a Rab family GTPase from P. juliflora (Pj Rab7) and the ability of this gene to confer salt stress tolerance in transgenic tobacco. Northern analysis for Pj Rab7 in P. juliflora leaf tissue revealed up-regulation of this gene under salt stress under the concentrations and time points analyzed. Pj Rab7 transgenic tobacco lines survived better under conditions of 150 mM NaCl stress compared to control un-transformed plants. Pj Rab7 transgenic plants were found to accumulate more sodium than control plants during salt stress. The results of our studies could be used as a starting point for generation of crop plants tolerant to abiotic stress.

  15. Subcellular localization analysis of the closely related Fps/Fes and Fer protein-tyrosine kinases suggests a distinct role for Fps/Fes in vesicular trafficking.

    Science.gov (United States)

    Zirngibl, R; Schulze, D; Mirski, S E; Cole, S P; Greer, P A

    2001-05-15

    The subcellular localizations of the Fps/Fes and closely related Fer cytoplasmic tyrosine kinases were studied using green fluorescent protein (GFP) fusions and confocal fluorescence microscopy. In contrast to previous reports, neither kinase localized to the nucleus. Fer was diffusely cytoplasmic throughout the cell cycle. Fps/Fes also displayed a diffuse cytoplasmic localization, but in addition it showed distinct accumulations in cytoplasmic vesicles as well as in a perinuclear region consistent with the Golgi. This localization was very similar to that of TGN38, a known marker of the trans Golgi. The localization of Fps/Fes and TGN38 were both perturbed by brefeldin A, a fungal metabolite that disrupts the Golgi apparatus. Fps/Fes was also found to colocalize to various extents with several Rab proteins, which are members of the monomeric G-protein superfamily involved in vesicular transport between specific subcellular compartments. Using Rabs that are involved in endocytosis (Rab5B and Rab7) or exocytosis (Rab1A and Rab3A), we showed that Fps/Fes is localized in both pathways. These results suggest that Fps/Fes may play a general role in the regulation of vesicular trafficking. Copyright 2001 Academic Press.

  16. Yarrowia lipolytica vesicle-mediated protein transport pathways

    Directory of Open Access Journals (Sweden)

    Beckerich Jean-Marie

    2007-11-01

    Full Text Available Abstract Background Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway. Results We identified S. cerevisiae proteins involved in vesicular secretion and these protein sequences were used for the BLAST searches against Génolevures protein database (Y. lipolytica, C. glabrata, K. lactis and D. hansenii. These proteins are well conserved between these yeasts and Saccharomyces cerevisiae. We note several specificities of Y. lipolytica which may be related to its good protein secretion capacities and to its dimorphic aspect. An expansion of the Y. lipolytica Rab protein family was observed with autoBLAST and the Rab2- and Rab4-related members were identified with BLAST against NCBI protein database. An expansion of this family is also found in filamentous fungi and may reflect the greater complexity of the Y. lipolytica secretion pathway. The Rab4p-related protein may play a role in membrane recycling as rab4 deleted strain shows a modification of colony morphology, dimorphic transition and permeability. Similarly, we find three copies of the gene (SSO encoding the plasma membrane SNARE protein. Quantification of the percentages of proteins with the greatest homology between S. cerevisiae, Y. lipolytica and animal homologues involved in vesicular

  17. [6]-Gingerol, from Zingiber officinale, potentiates GLP-1 mediated glucose-stimulated insulin secretion pathway in pancreatic β-cells and increases RAB8/RAB10-regulated membrane presentation of GLUT4 transporters in skeletal muscle to improve hyperglycemia in Leprdb/db type 2 diabetic mice.

    Science.gov (United States)

    Samad, Mehdi Bin; Mohsin, Md Nurul Absar Bin; Razu, Bodiul Alam; Hossain, Mohammad Tashnim; Mahzabeen, Sinayat; Unnoor, Naziat; Muna, Ishrat Aklima; Akhter, Farjana; Kabir, Ashraf Ul; Hannan, J M A

    2017-08-09

    [6]-Gingerol, a major component of Zingiber officinale, was previously reported to ameliorate hyperglycemia in type 2 diabetic mice. Endocrine signaling is involved in insulin secretion and is perturbed in db/db Type-2 diabetic mice. [6]-Gingerol was reported to restore the disrupted endocrine signaling in rodents. In this current study on Lepr db/db diabetic mice, we investigated the involvement of endocrine pathway in the insulin secretagogue activity of [6]-Gingerol and the mechanism(s) through which [6]-Gingerol ameliorates hyperglycemia. Lepr db/db type 2 diabetic mice were orally administered a daily dose of [6]-Gingerol (200 mg/kg) for 28 days. We measured the plasma levels of different endocrine hormones in fasting and fed conditions. GLP-1 levels were modulated using pharmacological approaches, and cAMP/PKA pathway for insulin secretion was assessed by qRT-PCR and ELISA in isolated pancreatic islets. Total skeletal muscle and its membrane fractions were used to measure glycogen synthase 1 level and Glut4 expression and protein levels. 4-weeks treatment of [6]-Gingerol dramatically increased glucose-stimulated insulin secretion and improved glucose tolerance. Plasma GLP-1 was found to be significantly elevated in the treated mice. Pharmacological intervention of GLP-1 levels regulated the effect of [6]-Gingerol on insulin secretion. Mechanistically, [6]-Gingerol treatment upregulated and activated cAMP, PKA, and CREB in the pancreatic islets, which are critical components of GLP-1-mediated insulin secretion pathway. [6]-Gingerol upregulated both Rab27a GTPase and its effector protein Slp4-a expression in isolated islets, which regulates the exocytosis of insulin-containing dense-core granules. [6]-Gingerol treatment improved skeletal glycogen storage by increased glycogen synthase 1 activity. Additionally, GLUT4 transporters were highly abundant in the membrane of the skeletal myocytes, which could be explained by the increased expression of Rab8 and Rab

  18. Functional Characterization of Rare RAB12 Variants and Their Role in Musician’s and Other Dystonias

    Directory of Open Access Journals (Sweden)

    Eva Hebert

    2017-10-01

    Full Text Available Mutations in RAB (member of the Ras superfamily genes are increasingly recognized as cause of a variety of disorders including neurological conditions. While musician’s dystonia (MD and writer’s dystonia (WD are task-specific movement disorders, other dystonias persistently affect postures as in cervical dystonia. Little is known about the underlying etiology. Next-generation sequencing revealed a rare missense variant (c.586A>G; p.Ile196Val in RAB12 in two of three MD/WD families. Next, we tested 916 additional dystonia patients; 512 Parkinson’s disease patients; and 461 healthy controls for RAB12 variants and identified 10 additional carriers of rare missense changes among dystonia patients (1.1% but only one carrier in non-dystonic individuals (0.1%; p = 0.005. The detected variants among index patients comprised p.Ile196Val (n = 6; p.Ala174Thr (n = 3; p.Gly13Asp; p.Ala148Thr; and p.Arg181Gln in patients with MD; cervical dystonia; or WD. Two relatives of MD patients with WD also carried p.Ile196Val. The two variants identified in MD patients (p.Ile196Val; p.Gly13Asp were characterized on endogenous levels in patient-derived fibroblasts and in two RAB12-overexpressing cell models. The ability to hydrolyze guanosine triphosphate (GTP, so called GTPase activity, was increased in mutants compared to wildtype. Furthermore, subcellular distribution of RAB12 in mutants was altered in fibroblasts. Soluble Transferrin receptor 1 levels were reduced in the blood of all three tested p.Ile196Val carriers. In conclusion, we demonstrate an enrichment of missense changes among dystonia patients. Functional characterization revealed altered enzyme activity and lysosomal distribution in mutants suggesting a contribution of RAB12 variants to MD and other dystonias.

  19. High RAB25 expression is associated with good clinical outcome in patients with locally advanced head and neck squamous cell carcinoma

    International Nuclear Information System (INIS)

    Téllez-Gabriel, Marta; Arroyo-Solera, Irene; León, Xavier; Gallardo, Alberto; López, Montserrat; Céspedes, Maria V; Casanova, Isolda; López-Pousa, Antonio; Quer, Miquel; Mangues, Maria A; Barnadas, Agustí; Mangues, Ramón; Pavón, Miguel A

    2013-01-01

    Currently there are no molecular markers able to predict clinical outcome in locally advanced head and neck squamous cell carcinoma (HNSCC). In a previous microarray study, RAB25 was identified as a potential prognostic marker. The aim of this study was to analyze the association between RAB25 expression and clinical outcome in patients with locally advanced HNSCC treated with standard therapy. In a retrospective immunohistochemical study (n = 97), we observed that RAB25-negative tumors had lower survival (log-rank, P = 0.01) than patients bearing positive tumors. In an independent prospective mRNA study (n = 117), low RAB25 mRNA expression was associated with poor prognosis. Using classification and regression tree analysis (CART) we established two groups of patients according to their RAB25 mRNA level and their risk of death. Low mRNA level was associated with poor local recurrence-free (log-rank, P = 0.005), progression-free (log-rank, P = 0.002) and cancer-specific (log-rank, P < 0.001) survival. Multivariate Cox model analysis showed that low expression of RAB25 was an independent poor prognostic factor for survival (hazard ratio: 3.84, 95% confidence interval: 1.93–7.62, P < 0.001). Patients whose tumors showed high RAB25 expression had a low probability of death after treatment. We also found lower RAB25 expression in tumors than in normal tissue (Mann–Whitney U, P < 0.001). Moreover, overexpression of RAB25 in the UM-SCC-74B HNSCC cell line increased cisplatin sensitivity, and reduced cell migration and invasion. Our findings support a tumor suppressor role for RAB25 in HNSCC and its potential use to identify locally advanced patients with a high probability of survival after genotoxic treatment

  20. Kinesin-73 is a processive motor that localizes to Rab5-containing organelles.

    Science.gov (United States)

    Huckaba, Thomas M; Gennerich, Arne; Wilhelm, James E; Chishti, Athar H; Vale, Ronald D

    2011-03-04

    Drosophila Kinesin-73 (Khc-73), which plays a role in mitotic spindle polarity in neuroblasts, is a metazoan-specific member of the Kinesin-3 family of motors, which includes mammalian KIF1A and Caenorhabditis elegans Unc-104. The mechanism of Kinesin-3 motors has been controversial because some studies have reported that they transport cargo as monomers whereas other studies have suggested a dimer mechanism. Here, we have performed single-molecule motility and cell biological studies of Khc-73. We find that constructs containing the motor and the conserved short stretches of putative coiled-coil-forming regions are predominantly monomeric in vitro, but that dimerization allows for fast, processive movement and high force production (7 piconewtons). In Drosophila cell lines, we present evidence that Khc-73 can dimerize in vivo. We also show that Khc-73 is recruited specifically to Rab5-containing endosomes through its "tail" domain. Our results suggest that the N-terminal half of Khc-73 can undergo a monomer-dimer transition to produce a fast processive motor and that its C-terminal half possesses a specific Rab5-vesicle binding domain.

  1. Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV) in Shrimp.

    Science.gov (United States)

    Ananphongmanee, Vorawit; Srisala, Jiraporn; Sritunyalucksana, Kallaya; Boonchird, Chuenchit

    2015-01-01

    Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7) and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV) could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1) promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7) and partial VP28 (pVP28) were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA) using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against WSSV by oral

  2. Yeast Surface Display of Two Proteins Previously Shown to Be Protective Against White Spot Syndrome Virus (WSSV in Shrimp.

    Directory of Open Access Journals (Sweden)

    Vorawit Ananphongmanee

    Full Text Available Cell surface display using the yeasts Saccharomyces cerevisiae and Pichia pastoris has been extensively developed for application in bioindustrial processes. Due to the rigid structure of their cell walls, a number of proteins have been successfully displayed on their cell surfaces. It was previously reported that the viral binding protein Rab7 from the giant tiger shrimp Penaeus monodon (PmRab7 and its binding partner envelope protein VP28 of white spot syndrome virus (WSSV could independently protect shrimp against WSSV infection. Thus, we aimed to display these two proteins independently on the cell surfaces of 2 yeast clones with the ultimate goal of using a mixture of the two clones as an orally deliverable, antiviral agent to protect shrimp against WSSV infection. PmRab7 and VP28 were modified by N-terminal tagging to the C-terminal half of S. cerevisiae α-agglutinin. DNA fragments, harboring fused-gene expression cassettes under control of an alcohol oxidase I (AOX1 promoter were constructed and used to transform the yeast cells. Immunofluorescence microscopy with antibodies specific to both proteins demonstrated that mutated PmRab7 (mPmRab7 and partial VP28 (pVP28 were localized on the cell surfaces of the respective clones, and fluorescence intensity for each was significantly higher than that of control cells by flow cytometry. Enzyme-linked immunosorbant assay (ELISA using cells displaying mPmRab7 or pVP28 revealed that the binding of specific antibodies for each was dose-dependent, and could be saturated. In addition, the binding of mPmRab7-expressing cells with free VP28, and vice versa was dose dependent. Binding between the two surface-expressed proteins was confirmed by an assay showing agglutination between cells expressing complementary mPmRab7 and pVP28. In summary, our genetically engineered P. pastoris can display biologically active mPmRab7 and pVP28 and is now ready for evaluation of efficacy in protecting shrimp against

  3. A Different Curriculum of Preparation for Work: Commentary on Mike Rose, Sara Goldrick-Rab, Kris Gutierrez and Norton Grubb

    Science.gov (United States)

    Worthen, Helena Harlow

    2012-01-01

    The January 2012 issue of "Mind, Culture, and Activity" published the Invited Presidential Address "Rethinking Remedial Education and the Academic-Vocational Divide," given by Mike Rose at the 2011 meeting of the American Educational Research Association in New Orleans, along with responses and commentary by Sara Goldrick-Rab, Kris Gutierrez, and…

  4. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

    International Nuclear Information System (INIS)

    Hu, Dong; Wu, Jing; Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan; Xiao, Jian; Hu, Fengyu; Yang, Yabo; Zhang, Rongbo

    2015-01-01

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy

  5. Should Community College Be Free? Forum. "Education Next" Talks with Sara Goldrick-Rab and Andrew P. Kelly

    Science.gov (United States)

    Goldrick-Rab, Sara; Kelly, Andrew P.

    2016-01-01

    In this article, "Education Next" talks with Sara Goldrick-Rab and Andrew Kelly. President Obama's proposal for tuition-free community college, seems to have laid down a marker for the Democratic Party. Vermont senator Bernie Sanders is touting his plan for free four-year public college on the primary trail; Massachusetts senator…

  6. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Dong, E-mail: austhudong@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wu, Jing, E-mail: wujing8008@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Hu, Fengyu; Yang, Yabo [Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Rongbo, E-mail: lory456@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China)

    2015-05-29

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.

  7. Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.

    Directory of Open Access Journals (Sweden)

    Fabienne Malagnac

    Full Text Available In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium. The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.

  8. Rab-GDI complex dissociation factor expressed through translational frameshifting in filamentous ascomycetes.

    Science.gov (United States)

    Malagnac, Fabienne; Fabret, Céline; Prigent, Magali; Rousset, Jean-Pierre; Namy, Olivier; Silar, Philippe

    2013-01-01

    In the model fungus Podospora anserina, the PaYIP3 gene encoding the orthologue of the Saccharomyces cerevisiae YIP3 Rab-GDI complex dissociation factor expresses two polypeptides, one of which, the long form, is produced through a programmed translation frameshift. Inactivation of PaYIP3 results in slightly delayed growth associated with modification in repartition of fruiting body on the thallus, along with reduced ascospore production on wood. Long and short forms of PaYIP3 are expressed in the mycelium, while only the short form appears expressed in the maturing fruiting body (perithecium). The frameshift has been conserved over the evolution of the Pezizomycotina, lasting for over 400 million years, suggesting that it has an important role in the wild.

  9. Gαs regulates Glucagon-Like Peptide 1 Receptor-mediated cyclic AMP generation at Rab5 endosomal compartment.

    Science.gov (United States)

    Girada, Shravan Babu; Kuna, Ramya S; Bele, Shilpak; Zhu, Zhimeng; Chakravarthi, N R; DiMarchi, Richard D; Mitra, Prasenjit

    2017-10-01

    Upon activation, G protein coupled receptors (GPCRs) associate with heterotrimeric G proteins at the plasma membrane to initiate second messenger signaling. Subsequently, the activated receptor experiences desensitization, internalization, and recycling back to the plasma membrane, or it undergoes lysosomal degradation. Recent reports highlight specific cases of persistent cyclic AMP generation by internalized GPCRs, although the functional significance and mechanistic details remain to be defined. Cyclic AMP generation from internalized Glucagon-Like Peptide-1 Receptor (GLP-1R) has previously been reported from our laboratory. This study aimed at deciphering the molecular mechanism by which internalized GLP-R supports sustained cyclic AMP generation upon receptor activation in pancreatic beta cells. We studied the time course of cyclic AMP generation following GLP-1R activation with particular emphasis on defining the location where cyclic AMP is generated. Detection involved a novel GLP-1 conjugate coupled with immunofluorescence using specific endosomal markers. Finally, we employed co-immunoprecipitation as well as immunofluorescence to assess the protein-protein interactions that regulate GLP-1R mediated cyclic AMP generation at endosomes. Our data reveal that prolonged association of G protein α subunit Gαs with activated GLP-1R contributed to sustained cyclic AMP generation at Rab 5 endosomal compartment. The findings provide the mechanism of endosomal cyclic AMP generation following GLP-1R activation. We identified the specific compartment that serves as an organizing center to generate endosomal cyclic AMP by internalized activated receptor complex. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

  10. PDGF-regulated rab4-dependent recycling of alphavbeta3 integrin from early endosomes is necessary for cell adhesion and spreading.

    Science.gov (United States)

    Roberts, M; Barry, S; Woods, A; van der Sluijs, P; Norman, J

    2001-09-18

    It has been postulated that the regulation of integrin vesicular traffic facilitates cell migration by internalizing integrins at the rear of the cell and transporting them forward within vesicles for exocytosis at the leading edge to form new contacts with the extracellular matrix. The rab family of GTPases control key targeting events in the endo/exocytic pathway; therefore, these GTPases may be involved in the regulation of cell-matrix contact assembly. The endo/exocytic cycle of alphavbeta3 and alpha5beta1 integrins was studied using mouse 3T3 fibroblast cell lines. In serum-starved cells, internalized integrins were transported through rab4-positive, early endosomes and arrived at the rab11-positive, perinuclear recycling compartment approximately 30 min after endocytosis. From the recycling compartment, integrins were recycled to the plasma membrane in a rab11-dependent fashion. Following treatment with PDGF, alphavbeta3 integrin, but not alpha5beta1, was rapidly recycled directly back to the plasma membrane from the early endosomes via a rab4-dependent mechanism without the involvement of rab11. This rapid recycling pathway directed alphavbeta3 to numerous small puncta distributed evenly across the dorsal surface of the cell, and the integrin only became localized into focal complexes at later times following PDGF addition. Interestingly, inhibition of PDGF-stimulated alphavbeta3 recycling using dominant-negative rab4 mutants compromised cell adhesion and spreading on vitronectin (a ligand for alphavbeta3), but adhesion to fibronectin (a ligand for alpha5beta1 and alphavbeta3) was unchanged. We propose that growth factor-regulated, rab4-dependent recycling of alphavbeta3 integrin from early endosomes to the plasma membrane is a critical upstream event in the assembly of cell-matrix contacts.

  11. Leishmania donovani resides in modified early endosomes by upregulating Rab5a expression via the downregulation of miR-494

    Science.gov (United States)

    Verma, Jitender Kumar; Rastogi, Ruchir

    2017-01-01

    Several intracellular pathogens arrest the phagosome maturation in the host cells to avoid transport to lysosomes. In contrast, the Leishmania containing parasitophorous vacuole (PV) is shown to recruit lysosomal markers and thus Leishmania is postulated to be residing in the phagolysosomes in macrophages. Here, we report that Leishmania donovani specifically upregulates the expression of Rab5a by degrading c-Jun via their metalloprotease gp63 to downregulate the expression of miR-494 in THP-1 differentiated human macrophages. Our results also show that miR-494 negatively regulates the expression of Rab5a in cells. Subsequently, L. donovani recruits and retains Rab5a and EEA1 on PV to reside in early endosomes and inhibits transport to lysosomes in human macrophages. Similarly, we have also observed that Leishmania PV also recruits Rab5a by upregulating its expression in human PBMC differentiated macrophages. However, the parasite modulates the endosome by recruiting Lamp1 and inactive pro-CathepsinD on PV via the overexpression of Rab5a in infected cells. Furthermore, siRNA knockdown of Rab5a or overexpression of miR-494 in human macrophages significantly inhibits the survival of the parasites. These results provide the first mechanistic insights of parasite-mediated remodeling of endo-lysosomal trafficking to reside in a specialized early endocytic compartment. PMID:28650977

  12. Activation of Rab GTPase Sec4 by its GEF Sec2 is required for prospore membrane formation during sporulation in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Suda, Yasuyuki; Tachikawa, Hiroyuki; Inoue, Ichiro; Kurita, Tomokazu; Saito, Chieko; Kurokawa, Kazuo; Nakano, Akihiko; Irie, Kenji

    2018-02-01

    Sec2 activates Sec4 Rab GTPase as a guanine nucleotide exchange factor for the recruitment of downstream effectors to facilitate tethering and fusion of post-Golgi vesicles at the plasma membrane. During the meiosis and sporulation of budding yeast, post-Golgi vesicles are transported to and fused at the spindle pole body (SPB) to form a de novo membrane, called the prospore membrane. Previous studies have revealed the role of the SPB outer surface called the meiotic outer plaque (MOP) in docking and fusion of post-Golgi vesicles. However, the upstream molecular machinery for post-Golgi vesicular fusion that facilitates prospore membrane formation remains enigmatic. Here, we demonstrate that the GTP exchange factor for Sec4, Sec2, participates in the formation of the prospore membrane. A conditional mutant in which the SEC2 expression is shut off during sporulation showed sporulation defects. Inactivation of Sec2 caused Sec4 targeting defects along the prospore membranes, thereby causing insufficient targeting of downstream effectors and cargo proteins to the prospore membrane. These results suggest that the activation of Sec4 by Sec2 is required for the efficient supply of post-Golgi vesicles to the prospore membrane and thus for prospore membrane formation/extension and subsequent deposition of spore wall materials. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  13. Vesicular PtdIns(3,4,5)P3 and Rab7 are key effectors of sea urchin zygote nuclear membrane fusion.

    Science.gov (United States)

    Lete, Marta G; Byrne, Richard D; Alonso, Alicia; Poccia, Dominic; Larijani, Banafshé

    2017-01-15

    Regulation of nuclear envelope dynamics is an important example of the universal phenomena of membrane fusion. The signalling molecules involved in nuclear membrane fusion might also be conserved during the formation of both pronuclear and zygote nuclear envelopes in the fertilised egg. Here, we determine that class-I phosphoinositide 3-kinases (PI3Ks) are needed for in vitro nuclear envelope formation. We show that, in vivo, PtdIns(3,4,5)P 3 is transiently located in vesicles around the male pronucleus at the time of nuclear envelope formation, and around male and female pronuclei before membrane fusion. We illustrate that class-I PI3K activity is also necessary for fusion of the female and male pronuclear membranes. We demonstrate, using coincidence amplified Förster resonance energy transfer (FRET) monitored using fluorescence lifetime imaging microscopy (FLIM), a protein-lipid interaction of Rab7 GTPase and PtdIns(3,4,5)P 3 that occurs during pronuclear membrane fusion to create the zygote nuclear envelope. We present a working model, which includes several molecular steps in the pathways controlling fusion of nuclear envelope membranes. © 2017. Published by The Company of Biologists Ltd.

  14. [Clinical decision making with regard to the granting of escorted leave for forensic patients detained by court order in a Dutch psychiatric clinic. Role of gender, disorder and the type of offence in the procedure].

    Science.gov (United States)

    Ter Horst, P; Jessen, A; Bogaerts, S; Spreen, M

    2015-01-01

    An increase in the length of time until the first escorted leave is granted to a patient detained by court order (tbs) results in a longer period of treatment. Physicians involved in the treatment and clinic managers are striving to reduce, in a responsible manner, the length of the period of treatment preceding the patient's first escorted leave. Forensic Psychiatric Clinic (fpk) 'De Woenselse Poort' aims to find out to what extent gender, pathology and the type of offence committed by the detainee influence the length of time that elapses before the patient's first leave is granted. We conducted a retrospective study based on patients' records. Although men use physical aggression more often than women, we found that gender, pathology and the type of offence had no influence on the length of the treatment period that preceded the granting of the patient's first escorted leave. Partly on the basis of risk management scales, clinicians judge whether the patient has adopted a more positive or a more negative attitude to risk factors relating to his or her offence. If the risk factors have become more positive, one would expect the application for leave to be made earlier. Surprisingly, this was not the case. In order to speed up the decision-making process regarding the application for leave, a clinical method for evaluating risk related treatment needs to be developed in which offence related risk factors are identified and the patient's positive or negative attitude to these risks are measured and monitored. At each treatment evaluation practitioners should be required to produce arguments that determine whether or not the patient is to be granted permission to go on leave at a particular moment.

  15. The monomeric GTPase RabA2 is required for progression and maintenance of membrane integrity of infection threads during root nodule symbiosis.

    Science.gov (United States)

    Dalla Via, Virginia; Traubenik, Soledad; Rivero, Claudio; Aguilar, O Mario; Zanetti, María Eugenia; Blanco, Flavio Antonio

    2017-04-01

    Progression of the infection canal that conducts rhizobia to the nodule primordium requires a functional Rab GTPase located in Golgi/trans-Golgi that also participate in root hair polar growth. Common bean (Phaseolus vulgaris) symbiotically associates with its partner Rhizobium etli, resulting in the formation of root nitrogen-fixing nodules. Compatible bacteria can reach cortical cells in a tightly regulated infection process, in which the specific recognition of signal molecules is a key step to select the symbiotic partner. In this work, we show that RabA2, a monomeric GTPase from common bean, is required for the progression of the infection canal, referred to as the infection thread (IT), toward the cortical cells. Expression of miss-regulated mutant variants of RabA2 resulted in an increased number of abortive infection events, including bursting of ITs and a reduction in the number of nodules. Nodules formed in these plants were small and contained infected cells with disrupted symbiosome membranes, indicating either early senescence of these cells or defects in the formation of the symbiosome membrane during bacterial release. RabA2 localized to mobile vesicles around the IT, but mutations that affect GTP hydrolysis or GTP/GDP exchange modified this localization. Colocalization of RabA2 with ArfA1 and a Golgi marker indicates that RabA2 localizes in Golgi stacks and the trans-Golgi network. Our results suggest that RabA2 is part of the vesicle transport events required to maintain the integrity of the membrane during IT progression.

  16. Protein mobilities and P-selectin storage in Weibel–Palade bodies

    OpenAIRE

    Kiskin, Nikolai I.; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J.; Carter, Tom

    2010-01-01

    Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel–Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P...

  17. A RAB3GAP1 SINE Insertion in Alaskan Huskies with Polyneuropathy, Ocular Abnormalities, and Neuronal Vacuolation (POANV Resembling Human Warburg Micro Syndrome 1 (WARBM1

    Directory of Open Access Journals (Sweden)

    Michaela Wiedmer

    2016-02-01

    Full Text Available We observed a hereditary phenotype in Alaskan Huskies that was characterized by polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV. The affected dogs developed a progressive severe ataxia, which led to euthanasia between 8 and 16 months of age. The pedigrees were consistent with a monogenic autosomal recessive inheritance. We localized the causative genetic defect to a 4 Mb interval on chromosome 19 by a combined linkage and homozygosity mapping approach. Whole genome sequencing of one affected dog, an obligate carrier, and an unrelated control revealed a 218-bp SINE insertion into exon 7 of the RAB3GAP1 gene. The SINE insertion was perfectly associated with the disease phenotype in a cohort of 43 Alaskan Huskies, and it was absent from 541 control dogs of diverse other breeds. The SINE insertion induced aberrant splicing and led to a transcript with a greatly altered exon 7. RAB3GAP1 loss-of-function variants in humans cause Warburg Micro Syndrome 1 (WARBM1, which is characterized by additional developmental defects compared to canine POANV, whereas Rab3gap1-deficient mice have a much milder phenotype than either humans or dogs. Thus, the RAB3GAP1 mutant Alaskan Huskies provide an interesting intermediate phenotype that may help to better understand the function of RAB3GAP1 in development. Furthermore, the identification of the presumed causative genetic variant will enable genetic testing to avoid the nonintentional breeding of affected dogs.

  18. A potential link between insulin signaling and GLUT4 translocation: Association of Rab10-GTP with the exocyst subunit Exoc6/6b

    Energy Technology Data Exchange (ETDEWEB)

    Sano, Hiroyuki; Peck, Grantley R. [Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 (United States); Blachon, Stephanie [Hybrigenics Services SAS, 3-5 Impasse Reille, 75014 Paris (France); Lienhard, Gustav E., E-mail: gustav.e.lienhard@dartmouth.edu [Department of Biochemistry, Geisel School of Medicine at Dartmouth, Hanover, NH 03755 (United States)

    2015-09-25

    Insulin increases glucose transport in fat and muscle cells by stimulating the exocytosis of specialized vesicles containing the glucose transporter GLUT4. This process, which is referred to as GLUT4 translocation, increases the amount of GLUT4 at the cell surface. Previous studies have provided evidence that insulin signaling increases the amount of Rab10-GTP in the GLUT4 vesicles and that GLUT4 translocation requires the exocyst, a complex that functions in the tethering of vesicles to the plasma membrane, leading to exocytosis. In the present study we show that Rab10 in its GTP form binds to Exoc6 and Exoc6b, which are the two highly homologous isotypes of an exocyst subunit, that both isotypes are found in 3T3-L1 adipocytes, and that knockdown of Exoc6, Exoc6b, or both inhibits GLUT4 translocation in 3T3-L1 adipocytes. These results suggest that the association of Rab10-GTP with Exoc6/6b is a molecular link between insulin signaling and the exocytic machinery in GLUT4 translocation. - Highlights: • Insulin stimulates the fusion of vesicles containing GLUT4 with the plasma membrane. • This requires vesicular Rab10-GTP and the exocyst plasma membrane tethering complex. • We find that Rab10-GTP associates with the Exoc6 subunit of the exocyst. • We find that knockdown of Exoc6 inhibits fusion of GLUT4 vesicles with the membrane. • The interaction of Rab10-GTP with Exoc6 potentially links signaling to exocytosis.

  19. Secretion of soluble vascular endothelial growth factor receptor 1 (sVEGFR1/sFlt1 requires Arf1, Arf6, and Rab11 GTPases.

    Directory of Open Access Journals (Sweden)

    Jae-Joon Jung

    Full Text Available The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1 is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA, a drug which blocks trafficking between the endoplasmic reticulum (ER and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.

  20. Respiration-dependent proton translocation in alkalophilic Bacillus firmus RAB and its non-alkalophilic mutant derivative.

    Science.gov (United States)

    Lewis, R J; Krulwich, T A; Reynafarje, B; Lehninger, A L

    1983-02-25

    Obligately alkalophilic Bacillus firmus RAB had a higher molar growth yield on L-malate (Ymal = 38 mg, dry weight/mmol of L-malate) than its non-alkalophilic mutant derivative, strain RABN (Ymal = 12 mg, dry weight/mmol of L-malate). Measurements of respiration dependent proton translocation by the two strains in the presence of K+ and valinomycin showed that the alkalophile also has much higher H+/O stoichiometries (at pH 9.0) than does the mutant (at pH 7.0). H+/O ratios for B. firmus RAB at pH 9.0 were as high as 13, with a frequently observed value of 9. These high values were observed in the first phase of a set of biphasic curves for both oxygen consumption and proton ejection. At pH 7.0, both the wild type and the mutant exhibited H+/O ratios near 4 in a single phase of oxygen consumption and proton ejection. The results are consistent with suggestions that the alkalophilic respiratory chain is especially well adapted for effective energy transduction at alkaline but not neutral pH.

  1. Knocking out Bcsas1 in Botrytis cinerea impacts growth, development, and secretion of extracellular proteins, which decreases virulence.

    Science.gov (United States)

    Zhang, Zhanquan; Qin, Guozheng; Li, Boqiang; Tian, Shiping

    2014-06-01

    Pathogenic fungi usually secrete a series of virulence factors to the extracellular environment to facilitate infection. Rab GTPases play a central role in the secretory pathway. To explore the function of Rab/GTPase in filamentous fungi, we knocked out a Rab/GTPase family gene, Bcsas1, in Botrytis cinerea, an aggressive fungal pathogen that infects more than 200 plant species. A detailed analysis was conducted on the virulence and the secretory capability of the mutants. The results indicated that knockout of Bcsas1 inhibited hyphal development and reduced sporulation of B. cinerea on potato dextrose agar plates resulting in reduced virulence on various fruit hosts. Knocking out the Bcsas1 gene led to an accumulation of transport vesicles at the hyphal tip, significantly reduced extracellular protein content, and lowered the activity of polygalacturonase and xylanase in the extracellular medium. However, mutation of Bcsas1 did not affect the expression of genes encoding polygalacturonase and xylanase, suggesting the secretion of these two family enzymes was suppressed in the mutant. Moreover, a comparative analysis of the secretome provided further evidence that the disruption of Bcsas1 in mutant strains significantly depressed the secretion of polysaccharide hydrolases and proteases. The results indicate that Bcsas1, the Rab8/SEC4-like gene, plays a crucial role in development, protein secretion, and virulence of B. cinerea.

  2. EHD proteins: Key conductors of endocytic transport

    Science.gov (United States)

    Naslavsky, Naava; Caplan, Steve

    2010-01-01

    Regulation of endocytic transport is controlled by an elaborate network of proteins. Rab GTP-binding proteins and their effectors have well-defined roles in mediating specific endocytic transport steps, but until recently, less was known about the four mammalian dynamin-like C-terminal Eps15 Homology Domain (EHD) proteins that also regulate endocytic events. In recent years, however, great strides have been made in understanding the structure and function of these unique proteins. Indeed, a growing body of literature addresses EHD protein structure, interactions with binding partners, functions in mammalian cells, and the generation of various new model systems. Accordingly, this is now an opportune time to pause and review the function and mechanisms of action of EHD proteins, and to highlight some of the challenges and future directions for the field. PMID:21067929

  3. Host cell virus entry mediated by Australian bat lyssavirus G envelope glycoprotein occurs through a clathrin-mediated endocytic pathway that requires actin and Rab5.

    Science.gov (United States)

    Weir, Dawn L; Laing, Eric D; Smith, Ina L; Wang, Lin-Fa; Broder, Christopher C

    2014-02-27

    Australian bat lyssavirus (ABLV), a rhabdovirus of the genus Lyssavirus which circulates in both pteropid fruit bats and insectivorous bats in mainland Australia, has caused three fatal human infections, the most recent in February 2013, manifested as acute neurological disease indistinguishable from clinical rabies. Rhabdoviruses infect host cells through receptor-mediated endocytosis and subsequent pH-dependent fusion mediated by their single envelope glycoprotein (G), but the specific host factors and pathways involved in ABLV entry have not been determined. ABLV internalization into HEK293T cells was examined using maxGFP-encoding recombinant vesicular stomatitis viruses (rVSV) that express ABLV G glycoproteins. A combination of chemical and molecular approaches was used to investigate the contribution of different endocytic pathways to ABLV entry. Dominant negative Rab GTPases were used to identify the endosomal compartment utilized by ABLV to gain entry into the host cell cytosol. Here we show that ABLV G-mediated entry into HEK293T cells was significantly inhibited by the dynamin-specific inhibitor dynasore, chlorpromazine, a drug that blocks clathrin-mediated endocytosis, and the actin depolymerizing drug latrunculin B. Over expression of dominant negative mutants of Eps15 and Rab5 also significantly reduced ABLV G-mediated entry into HEK293T cells. Chemical inhibitors of caveolae-dependent endocytosis and macropinocytosis and dominant negative mutants of Rab7 and Rab11 had no effect on ABLV entry. The predominant pathway utilized by ABLV for internalization into HEK293T cells is clathrin-and actin-dependent. The requirement of Rab5 for productive infection indicates that ABLV G-mediated fusion occurs within the early endosome compartment.

  4. Transcytosis of immunoglobulin A in the mouse enterocyte occurs through glycolipid raft- and rab17-containing compartments

    DEFF Research Database (Denmark)

    Hansen, Gert Helge; Niels-Christiansen, L L; Immerdal, Lissi

    1999-01-01

    BACKGROUND & AIMS: Glycolipid "rafts" have been shown to play a role in apical membrane trafficking in the enterocyte. The present study characterized the membrane compartments of the enterocyte involved in transepithelial transport of small intestinal immunoglobulin A (IgA). Methods: Immunogold...... electron microscopy and radioactive labeling of mouse small intestinal explants were performed. RESULTS: IgA and the polymeric immunoglobulin receptor/secretory component were present in a raft compartment. Raft association occurred posttranslationally within 30 minutes, preceding secretion...... and were also frequently seen associated with the same vesicular profiles of glycolipid rafts. Colocalization of IgA and rab17, a small guanosine triphosphatase involved in transcytosis, was seen mainly along the basolateral plasma membrane and over basolateral endosomes and vesicles, but also...

  5. Norma y legitimación del conocimiento rabínico: ángeles, hombres y vulgares burros

    Directory of Open Access Journals (Sweden)

    de Prado Plumed, Jesús

    2008-06-01

    Full Text Available This article presents an examination of two major working ideas of rabbinical Judaism: a humankind lives through an endless spiritual decline since the days of our pious forefathers (yĕridat ha-dorot; b in matters of legal adjudication, the opinion of active legal decisors (posĕqim is the one and only to be followed, even if divergent from earlier authoritative sources (hilxeta kĕ-vatraˀe. My examination of the nature and present currency of both ideas will be led by the poignant halakhic problem of the agunot (“chained women”. Rather than a thorough epistemology of both ideas, I will favour an argument which highlights the paradox of the link between authority and scholarship in rabbinical Judaism.En el presente artículo se analizan dos ideas comúnmente aceptadas en el judaísmo rabínico: la de que la humanidad vive en un continuo declive espiritual respecto de la época de nuestros piadosos antepasados (yĕridat ha-dorot y la de que la única voz autorizada a la hora de juzgar es la de los juristas en activo cuya opinión haya alcanzado rango vinculante (posĕqim, aunque diverja de la de autoridades precedentes (hilxeta kĕ-vatraˀe. Me serviré del acuciante problema halájico (“legal”, grosso modo de las “mujeres encadenadas” (agunot para trazar la naturaleza y extensión actual de ambas ideas. Antes que a llevar a cabo una completa epistemología de ambas ideas, me centraré en el análisis de la paradoja que resulta del vínculo existente entre reflexión académica y autoridad en el judaísmo rabínico.

  6. HIV-1 Nef binds with human GCC185 protein and regulates mannose 6 phosphate receptor recycling

    Energy Technology Data Exchange (ETDEWEB)

    Kumar, Manjeet; Kaur, Supinder; Nazir, Aamir; Tripathi, Raj Kamal, E-mail: rajkamalcdri@gmail.com

    2016-05-20

    HIV-1 Nef modulates cellular function that enhances viral replication in vivo which culminate into AIDS pathogenesis. With no enzymatic activity, Nef regulates cellular function through host protein interaction. Interestingly, trans-cellular introduction of recombinant Nef protein in Caenorhabditis elegans results in AIDS like pathogenesis which might share common pathophysiology because the gene sequence of C. elegans and humans share considerable homology. Therefore employing C. elegans based initial screen complemented with sequence based homology search we identified GCC185 as novel host protein interacting with HIV-1 Nef. The detailed molecular characterization revealed N-terminal EEEE{sub 65} acidic domain of Nef as key region for interaction. GCC185 is a tethering protein that binds with Rab9 transport vesicles. Our results show that Nef-GCC185 interaction disrupts Rab9 interaction resulting in delocalization of CI-MPR (cation independent Mannose 6 phosphate receptor) resulting in elevated secretion of hexosaminidase. In agreement with this, our studies identified novel host GCC185 protein that interacts with Nef EEEE65 acidic domain interfering GCC185-Rab9 vesicle membrane fusion responsible for retrograde vesicular transport of CI-MPR from late endosomes to TGN. In light of existing report suggesting critical role of Nef-GCC185 interaction reveals valuable mechanistic insights affecting specific protein transport pathway in docking of late endosome derived Rab9 bearing transport vesicle at TGN elucidating role of Nef during viral pathogenesis. -- Highlights: •Nef, an accessory protein of HIV-1 interacts with host factor and culminates into AIDS pathogenesis. •Using Caenorhabditis elegans based screen system, novel Nef interacting cellular protein GCC185 was identified. •Molecular characterization of Nef and human protein GCC185 revealed Nef EEEE{sub 65} key region interacted with full length GCC185. •Nef impeded the GCC185-Rab 9 interaction and

  7. The Role of the Rab Coupling Protein in ErbB2-Driven Mammary Tumorigenesis and Metastasis

    Science.gov (United States)

    2015-10-01

    SOW-Task 1.Attain ethical approval from ACURO for the use of mice (Months 2-3) Evolution. Completion: 100%. To follow the ethical approval from...Western blotting were purchased from Biobasic, Bioshop and Sigma Aldrich respectively. Herceptin was purchased from Genetech. 7.16.4 murine antibody was...staining. Lung metastases were counted manually using Scanscope microscope software. DNA Plasmids and shRNAs— pcDNA3.0-His-RCP (a gift from Jim Norman

  8. Rapid production of functionalized recombinant proteins: marrying ligation independent cloning and in vitro protein ligation.

    Science.gov (United States)

    Kushnir, Susanna; Marsac, Yoann; Breitling, Reinhard; Granovsky, Igor; Brok-Volchanskaya, Vera; Goody, Roger S; Becker, Christian F W; Alexandrov, Kirill

    2006-01-01

    Functional genomics and proteomics have been very active fields since the sequencing of several genomes was completed. To assign a physiological role to the newly discovered coding genes with unknown function, new generic methods for protein production, purification, and targeted functionalization are needed. This work presents a new vector, pCYSLIC, that allows rapid generation of Escherichia coli expression constructs via ligation-independent cloning (LIC). The vector is designed to facilitate protein purification by either Ni-NTA or GSH affinity chromatography. Subsequent proteolytic removal of affinity tags liberates an N-terminal cysteine residue that is then used for covalent modification of the target protein with different biophysical probes via protein ligation. The described system has been tested on 36 mammalian Rab GTPases, and it was demonstrated that recombinant GTPases produced with pCYSLIC could be efficiently modified with fluorescein or biotin in vitro. Finally, LIC was compared with the recently developed In-Fusion cloning method, and it was demonstrated that In-Fusion provides superior flexibility in choice of expression vector. By the application of In-Fusion cloning Cys-Rab6A GTPase with an N-terminal cysteine residue was generated employing unmodified pET30a vector and TVMV protease.

  9. Influence of UV and blue-light sun radiation to developing of age-related macular degeneration on Croatian Island Rab

    International Nuclear Information System (INIS)

    Vojnikovic, Bozo; Coklo, Miran; Ranogajec-Komor, Maria

    2008-01-01

    Full text: Croatian island Rab has one of the highest solar radiation in geographical area of Europe. The aim of this clinical trial was to estimate correlation between incidence of age-related macular degeneration (AMD) and the exposure to sunlight in different population on island Rab. In the first group agriculturists and fishermen were collected, in the second group members of the urban population were involved. 1753 individuals were investigated. The age in this population was between 40 and 73 years. The incidence of AMD in the first group was 19% while in the urban population only 2% showed AMD. It can be concluded that a very significant incidence exists between chronic exposure to sunlight and appearance of age-related macular degeneration. (author)

  10. Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana

    Directory of Open Access Journals (Sweden)

    Diana Castaño

    2010-08-01

    La expresión de mutantes constitutivamente activos de las Rab de endosomas tempranos impide la maduración de fagosomas que contienen esferas de látex o micobacterias inactivadas por calor. Mientras que su silenciamiento, mediante ARN de interferencia o mediante dominantes negativos, induce la maduración de fagosomas micobacterianos. Los mecanismos exactos por los que las micobacterias alteran la dinámica de expresión de estas GTPasas, afectando la maduración fagolisosómica, no se han establecido. El problema podría explicarse por defectos en el reclutamiento de las proteínas que interactúan con Rab, como la cinasa-3 del fosfatidilinositol y el antígeno endosómico temprano 1. La identificación de los mecanismos empleados por Mycobacterium spp. para interrumpir el ciclo de activación de las Rab, será esencial para comprender la fisiopatología de la infección micobacteriana y útil como posibles blancos farmacológicos.

  11. Neuron specific Rab4 effector GRASP-1 coordinates membrane specialization and maturation of recycling endosomes

    NARCIS (Netherlands)

    C.C. Hoogenraad (Casper); I. Popa (Ioana); K. Futai (Kensuke); E. Sanchez-Martinez (Emma); P. Wulf (Phebe); T. van Vlijmen (Thijs); B.R. Dortland (Bjorn); V. Oorschot (Viola); R. Govers (Robert); M. Monti (Maria); A.J.R. Heck (Albert); M. Sheng (Morgan); J. Klumperman (Judith); H. Rehmann (Holger); D. Jaarsma (Dick); L.C. Kapitein (Lukas); P. van der Sluijs

    2010-01-01

    textabstractThe endosomal pathway in neuronal dendrites is essential for membrane receptor trafficking and proper synaptic function and plasticity. However, the molecular mechanisms that organize specific endocytic trafficking routes are poorly understood. Here, we identify GRIP-associated protein-1

  12. Developmentally regulated GTP-binding protein 2 is required for stabilization of Rac1-positive membrane tubules.

    Science.gov (United States)

    Mani, Muralidharan; Lee, Unn Hwa; Yoon, Nal Ae; Yoon, Eun Hye; Lee, Byung Ju; Cho, Wha Ja; Park, Jeong Woo

    2017-11-04

    Previously we have reported that developmentally regulated GTP-binding protein 2 (DRG2) localizes on Rab5 endosomes and plays an important role in transferrin (Tfn) recycling. We here identified DRG2 as a key regulator of membrane tubule stability. At 30 min after Tfn treatment, DRG2 localized to membrane tubules which were enriched with phosphatidylinositol 4-monophosphate [PI(4)P] and did not contain Rab5. DRG2 interacted with Rac1 more strongly with GTP-bound Rac1 and tubular localization of DRG2 depended on Rac1 activity. DRG2 depletion led to destabilization of membrane tubules, while ectopic expression of DRG2 rescued the stability of the membrane tubules in DRG2-depleted cells. Our results reveal a novel mechanism for regulation of membrane tubule stability mediated by DRG2. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Structural studies of human Naked2: A biologically active intrinsically unstructured protein

    International Nuclear Information System (INIS)

    Hu Tianhui; Krezel, Andrzej M.; Li Cunxi; Coffey, Robert J.

    2006-01-01

    Naked1 and 2 are two mammalian orthologs of Naked Cuticle, a canonical Wnt signaling antagonist in Drosophila. Naked2, but not Naked1, interacts with transforming growth factor-α (TGFα) and escorts TGFα-containing vesicles to the basolateral membrane of polarized epithelial cells. Full-length Naked2 is poorly soluble. Since most functional domains, including the Dishevelled binding region, EF-hand, vesicle recognition, and membrane targeting motifs, reside in the N-terminal half of the protein, we expressed and purified the first 217 residues of human Naked2 and performed a functional analysis of this fragment. Its circular dichroism (CD) and nuclear magnetic resonance (NMR) spectra showed no evidence of secondary and/or tertiary structure. The fragment did not bind calcium or zinc. These results indicate that the N-terminal half of Naked2 behaves as an intrinsically unstructured protein

  14. OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, You [Minerva Foundation Institute for Medical Research, Biomedicum 2U, and National Institute for Health and Welfare/Public Health Genomics Unit, Biomedicum 1, FI-00290, Helsinki (Finland); Li, Shiqian [Department of Biology, Jinan University, Guangzhou 510632 (China); Maeyraenpaeae, Mikko I. [Wihuri Research Institute, FI-00140 Helsinki, and the Department of Forensic Medicine, FI-00014 University of Helsinki (Finland); Zhong, Wenbin [Department of Biology, Jinan University, Guangzhou 510632 (China); Baeck, Nils [Institute of Biomedicine/Anatomy, FI-00014 University of Helsinki, Helsinki (Finland); Yan, Daoguang [Department of Biology, Jinan University, Guangzhou 510632 (China); Olkkonen, Vesa M., E-mail: vesa.olkkonen@helsinki.fi [Minerva Foundation Institute for Medical Research, Biomedicum 2U, and National Institute for Health and Welfare/Public Health Genomics Unit, Biomedicum 1, FI-00290, Helsinki (Finland); Institute of Biomedicine/Anatomy, FI-00014 University of Helsinki, Helsinki (Finland)

    2010-11-15

    We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, but displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.

  15. OSBP-related protein 11 (ORP11) dimerizes with ORP9 and localizes at the Golgi-late endosome interface

    International Nuclear Information System (INIS)

    Zhou, You; Li, Shiqian; Maeyraenpaeae, Mikko I.; Zhong, Wenbin; Baeck, Nils; Yan, Daoguang; Olkkonen, Vesa M.

    2010-01-01

    We characterize here ORP11, a member of the oxysterol-binding protein family. ORP11 is present at highest levels in human ovary, testis, kidney, liver, stomach, brain, and adipose tissue. Immunohistochemistry demonstrates abundant ORP11 in the epithelial cells of kidney tubules, testicular tubules, caecum, and skin. ORP11 in HEK293 cells resides on Golgi complex and LE, co-localizing with GFP-Rab9, TGN46, GFP-Rab7, and a fluorescent medial-trans-Golgi marker. Under electron microscopic observation, cells overexpressing ORP11 displayed lamellar lipid bodies associated with vacuolar structures or the Golgi complex, indicating a disturbance of lipid trafficking. N-terminal fragment of ORP11 (aa 1-292) localized partially to Golgi, but displayed enhanced localization on Rab7- and Rab9-positive LE, while the C-terminal ligand-binding domain (aa 273-747) was cytosolic, demonstrating that the membrane targeting determinants are N-terminal. Yeast two-hybrid screen revealed interaction of ORP11 with the related ORP9. The interacting region was delineated within aa 98-372 of ORP9 and aa 154-292 of ORP11. Overexpressed ORP9 was able to recruit EGFP-ORP11 to membranes, and ORP9 silencing inhibited ORP11 Golgi association. The results identify ORP11 as an OSBP homologue distributing at the Golgi-LE interface and define the ORP9-ORP11 dimer as a functional unit that may act as an intracellular lipid sensor or transporter.

  16. Cloning, expression, crystallization and preliminary X-ray analysis of a putative multiple antibiotic resistance repressor protein (MarR) from Xanthomonas campestris

    International Nuclear Information System (INIS)

    Tu, Zhi-Le; Li, Juo-Ning; Chin, Ko-Hsin; Chou, Chia-Cheng; Lee, Cheng-Chung; Shr, Hui-Lin; Lyu, Ping-Chiang; Gao, Fei Philip; Wang, Andrew H.-J.; Chou, Shan-Ho

    2005-01-01

    A putative repressor for the multiple antibiotic resistance operon from a plant pathogen X. campestris pv. campestris has been overexpressed in E. coli, purified and crystallized. The crystals diffracted to 2.3 Å with good quality. The multiple antibiotic resistance operon (marRAB) is a member of the multidrug-resistance system. When induced, this operon enhances resistance of bacteria to a variety of medically important antibiotics, causing a serious global health problem. MarR is a marR-encoded protein that represses the transcription of the marRAB operon. Through binding with salicylate and certain antibiotics, however, MarR can derepress and activate the marRAB operon. In this report, the cloning, expression, crystallization and preliminary X-ray analysis of XC1739, a putative MarR repressor protein present in the Xanthomonas campestris pv. campestris, a Gram-negative bacterium causing major worldwide disease of cruciferous crops, are described. The XC1739 crystals diffracted to a resolution of at least 1.8 Å. They are orthorhombic and belong to space group P2 1 2 1 2 1 , with unit-cell parameters a = 39.5, b = 54.2 and c = 139.5 Å, respectively. They contain two molecules in the asymmetric unit from calculation of the self-rotation function

  17. Canine hereditary ataxia in old english sheepdogs and gordon setters is associated with a defect in the autophagy gene encoding RAB24.

    Directory of Open Access Journals (Sweden)

    Caryline Agler

    2014-02-01

    Full Text Available Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.

  18. Increased Eps15 homology domain 1 and RAB11FIP3 expression regulate breast cancer progression via promoting epithelial growth factor receptor recycling.

    Science.gov (United States)

    Tong, Dandan; Liang, Ya-Nan; Stepanova, A A; Liu, Yu; Li, Xiaobo; Wang, Letian; Zhang, Fengmin; Vasilyeva, N V

    2017-02-01

    Recent research indicates that the C-terminal Eps15 homology domain 1 is associated with epithelial growth factor receptor-mediated endocytosis recycling in non-small-cell lung cancer. The aim of this study was to determine the clinical significance of Eps15 homology domain 1 gene expression in relation to phosphorylation of epithelial growth factor receptor expression in patients with breast cancer. Primary breast cancer samples from 306 patients were analyzed for Eps15 homology domain 1, RAB11FIP3, and phosphorylation of epithelial growth factor receptor expression via immunohistochemistry. The clinical significance was assessed via a multivariate Cox regression analysis, Kaplan-Meier curves, and the log-rank test. Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor were upregulated in 60.46% (185/306) and 53.92% (165/306) of tumor tissues, respectively, as assessed by immunohistochemistry. The statistical correlation analysis indicated that Eps15 homology domain 1 overexpression was positively correlated with the increases in phosphorylation of epithelial growth factor receptor ( r = 0.242, p breast cancer for the overall survival in the total, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. However, the use of combined expression of Eps15 homology domain 1 and phosphorylation of epithelial growth factor receptor markers is more effective for the disease-free survival in the overall population, chemotherapy, and human epidermal growth factor receptor 2 (-) groups. Moreover, the combined markers are also significant prognostic markers of breast cancer in the human epidermal growth factor receptor 2 (+), estrogen receptor (+), and estrogen receptor (-) groups. Eps15 homology domain 1 has a tumor suppressor function, and the combined marker of Eps15 homology domain 1/phosphorylation of epithelial growth factor receptor expression was identified as a better prognostic marker in breast cancer diagnosis

  19. A Novel Nuclear Trafficking Module Regulates the Nucleocytoplasmic Localization of the Rabies Virus Interferon Antagonist, P Protein*

    Science.gov (United States)

    Oksayan, Sibil; Wiltzer, Linda; Rowe, Caitlin L.; Blondel, Danielle; Jans, David A.; Moseley, Gregory W.

    2012-01-01

    Regulated nucleocytoplasmic transport of proteins is central to cellular function and dysfunction during processes such as viral infection. Active protein trafficking into and out of the nucleus is dependent on the presence within cargo proteins of intrinsic specific modular signals for nuclear import (nuclear localization signals, NLSs) and export (nuclear export signals, NESs). Rabies virus (RabV) phospho (P) protein, which is largely responsible for antagonising the host anti-viral response, is expressed as five isoforms (P1–P5). The subcellular trafficking of these isoforms is thought to depend on a balance between the activities of a dominant N-terminal NES (N-NES) and a distinct C-terminal NLS (C-NLS). Specifically, the N-NES-containing isoforms P1 and P2 are cytoplasmic, whereas the shorter P3–P5 isoforms, which lack the N-NES, are believed to be nuclear through the activity of the C-NLS. Here, we show for the first time that RabV P contains an additional strong NLS in the N-terminal region (N-NLS), which, intriguingly, overlaps with the N-NES. This arrangement represents a novel nuclear trafficking module where the N-NLS is inactive in P1 but becomes activated in P3, concomitant with truncation of the N-NES, to become the principal targeting signal conferring nuclear accumulation. Understanding this unique switch arrangement of overlapping, co-regulated NES/NLS sequences is vital to delineating the critical role of RabV P protein in viral infection. PMID:22700958

  20. The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion.

    Directory of Open Access Journals (Sweden)

    Chengliang Zhang

    Full Text Available Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4 and Endophilin B1 (Endo B1 that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺-ATPases (V-ATPases to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA, producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.

  1. Panel of monoclonal antibodies to sperm surface proteins as a tool for monitoring localization and identification of sperm-zona pellucida receptors

    Czech Academy of Sciences Publication Activity Database

    Zigo, Michal; Dorosh, Andriy; Pohlová, Alžběta; Jonáková, Věra; Šulc, Miroslav; Maňásková-Postlerová, Pavla

    March, č. 359 (2015), s. 895-908 ISSN 0302-766X R&D Projects: GA ČR(CZ) GA14-05547S; GA MŠk(CZ) ED1.1.00/02.0109; GA ČR(CZ) GAP503/12/1834 Institutional research plan: CEZ:AV0Z50520701 Institutional support: RVO:86652036 ; RVO:61388971 Keywords : zona pellucida-binding receptors * monoclonal antibodies against sperm surface proteins * sperm surface proteins * RAB-2A * lactahedrin P47 Subject RIV: CE - Biochemistry Impact factor: 2.948, year: 2015

  2. Roles of the Outer Membrane Protein AsmA of Salmonella enterica in the Control of marRAB Expression and Invasion of Epithelial Cells▿

    OpenAIRE

    Prieto, Ana I.; Hernández, Sara B.; Cota, Ignacio; Pucciarelli, M. Graciela; Orlov, Yuri; Ramos-Morales, Francisco; García-del Portillo, Francisco; Casadesús, Josep

    2009-01-01

    A genetic screen for suppressors of bile sensitivity in DNA adenine methylase (dam) mutants of Salmonella enterica serovar Typhimurium yielded insertions in an uncharacterized locus homologous to the Escherichia coli asmA gene. Disruption of asmA suppressed bile sensitivity also in phoP and wec mutants of S. enterica and increased the MIC of sodium deoxycholate for the parental strain ATCC 14028. Increased levels of marA mRNA were found in asmA, asmA dam, asmA phoP, and asmA wec strains of S....

  3. Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

    International Nuclear Information System (INIS)

    Born, E J; Hartman, S V; Holstein, S A

    2013-01-01

    Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways

  4. The Effect of Map Scale on the Determination of the Coastline Length and the Area of Islands in the Adriatic Sea - the Example of the Island of Rab

    Directory of Open Access Journals (Sweden)

    Nada Vučetić

    2006-12-01

    Full Text Available The procedure to determine the coastline length and the area of the island of Rab from the maps at the scales 1:25 000, 1:50 000, 1:100 000, 1:200 000, 1:300 000, 1:500 000, 1:1 000 000 and 1:2 000 000 is described. The map sheets at the scales 1:25 000, 1:100 000 and 1:200 000 were obtained already in a georeferenced raster format, and the others were scanned and georeferenced. This was followed by a manual vectorization of the coastline and a transformation of all coordinates into the 5th zone of the Gauss-Krüger projection. The length of the coastline and the area of the island were calculated in the Gauss-Krüger projection taking into account the deformations of the projection. The results are given in tables and represented graphically.

  5. Decreased expression of miR‑490‑3p in colorectal cancer predicts poor prognosis and promotes cell proliferation and invasion by targeting RAB14.

    Science.gov (United States)

    Wang, Bo; Yin, Mujun; Cheng, Cheng; Jiang, Hongpeng; Jiang, Kewei; Shen, Zhanlong; Ye, Yingjiang; Wang, Shan

    2018-06-19

    Growing evidence indicates a potential role for miR‑490‑3p in tumorigenesis. However, its function in colorectal carcinoma (CRC) remains undefined. In this study, miR‑490‑3p was markedly downregulated in fifty colorectal cancer tissue samples compared with the corresponding adjacent non‑cancerous specimens, by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The expression levels of miR‑490‑3p were closely associated with tumor differentiation and distant metastasis. In addition, both Kaplan-Meier and multivariate analyses indicated CRC patients with elevated miR‑490‑3p amounts had prolonged overall survival. Overexpression of miR‑490‑3p markedly reduced proliferation, colony formation and invasion in CRC cells by enhancing apoptosis and promoting G2/M phase arrest. Furthermore, ectopic expression of miR‑490‑3p resulted in decreased expression of RAB14, which was directly targeted by miR‑490‑3p, as shown by the dual-luciferase reporter gene assay. Finally, in a nude mouse model, miR‑490‑3p overexpression significantly suppressed the growth of CRC cells. The above results indicated that miR‑490‑3p might constitute a prognostic indicator and a novel molecular target for miRNA-based CRC therapy.

  6. GDP-bound and nucleotide-free intermediates of the guanine nucleotide exchange in the Rab5·Vps9 system.

    Science.gov (United States)

    Uejima, Tamami; Ihara, Kentaro; Goh, Tatsuaki; Ito, Emi; Sunada, Mariko; Ueda, Takashi; Nakano, Akihiko; Wakatsuki, Soichi

    2010-11-19

    Many GTPases regulate intracellular transport and signaling in eukaryotes. Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing the exchange of their GDP for GTP. Here we present crystallographic and biochemical studies of a GEF reaction with four crystal structures of Arabidopsis thaliana ARA7, a plant homolog of Rab5 GTPase, in complex with its GEF, VPS9a, in the nucleotide-free and GDP-bound forms, as well as a complex with aminophosphonic acid-guanylate ester and ARA7·VPS9a(D185N) with GDP. Upon complex formation with ARA7, VPS9 wedges into the interswitch region of ARA7, inhibiting the coordination of Mg(2+) and decreasing the stability of GDP binding. The aspartate finger of VPS9a recognizes GDP β-phosphate directly and pulls the P-loop lysine of ARA7 away from GDP β-phosphate toward switch II to further destabilize GDP for its release during the transition from the GDP-bound to nucleotide-free intermediates in the nucleotide exchange reaction.

  7. 3D time-lapse analysis of Rab11/FIP5 complex: spatiotemporal dynamics during apical lumen formation.

    Science.gov (United States)

    Mangan, Anthony; Prekeris, Rytis

    2015-01-01

    Fluorescent imaging of fixed cells grown in two-dimensional (2D) cultures is one of the most widely used techniques for observing protein localization and distribution within cells. Although this technique can also be applied to polarized epithelial cells that form three-dimensional (3D) cysts when grown in a Matrigel matrix suspension, there are still significant limitations in imaging cells fixed at a particular point in time. Here, we describe the use of 3D time-lapse imaging of live cells to observe the dynamics of apical membrane initiation site (AMIS) formation and lumen expansion in polarized epithelial cells.

  8. Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Yoo, Byong Chul [Colorectal Cancer Branch, Research Institute, National Cancer Center, Goyang (Korea, Republic of); Yeo, Seung Gu [Dept. of Radiation Oncology, Soonchunhyang University College of Medicine, Soonchunhyang University Hospital, Cheonan (Korea, Republic of)

    2017-09-15

    The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

  9. Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

    International Nuclear Information System (INIS)

    Yoo, Byong Chul; Yeo, Seung Gu

    2017-01-01

    The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins

  10. Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro

    Science.gov (United States)

    Koguchi, Yoshinobu; Gardell, Jennifer L.; Thauland, Timothy J.; Parker, David C.

    2011-01-01

    CD40L is critically important for the initiation and maintenance of adaptive immune responses. It is generally thought that CD40L expression in CD4+ T cells is regulated transcriptionally and made from new mRNA following antigen recognition. However, recent studies with two-photon microscopy revealed that the majority of cognate interactions between effector CD4+ T cells and APCs are too short for de novo synthesis of CD40L. Given that effector and memory CD4+ T cells store preformed CD40L (pCD40L) in lysosomal compartments and that pCD40L comes to the cell surface within minutes of antigenic stimulation, we and others have proposed that pCD40L might mediate T cell-dependent activation of cognate APCs during brief encounters in vivo. However, it has not been shown that this relatively small amount of pCD40L is sufficient to activate APCs, owing to the difficulty of separating the effects of pCD40L from those of de novo CD40L and other cytokines in vitro. Here we show that pCD40L surface mobilization is resistant to cyclosporine or FK506 treatment, while de novo CD40L and cytokine expression are completely inhibited. These drugs thus provide a tool to dissect the role of pCD40L in APC activation. We find that pCD40L mediates selective activation of cognate but not bystander APCs in vitro and that mobilization of pCD40L does not depend on Rab27a, which is required for mobilization of lytic granules. Therefore, effector CD4+ T cells deliver pCD40L specifically to APCs on the same time scale as the lethal hit of CTLs but with distinct molecular machinery. PMID:21677130

  11. Differential requirements of arrestin-3 and clathrin for ligand-dependent and -independent internalization of human G protein-coupled receptor 40.

    Science.gov (United States)

    Qian, Jing; Wu, Chun; Chen, Xiaopan; Li, Xiangmei; Ying, Guoyuan; Jin, Lili; Ma, Qiang; Li, Guo; Shi, Ying; Zhang, Guozheng; Zhou, Naiming

    2014-11-01

    G protein-coupled receptor 40 (GPR40) is believed to be an attractive target to enhance insulin secretion in patients with type 2 diabetes. GPR40 has been found to couple to Gq protein, leading to the activation of phospholipase C and subsequent increases in the intracellular Ca(2+) level. However, the underlying mechanisms that regulate the internalization and desensitization of GPR40 remain to be elucidated. In the present study, a construct of GPR40 fused with enhanced green fluorescent protein (EGFP) at its C-terminus was constructed for direct imaging of the localization and internalization of GPR40 by confocal microscopy. In stably transfected HEK-293 cells, GPR40 receptors underwent rapid agonist-induced internalization and constitutive ligand-independent internalization. Our data demonstrated that the agonist-mediated internalization of GPR40 was significantly blocked by hypertonic sucrose treatment and by siRNA mediated depletion of the heavy chain of clathrin. In contrast, constitutive GPR40 internalization was not affected by hypertonic sucrose or by knock-down of clathrin expression, but it was affected by treatment with methyl-β-cyclodextrin (MβCD) and nystatin. Furthermore, our results using an arrestin-3-EGFP redistribution assay and siRNA-mediated knock-down of arrestin-3 and GRK2 expression revealed that arrestin-3 and GRK2 play an essential role in the regulation of agonist-mediated GPR40 internalization, but are not involved in the regulation of constitutive GPR40 internalization. Additionally, our observation showed that upon activation by agonist, the internalized GPR40 receptors were rapidly recycled back to the plasma membrane via Rab4/Rab5 positive endosomes, whereas the constitutively internalized GPR40 receptors were recycled back to the cell surface through Rab5 positive endosomes. Because FFA receptors exhibit a high level of homology, our observations could be applicable to other members of this family. Copyright © 2014 Elsevier Inc

  12. Towards complete sets of farnesylated and geranylgeranylated proteins.

    Directory of Open Access Journals (Sweden)

    Sebastian Maurer-Stroh

    2007-04-01

    Full Text Available Three different prenyltransferases attach isoprenyl anchors to C-terminal motifs in substrate proteins. These lipid anchors serve for membrane attachment or protein-protein interactions in many pathways. Although well-tolerated selective prenyltransferase inhibitors are clinically available, their mode of action remains unclear since the known substrate sets of the various prenyltransferases are incomplete. The Prenylation Prediction Suite (PrePS has been applied for large-scale predictions of prenylated proteins. To prioritize targets for experimental verification, we rank the predictions by their functional importance estimated by evolutionary conservation of the prenylation motifs within protein families. The ranked lists of predictions are accessible as PRENbase (http://mendel.imp.univie.ac.at/sat/PrePS/PRENbase and can be queried for verification status, type of modifying enzymes (anchor type, and taxonomic distribution. Our results highlight a large group of plant metal-binding chaperones as well as several newly predicted proteins involved in ubiquitin-mediated protein degradation, enriching the known functional repertoire of prenylated proteins. Furthermore, we identify two possibly prenylated proteins in Mimivirus. The section HumanPRENbase provides complete lists of predicted prenylated human proteins-for example, the list of farnesyltransferase targets that cannot become substrates of geranylgeranyltransferase 1 and, therefore, are especially affected by farnesyltransferase inhibitors (FTIs used in cancer and anti-parasite therapy. We report direct experimental evidence verifying the prediction of the human proteins Prickle1, Prickle2, the BRO1 domain-containing FLJ32421 (termed BROFTI, and Rab28 (short isoform as exclusive farnesyltransferase targets. We introduce PRENbase, a database of large-scale predictions of protein prenylation substrates ranked by evolutionary conservation of the motif. Experimental evidence is presented for

  13. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    International Nuclear Information System (INIS)

    Ibrahim, Amr; Hutchens, Heather M.; Howard Berg, R.; Sue Loesch-Fries, L.

    2012-01-01

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  14. α-Synuclein-induced lysosomal dysfunction occurs through disruptions in protein trafficking in human midbrain synucleinopathy models.

    Science.gov (United States)

    Mazzulli, Joseph R; Zunke, Friederike; Isacson, Ole; Studer, Lorenz; Krainc, Dimitri

    2016-02-16

    Parkinson's disease (PD) is an age-related neurodegenerative disorder characterized by the accumulation of protein aggregates comprised of α-synuclein (α-syn). A major barrier in treatment discovery for PD is the lack of identifiable therapeutic pathways capable of reducing aggregates in human neuronal model systems. Mutations in key components of protein trafficking and cellular degradation machinery represent important risk factors for PD; however, their precise role in disease progression and interaction with α-syn remains unclear. Here, we find that α-syn accumulation reduced lysosomal degradation capacity in human midbrain dopamine models of synucleinopathies through disrupting hydrolase trafficking. Accumulation of α-syn at the cell body resulted in aberrant association with cis-Golgi-tethering factor GM130 and disrupted the endoplasmic reticulum-Golgi localization of rab1a, a key mediator of vesicular transport. Overexpression of rab1a restored Golgi structure, improved hydrolase trafficking and activity, and reduced pathological α-syn in patient neurons. Our work suggests that enhancement of lysosomal hydrolase trafficking may prove beneficial in synucleinopathies and indicates that human midbrain disease models may be useful for identifying critical therapeutic pathways in PD and related disorders.

  15. Alfalfa mosaic virus replicase proteins, P1 and P2, localize to the tonoplast in the presence of virus RNA

    Energy Technology Data Exchange (ETDEWEB)

    Ibrahim, Amr [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Present address: Genomics Facility, Agricultural Genetic Engineering Research Institute, Agricultural Research Center, Giza 12619 (Egypt); Hutchens, Heather M. [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States); Howard Berg, R. [Integrated Microscopy Facility, Donald Danforth Plant Science Center, Saint Louis, MO 63132 (United States); Sue Loesch-Fries, L., E-mail: loeschfr@purdue.edu [Department of Botany and Plant Pathology, Purdue University, West Lafayette, IN 47907 (United States)

    2012-11-25

    To identify the virus components important for assembly of the Alfalfa mosaic virus replicase complex, we used live cell imaging of Arabidopsis thaliana protoplasts that expressed various virus cDNAs encoding native and GFP-fusion proteins of P1 and P2 replicase proteins and full-length virus RNAs. Expression of P1-GFP alone resulted in fluorescent vesicle-like bodies in the cytoplasm that colocalized with FM4-64, an endocytic marker, and RFP-AtVSR2, RabF2a/Rha1-mCherry, and RabF2b/Ara7-mCherry, all of which localize to multivesicular bodies (MVBs), which are also called prevacuolar compartments, that mediate traffic to the lytic vacuole. GFP-P2 was driven from the cytosol to MVBs when expressed with P1 indicating that P1 recruited GFP-P2. P1-GFP localized on the tonoplast, which surrounds the vacuole, in the presence of infectious virus RNA, replication competent RNA2, or P2 and replication competent RNA1 or RNA3. This suggests that a functional replication complex containing P1, P2, and a full-length AMV RNA assembles on MVBs to traffic to the tonoplast.

  16. The cellular prion protein negatively regulates phagocytosis and cytokine expression in murine bone marrow-derived macrophages.

    Directory of Open Access Journals (Sweden)

    Min Wang

    Full Text Available The cellular prion protein (PrP(C is a glycosylphosphatidylinositol (GPI-anchored glycoprotein on the cell surface. Previous studies have demonstrated contradictory roles for PrP(C in connection with the phagocytic ability of macrophages. In the present work, we investigated the function of PrP(C in phagocytosis and cytokine expression in bone marrow-derived macrophages infected with Escherichia coli. E. coli infection induced an increase in the PRNP mRNA level. Knockout of PrP(C promoted bacterial uptake; upregulated Rab5, Rab7, and Eea1 mRNA expression; and increased the recruitment of lysosomal-associated membrane protein-2 to phagosomes, suggesting enhanced microbicidal activity. Remarkably, knockout of PrP(C suppressed the proliferation of internalized bacteria and increased the expression of cytokines such as interleukin-1β. Collectively, our data reveal an important role of PrP(C as a negative regulator for phagocytosis, phagosome maturation, cytokine expression, and macrophage microbicidal activity.

  17. FYVE zinc-finger proteins in the plant model Arabidopsis thaliana

    DEFF Research Database (Denmark)

    Jensen, R B; La Cour, T; Albrethsen, J

    2001-01-01

    Classic FYVE zinc-finger domains recognize the phosphoinositide signal PtdIns3P and share the basic (R/K)(1)(R/K)HHCR(6) (single-letter amino acid codes) consensus sequence. This domain is present in predicted PtdIns3P 5-kinases and lipases from Arabidopsis thaliana. Other Arabidopsis proteins......) of the basic motif. Dot-blot and liposome-binding assays were used in vitro to examine the phospholipid-binding ability of isolated PRAF domains. Whereas the PH domain preferentially bound PtdIns(4,5)P(2), the variant FYVE domain showed a weaker charge-dependent binding of phosphoinositides. In contrast....... A biochemical function for PRAF was indicated by its ability to catalyse guanine nucleotide exchange on some of the small GTPases of the Rab family, permitting a discussion of the biological roles of plant FYVE proteins and their regulation by phosphoinositides....

  18. Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.

    Directory of Open Access Journals (Sweden)

    Hayley J Newton

    Full Text Available The human pathogen Coxiella burnetii encodes a type IV secretion system called Dot/Icm that is essential for intracellular replication. The Dot/Icm system delivers bacterial effector proteins into the host cytosol during infection. The effector proteins delivered by C. burnetii are predicted to have important functions during infection, but when these proteins are needed during infection has not been clearly defined. Here, we use a reporter system consisting of fusion proteins that have a β-lactamase enzyme (BlaM fused to C. burnetii effector proteins to study protein translocation by the Dot/Icm system. Translocation of BlaM fused to the effector proteins CBU0077, CBU1823 and CBU1524 was not detected until 8-hours after infection of HeLa cells, which are permissive for C. burnetii replication. Translocation of these effector fusion proteins by the Dot/Icm system required acidification of the Coxiella-containing vacuole. Silencing of the host genes encoding the membrane transport regulators Rab5 or Rab7 interfered with effector translocation, which indicates that effectors are not translocated until bacteria traffic to a late endocytic compartment in the host cell. Similar requirements for effector translocation were discerned in bone marrow macrophages derived from C57BL/6 mice, which are primary cells that restrict the intracellular replication of C. burnetii. In addition to requiring endocytic maturation of the vacuole for Dot/Icm-mediated translocation of effectors, bacterial transcription was required for this process. Thus, translocation of effector proteins by the C. burnetii Dot/Icm system occurs after acidification of the CCV and maturation of this specialized organelle to a late endocytic compartment. This indicates that creation of the specialized vacuole in which C. burnetii replicates represents a two-stage process mediated initially by host factors that regulate endocytic maturation and then by bacterial effectors delivered into

  19. Opinião de acompanhantes de crianças em quimioterapia ambulatorial sobre uma quimioteca no Município de São Paulo Opinión de acompañantes de niños sometidos a quimioterapia en ambulatorio sobre una quimioteca en el municipio de Sao Paulo Opinion of adults escorting children on an outpatient chemotherapy service about a "Chemo-teca" in a municipality of Sao Paulo

    Directory of Open Access Journals (Sweden)

    Ione Queiroz de Jesus

    2010-04-01

    de los niños en tratamiento. La Quimioteca es una experiencia innovadora que puede servir de ejemplo para el desarrollo de tecnologías creativas de cuidado a niños con cáncer y a sus familias, contribuyendo para la mejoría de la asistencia a esa clientela.OBJECTIVE: To know the opinion of adults escorting children, from 2 to 12 years, undergoing chemotherapy about the Orsa Foundation "Chemo-teca". METHODS: This is a descriptive quantitative study. Were conducted semi-structured interviews with 54 children's escorting companions during an outpatient chemotherapy treatment. RESULTS: In the opinion of those escorting companions, the colorful environment, the toys and the leisure activities, offered during the administration of chemotherapy, has positive influence on the quality of treatment and the state of child welfare. Despite the very positive evaluation of the service, were mentioned areas requiring improvement, particularly in relation to the attention care. CONCLUSION: The companions escorting the children perceived that the happy environment collaborated on the wellbeing of children in treatment. The "Chemo-teca" is an innovative experience that can serve as an example for the development of creative technologies to care for children with cancer and their families, contributing to the improvement of care assistance to this clientele.

  20. Differentially Regulated Host Proteins Associated with Chronic Rhinosinusitis Are Correlated with the Sinonasal Microbiome

    Directory of Open Access Journals (Sweden)

    Kristi Biswas

    2017-12-01

    Full Text Available The chronic inflammatory nature of chronic rhinosinusitis (CRS makes it a morbid condition for individuals with the disease and one whose pathogenesis is poorly understood. To date, proteomic approaches have been applied successfully in a handful of CRS studies. In this study we use a multifaceted approach, including proteomics (iTRAQ labeling and microbiome (bacterial 16S rRNA gene sequencing analyses of middle meatus swabs, as well as immune cell analysis of the underlying tissue, to investigate the host-microbe interaction in individuals with CRS (n = 10 and healthy controls (n = 9. Of the total 606 proteins identified in this study, seven were significantly (p < 0.05 more abundant and 104 were significantly lower in the CRS cohort compared with healthy controls. The majority of detected proteins (82% of proteins identified were not significantly correlated with disease status. Elevated levels of blood and immune cell proteins in the CRS cohort, together with significantly higher numbers of B-cells and macrophages in the underlying tissue, confirmed the inflammatory status of CRS individuals. Protein PRRC2C and Ras-related protein (RAB14 (two of the seven elevated proteins showed the biggest fold difference between the healthy and CRS groups. Validation of the elevated levels of these two proteins in CRS samples was provided by immunohistochemistry. Members of the bacterial community in the two study cohorts were not associated with PRRC2C, however members of the genus Moraxella did correlate with RAB14 (p < 0.0001, rho = −0.95, which is a protein involved in the development of basement membrane. In addition, significant correlations between certain members of the CRS bacterial community and 33 lower abundant proteins in the CRS cohort were identified. Members of the genera Streptococcus, Haemophilus and Veillonella were strongly correlated with CRS and were significantly associated with a number of proteins with varying functions. The

  1. An engineered genetic selection for ternary protein complexes inspired by a natural three-component hitchhiker mechanism.

    Science.gov (United States)

    Lee, Hyeon-Cheol; Portnoff, Alyse D; Rocco, Mark A; DeLisa, Matthew P

    2014-12-22

    The bacterial twin-arginine translocation (Tat) pathway is well known to translocate correctly folded monomeric and dimeric proteins across the tightly sealed cytoplasmic membrane. We identified a naturally occurring heterotrimer, the Escherichia coli aldehyde oxidoreductase PaoABC, that is co-translocated by the Tat translocase according to a ternary "hitchhiker" mechanism. Specifically, the PaoB and PaoC subunits, each devoid of export signals, are escorted to the periplasm in a piggyback fashion by the Tat signal peptide-containing subunit PaoA. Moreover, export of PaoA was blocked when either PaoB or PaoC was absent, revealing a surprising interdependence for export that is not seen for classical secretory proteins. Inspired by this observation, we created a bacterial three-hybrid selection system that links the formation of ternary protein complexes with antibiotic resistance. As proof-of-concept, a bispecific antibody was employed as an adaptor that physically crosslinked one antigen fused to a Tat export signal with a second antigen fused to TEM-1 β-lactamase (Bla). The resulting non-covalent heterotrimer was exported in a Tat-dependent manner, delivering Bla to the periplasm where it hydrolyzed β-lactam antibiotics. Collectively, these results highlight the remarkable flexibility of the Tat system and its potential for studying and engineering ternary protein interactions in living bacteria.

  2. HMM Logos for visualization of protein families

    Directory of Open Access Journals (Sweden)

    Schultz Jörg

    2004-01-01

    Full Text Available Abstract Background Profile Hidden Markov Models (pHMMs are a widely used tool for protein family research. Up to now, however, there exists no method to visualize all of their central aspects graphically in an intuitively understandable way. Results We present a visualization method that incorporates both emission and transition probabilities of the pHMM, thus extending sequence logos introduced by Schneider and Stephens. For each emitting state of the pHMM, we display a stack of letters. The stack height is determined by the deviation of the position's letter emission frequencies from the background frequencies. The stack width visualizes both the probability of reaching the state (the hitting probability and the expected number of letters the state emits during a pass through the model (the state's expected contribution. A web interface offering online creation of HMM Logos and the corresponding source code can be found at the Logos web server of the Max Planck Institute for Molecular Genetics http://logos.molgen.mpg.de. Conclusions We demonstrate that HMM Logos can be a useful tool for the biologist: We use them to highlight differences between two homologous subfamilies of GTPases, Rab and Ras, and we show that they are able to indicate structural elements of Ras.

  3. Protein mobilities and P-selectin storage in Weibel-Palade bodies.

    Science.gov (United States)

    Kiskin, Nikolai I; Hellen, Nicola; Babich, Victor; Hewlett, Lindsay; Knipe, Laura; Hannah, Matthew J; Carter, Tom

    2010-09-01

    Using fluorescence recovery after photobleaching (FRAP) we measured the mobilities of EGFP-tagged soluble secretory proteins in the endoplasmic reticulum (ER) and in individual Weibel-Palade bodies (WPBs) at early (immature) and late (mature) stages in their biogenesis. Membrane proteins (P-selectin, CD63, Rab27a) were also studied in individual WPBs. In the ER, soluble secretory proteins were mobile; however, following insertion into immature WPBs larger molecules (VWF, Proregion, tPA) and P-selectin became immobilised, whereas small proteins (ssEGFP, eotaxin-3) became less mobile. WPB maturation led to further decreases in mobility of small proteins and CD63. Acute alkalinisation of mature WPBs selectively increased the mobilities of small soluble proteins without affecting larger molecules and the membrane proteins. Disruption of the Proregion-VWF paracrystalline core by prolonged incubation with NH(4)Cl rendered P-selectin mobile while VWF remained immobile. FRAP of P-selectin mutants revealed that immobilisation most probably involves steric entrapment of the P-selectin extracellular domain by the Proregion-VWF paracrystal. Significantly, immobilisation contributed to the enrichment of P-selectin in WPBs; a mutation of P-selectin preventing immobilisation led to a failure of enrichment. Together these data shed new light on the transitions that occur for soluble and membrane proteins following their entry and storage into post-Golgi-regulated secretory organelles.

  4. Spatiotemporal dynamics of membrane remodeling and fusion proteins during endocytic transport.

    Science.gov (United States)

    Arlt, Henning; Auffarth, Kathrin; Kurre, Rainer; Lisse, Dominik; Piehler, Jacob; Ungermann, Christian

    2015-04-01

    Organelles of the endolysosomal system undergo multiple fission and fusion events to combine sorting of selected proteins to the vacuole with endosomal recycling. This sorting requires a consecutive remodeling of the organelle surface in the course of endosomal maturation. Here we dissect the remodeling and fusion machinery on endosomes during the process of endocytosis. We traced selected GFP-tagged endosomal proteins relative to exogenously added fluorescently labeled α-factor on its way from the plasma membrane to the vacuole. Our data reveal that the machinery of endosomal fusion and ESCRT proteins has similar temporal localization on endosomes, whereas they precede the retromer cargo recognition complex. Neither deletion of retromer nor the fusion machinery with the vacuole affects this maturation process, although the kinetics seems to be delayed due to ESCRT deletion. Of importance, in strains lacking the active Rab7-like Ypt7 or the vacuolar SNARE fusion machinery, α-factor still proceeds to late endosomes with the same kinetics. This indicates that endosomal maturation is mainly controlled by the early endosomal fusion and remodeling machinery but not the downstream Rab Ypt7 or the SNARE machinery. Our data thus provide important further understanding of endosomal biogenesis in the context of cargo sorting. © 2015 Arlt et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  5. Characterization of the human GARP (Golgi associated retrograde protein) complex

    International Nuclear Information System (INIS)

    Liewen, Heike; Meinhold-Heerlein, Ivo; Oliveira, Vasco; Schwarzenbacher, Robert; Luo Guorong; Wadle, Andreas; Jung, Martin; Pfreundschuh, Michael; Stenner-Liewen, Frank

    2005-01-01

    The Golgi associated retrograde protein complex (GARP) or Vps fifty-three (VFT) complex is part of cellular inter-compartmental transport systems. Here we report the identification of the VFT tethering factor complex and its interactions in mammalian cells. Subcellular fractionation shows that human Vps proteins are found in the smooth membrane/Golgi fraction but not in the cytosol. Immunostaining of human Vps proteins displays a vesicular distribution most concentrated at the perinuclear envelope. Co-staining experiments with endosomal markers imply an endosomal origin of these vesicles. Significant accumulation of VFT complex positive endosomes is found in the vicinity of the Trans Golgi Network area. This is in accordance with a putative role in Golgi associated transport processes. In Saccharomyces cerevisiae, GARP is the main effector of the small GTPase Ypt6p and interacts with the SNARE Tlg1p to facilitate membrane fusion. Accordingly, the human homologue of Ypt6p, Rab6, specifically binds hVps52. In human cells, the 'orphan' SNARE Syntaxin 10 is the genuine binding partner of GARP mediated by hVps52. This reveals a previously unknown function of human Syntaxin 10 in membrane docking and fusion events at the Golgi. Taken together, GARP shows significant conservation between various species but diversification and specialization result in important differences in human cells

  6. Diagnostic potential of recombinant scFv antibodies generated against hemagglutinin protein of influenza A virus

    Directory of Open Access Journals (Sweden)

    Roopali eRajput

    2015-09-01

    Full Text Available Human influenza A viruses have been the cause of enormous socio-economic losses worldwide. In order to combat such a notorious pathogen, hemagglutinin protein (HA has been a preferred target for generation of neutralizing-antibodies, as potent therapeutic/ diagnostic agents. In the present study, recombinant anti-HA single chain variable fragment (scFv antibodies were constructed using the phage display technology to aid in diagnosis and treatment of human influenza A virus infections. Spleen cells of mice hyper-immunized with A/New Caledonia/20/99 (H1N1 virus were used as the source for recombinant antibody (rAb production. The antigen-binding phages were quantified after 6 rounds of bio-panning against A/New Caledonia/20/99 (H1N1, A/California/07/2009 (H1N1-like, or A/Udorn/307/72(H3N2 viruses. The phage yield was maximum for the A/New Caledonia/20/99 (H1N1, however, considerable cross-reactivity was observed for the other virus strains as well. The HA-specific polyclonal rAb preparation was subjected to selection of single clones for identification of high reactive relatively conserved epitopes. The high affinity rAbs were tested against certain known conserved HA epitopes by peptide ELISA. Three recombinant mAbs showed reactivity with both the H1N1 strains and one (C5 showed binding with all the three viral strains. The C5 antibody was thus used for development of an ELISA test for diagnosis of influenza virus infection. Based on the sample size in the current analysis, the ELISA test demonstrated 83.9% sensitivity and 100% specificity. Thus, the ELISA, developed in our study, may prove as a cheaper alternative to the presently used real time RT-PCR test for detection of human influenza A viruses in clinical specimens, which will be beneficial, especially in the developing countries. Since, the two antibodies identified in this study are reactive to conserved HA epitopes; these may prove as potential therapeutic agents as well.

  7. A DEAD box protein facilitates HIV-1 replication as a cellular co-factor of Rev

    International Nuclear Information System (INIS)

    Fang Jianhua; Kubota, Satoshi; Yang Bin; Zhou Naiming; Zhang Hui; Godbout, Roseline; Pomerantz, Roger J.

    2004-01-01

    HIV-1 Rev escorts unspliced viral mRNAs out of the nucleus of infected cells, which allows formation of infectious HIV-1 virions. We have identified a putative DEAD box (Asp-Glu-Ala-Asp) RNA helicase, DDX1, as a cellular co-factor of Rev, through yeast and mammalian two-hybrid systems using the N-terminal motif of Rev as 'bait'. DDX1 is not a functional homolog of HIV-1 Rev, but down-regulation of DDX1 resulted in an alternative splicing pattern of Rev-responsive element (RRE)-containing mRNA, and attenuation of Gag p24 antigen production from HLfb rev(-) cells rescued by exogenous Rev. Co-transfection of a DDX1 expression vector with HIV-1 significantly increased viral production. DDX1 binding to Rev, as well as to the RRE, strongly suggest that DDX1 affects Rev function through the Rev-RRE axis. Moreover, down-regulation of DDX1 altered the steady state subcellular distribution of Rev, from nuclear/nucleolar to cytoplasmic dominance. These findings indicate that DDX1 is a critical cellular co-factor for Rev function, which maintains the proper subcellular distribution of this lentiviral regulatory protein. Therefore, alterations in DDX1-Rev interactions could induce HIV-1 persistence and targeting DDX1 may lead to rationally designed and novel anti-HIV-1 strategies and therapeutics

  8. Genetics Home Reference: beta-propeller protein-associated neurodegeneration

    Science.gov (United States)

    ... signs and symptoms, affected individuals can live into middle age. Death may result from complications of dementia or ... What is precision medicine? What is newborn screening? New Pages RAB18 deficiency Depression Pelizaeus-Merzbacher-like disease ...

  9. Characterization of a Fasciola gigantica protein carrying two DM9 domains reveals cellular relocalization property.

    Science.gov (United States)

    Phadungsil, Wansika; Smooker, Peter M; Vichasri-Grams, Suksiri; Grams, Rudi

    2016-01-01

    Even at the present age of whole-organism analysis, e.g., genomics, transcriptomics, and proteomics, the biological roles of many proteins remain unresolved. Classified among the proteins of unknown function is a family of proteins harboring repeats of the DM9 domain, a 60-75 amino acids motif first described in a small number of Drosophila melanogaster proteins. Proteins may carry two or more DM9 domains either in combination with other domains or as their sole constituent. Here we have characterized a 16.8 kDa Fasciola gigantica protein comprising two tandem repeated DM9 domains (FgDM9-1). The protein was located in the parenchyma of the immature and mature parasite and consequently it was not detected in the ES product of the parasite but only in the whole worm extract. Interestingly, extraction with SDS yielded a substantially higher amount of the protein suggesting association with insoluble cell components. In Sf9 insect cells a heterologously expressed EGFP-FgDM9-1 chimera showed cell-wide distribution but relocated to vesicle-like structures in the cytoplasm after stimulating cellular stress by bacteria, heat shock or chloroquine. These structures did not colocalize with the markers of endocytosis/phagocytosis ubiquitin, RAB7, GABARAP. The same behavior was noted for Aedes aegypti PRS1, a homologous mosquito DM9 protein as a positive control while EGFP did not exhibit such relocation in the insect cells. Cross-linking experiments on soluble recombinant FgDM9-1 indicated that the protein can undergo specific oligomerization. It is speculated that proteins carrying the DM9 domain have a role in vesicular transport in flatworms and insects. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1

    Science.gov (United States)

    Renigunta, Vijay; Fischer, Thomas; Zuzarte, Marylou; Kling, Stefan; Zou, Xinle; Siebert, Kai; Limberg, Maren M.; Rinné, Susanne; Decher, Niels; Schlichthörl, Günter; Daut, Jürgen

    2014-01-01

    The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion. PMID:24743596

  11. 28 CFR 570.42 - Non-medical escorted trips.

    Science.gov (United States)

    2010-07-01

    ... member of an inmate's immediate family. For purposes of this rule, immediate family refers to mother, father, brother, sister, spouse, children, step-parents, and foster parents. (2) Non-emergency, non... persons (e.g., attending physician, hospital staff, funeral home staff, family members, U.S. Probation...

  12. Total protein

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003483.htm Total protein To use the sharing features on this page, please enable JavaScript. The total protein test measures the total amount of two classes ...

  13. Proteins engineering

    International Nuclear Information System (INIS)

    2000-01-01

    At the - Departement d'Ingenierie et d'etudes de proteines (Deip) of the CEA more than seventy researchers are working hard to understand the function of proteins. For that they use the molecular labelling technique (F.M.)

  14. Whey Protein

    Science.gov (United States)

    ... reliable information about the safety of taking whey protein if you are pregnant or breast feeding. Stay on the safe side and avoid use. Milk allergy: If you are allergic to cow's milk, avoid using whey protein.

  15. Analysis of differentially expressed proteins in Yersinia enterocolitica-infected HeLa cells.

    Science.gov (United States)

    Alugubelly, Navatha; Hercik, Kamil; Kibler, Peter; Nanduri, Bindu; Edelmann, Mariola J

    2016-05-01

    Yersinia enterocolitica is a facultative intracellular pathogen and a causative agent of yersiniosis, which can be contracted by ingestion of contaminated food. Yersinia secretes virulence factors to subvert critical pathways in the host cell. In this study we utilized shotgun label-free proteomics to study differential protein expression in epithelial cells infected with Y.enterocolitica. We identified a total of 551 proteins, amongst which 42 were downregulated (including Prostaglandin E Synthase 3, POH-1 and Karyopherin alpha) and 22 were upregulated (including Rab1 and RhoA) in infected cells. We validated some of these results by western blot analysis of proteins extracted from Caco-2 and HeLa cells. The proteomic dataset was used to identify host canonical pathways and molecular functions modulated by this infection in the host cells. This study constitutes a proteome of Yersinia-infected cells and can support new discoveries in the area of host-pathogen interactions. We describe a proteome of Yersinia enterocolitica-infected HeLa cells, including a description of specific proteins differentially expressed upon infection, molecular functions as well as pathways altered during infection. This proteomic study can lead to a better understanding of Y. enterocolitica pathogenesis in human epithelial cells. Copyright © 2016. Published by Elsevier B.V.

  16. Topological and functional properties of the small GTPases protein interaction network.

    Directory of Open Access Journals (Sweden)

    Anna Delprato

    Full Text Available Small GTP binding proteins of the Ras superfamily (Ras, Rho, Rab, Arf, and Ran regulate key cellular processes such as signal transduction, cell proliferation, cell motility, and vesicle transport. A great deal of experimental evidence supports the existence of signaling cascades and feedback loops within and among the small GTPase subfamilies suggesting that these proteins function in a coordinated and cooperative manner. The interplay occurs largely through association with bi-partite regulatory and effector proteins but can also occur through the active form of the small GTPases themselves. In order to understand the connectivity of the small GTPases signaling routes, a systems-level approach that analyzes data describing direct and indirect interactions was used to construct the small GTPases protein interaction network. The data were curated from the Search Tool for the Retrieval of Interacting Genes (STRING database and include only experimentally validated interactions. The network method enables the conceptualization of the overall structure as well as the underlying organization of the protein-protein interactions. The interaction network described here is comprised of 778 nodes and 1943 edges and has a scale-free topology. Rac1, Cdc42, RhoA, and HRas are identified as the hubs. Ten sub-network motifs are also identified in this study with themes in apoptosis, cell growth/proliferation, vesicle traffic, cell adhesion/junction dynamics, the nicotinamide adenine dinucleotide phosphate (NADPH oxidase response, transcription regulation, receptor-mediated endocytosis, gene silencing, and growth factor signaling. Bottleneck proteins that bridge signaling paths and proteins that overlap in multiple small GTPase networks are described along with the functional annotation of all proteins in the network.

  17. MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

    Science.gov (United States)

    Shelby, Shameka J; Colwill, Karen; Dhe-Paganon, Sirano; Pawson, Tony; Thompson, Debra A

    2013-01-01

    The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE). A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2)-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999)), purified and phosphorylated. Ni(2+)-NTA pull downs were performed using 6xHis-rMERTK(571-999) in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999) and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α), VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS), siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

  18. MERTK interactions with SH2-domain proteins in the retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Shameka J Shelby

    Full Text Available The receptor tyrosine kinase MERTK plays an essential role in the phagocytic uptake of shed photoreceptor membranes by the retinal pigment epithelium (RPE. A fundamental aspect of signal transduction by receptor tyrosine kinases involves autophosphorylation of tyrosine residues that recruit Src-homology 2 (SH2-domain proteins to the receptor intracellular domain. The goal of the current study was to evaluate the interactions of human MERTK with SH2-domain proteins present in the RPE. The MERTK intracellular domain was expressed as a 6xHis-fusion protein (6xHis-rMERTK(571-999, purified and phosphorylated. Ni(2+-NTA pull downs were performed using 6xHis-rMERTK(571-999 in incubations with recombinant phosphotyrosine-recognition sequences expressed as GST-fusion proteins. In addition, pull downs of native SH2-domain proteins were performed using 6xHis-rMERTK(571-999 and protein homogenates from rat RPE/choroid. For both recombinant and native proteins, western analysis detected MERTK interactions with GRB2, PIK3R1 (P85α, VAV3, and SRC. Immunohistochemical analysis localized each protein to mouse RPE. In cultured RPE-J cells incubated with rod outer segments (OS, siRNA knockdown of Grb2 had no effect on OS binding, but significantly reduced OS uptake. Pik3r1 localized to early phagosomes along with Rab5 and Eea1. Phosphorylation and activation of Src was detected downstream of phagocytosis and Mertk activation. These findings suggest that MERTK signaling in the RPE involves a cohort of SH2-domain proteins with the potential to regulate both cytoskeletal rearrangement and membrane movement. Identification of the SH2-domain signaling partners of MERTK is an important step toward further defining the mechanism of RPE phagocytosis that is central to the function and survival of the retina.

  19. Sterol regulatory element binding protein and dietary lipid regulation of fatty acid synthesis in the mammary epithelium.

    Science.gov (United States)

    Rudolph, Michael C; Monks, Jenifer; Burns, Valerie; Phistry, Meridee; Marians, Russell; Foote, Monica R; Bauman, Dale E; Anderson, Steven M; Neville, Margaret C

    2010-12-01

    The lactating mammary gland synthesizes large amounts of triglyceride from fatty acids derived from the blood and from de novo lipogenesis. The latter is significantly increased at parturition and decreased when additional dietary fatty acids become available. To begin to understand the molecular regulation of de novo lipogenesis, we tested the hypothesis that the transcription factor sterol regulatory element binding factor (SREBF)-1c is a primary regulator of this system. Expression of Srebf1c mRNA and six of its known target genes increased ≥2.5-fold at parturition. However, Srebf1c-null mice showed only minor deficiencies in lipid synthesis during lactation, possibly due to compensation by Srebf1a expression. To abrogate the function of both isoforms of Srebf1, we bred mice to obtain a mammary epithelial cell-specific deletion of SREBF cleavage-activating protein (SCAP), the SREBF escort protein. These dams showed a significant lactation deficiency, and expression of mRNA for fatty acid synthase (Fasn), insulin-induced gene 1 (Insig1), mitochondrial citrate transporter (Slc25a1), and stearoyl-CoA desaturase 2 (Scd2) was reduced threefold or more; however, the mRNA levels of acetyl-CoA carboxylase-1α (Acaca) and ATP citrate lyase (Acly) were unchanged. Furthermore, a 46% fat diet significantly decreased de novo fatty acid synthesis and reduced the protein levels of ACACA, ACLY, and FASN significantly, with no change in their mRNA levels. These data lead us to conclude that two modes of regulation exist to control fatty acid synthesis in the mammary gland of the lactating mouse: the well-known SREBF1 system and a novel mechanism that acts at the posttranscriptional level in the presence of SCAP deletion and high-fat feeding to alter enzyme protein.

  20. Agonist-induced down-regulation of endogenous protein kinase c α through an endolysosomal mechanism.

    Science.gov (United States)

    Lum, Michelle A; Pundt, Krista E; Paluch, Benjamin E; Black, Adrian R; Black, Jennifer D

    2013-05-03

    Protein kinase C (PKC) isozymes undergo down-regulation upon sustained stimulation. Previous studies have pointed to the existence of both proteasome-dependent and -independent pathways of PKCα processing. Here we demonstrate that these down-regulation pathways are engaged in different subcellular compartments; proteasomal degradation occurs mainly at the plasma membrane, whereas non-proteasomal processing occurs in the perinuclear region. Using cholesterol depletion, pharmacological inhibitors, RNA interference, and dominant-negative mutants, we define the mechanisms involved in perinuclear accumulation of PKCα and identify the non-proteasomal mechanism mediating its degradation. We show that intracellular accumulation of PKCα involves at least two clathrin-independent, cholesterol/lipid raft-mediated pathways that do not require ubiquitination of the protein; one is dynamin-dependent and likely involves caveolae, whereas the other is dynamin- and small GTPase-independent. Internalized PKCα traffics through endosomes and is delivered to the lysosome for degradation. Supportive evidence includes (a) detection of the enzyme in EEA1-positive early endosomes, Rab7-positive late endosomes/multivesicular bodies, and LAMP1-positive lysosomes and (b) inhibition of its down-regulation by lysosome-disrupting agents and leupeptin. Only limited dephosphorylation of PKCα occurs during trafficking, with fully mature enzyme being the main target for lysosomal degradation. These studies define a novel and widespread mechanism of desensitization of PKCα signaling that involves endocytic trafficking and lysosome-mediated degradation of the mature, fully phosphorylated protein.

  1. Role for protein geranylgeranylation in adult T-cell leukemia cell survival

    International Nuclear Information System (INIS)

    Nonaka, Mizuho; Uota, Shin; Saitoh, Yasunori; Takahashi, Mayumi; Sugimoto, Haruyo; Amet, Tohti; Arai, Ayako; Miura, Osamu; Yamamoto, Naoki; Yamaoka, Shoji

    2009-01-01

    Adult T-cell leukemia (ATL) is a fatal lymphoproliferative disease that develops in human T-cell leukemia virus type I (HTLV-I)-infected individuals. Despite the accumulating knowledge of the molecular biology of HTLV-I-infected cells, effective therapeutic strategies remain to be established. Recent reports showed that the hydroxyl-3-methylglutaryl (HMG)-CoA reductase inhibitor statins have anti-proliferative and apoptotic effects on certain tumor cells through inhibition of protein prenylation. Here, we report that statins hinder the survival of ATL cells and induce apoptotic cell death. Inhibition of protein geranylgeranylation is responsible for these effects, since simultaneous treatment with isoprenoid precursors, geranylgeranyl pyrophosphate or farnesyl pyrophosphate, but not a cholesterol precursor squalene, restored the viability of ATL cells. Simvastatin inhibited geranylgeranylation of small GTPases Rab5B and Rac1 in ATL cells, and a geranylgeranyl transferase inhibitor GGTI-298 reduced ATL cell viability more efficiently than a farnesyl transferase inhibitor FTI-277. These results not only unveil an important role for protein geranylgeranylation in ATL cell survival, but also implicate therapeutic potentials of statins in the treatment of ATL

  2. Haploinsufficiency of Dmxl2, encoding a synaptic protein, causes infertility associated with a loss of GnRH neurons in mouse.

    Directory of Open Access Journals (Sweden)

    Brooke Tata

    2014-09-01

    Full Text Available Characterization of the genetic defects causing gonadotropic deficiency has made a major contribution to elucidation of the fundamental role of Kisspeptins and Neurokinin B in puberty onset and reproduction. The absence of puberty may also reveal neurodevelopmental disorders caused by molecular defects in various cellular pathways. Investigations of these neurodevelopmental disorders may provide information about the neuronal processes controlling puberty onset and reproductive capacity. We describe here a new syndrome observed in three brothers, which involves gonadotropic axis deficiency, central hypothyroidism, peripheral demyelinating sensorimotor polyneuropathy, mental retardation, and profound hypoglycemia, progressing to nonautoimmune insulin-dependent diabetes mellitus. High-throughput sequencing revealed a homozygous in-frame deletion of 15 nucleotides in DMXL2 in all three affected patients. This homozygous deletion was associated with lower DMXL2 mRNA levels in the blood lymphocytes of the patients. DMXL2 encodes the synaptic protein rabconnectin-3α, which has been identified as a putative scaffold protein for Rab3-GAP and Rab3-GEP, two regulators of the GTPase Rab3a. We found that rabconnectin-3α was expressed in exocytosis vesicles in gonadotropin-releasing hormone (GnRH axonal extremities in the median eminence of the hypothalamus. It was also specifically expressed in cells expressing luteinizing hormone (LH and follicle-stimulating hormone (FSH within the pituitary. The conditional heterozygous deletion of Dmxl2 from mouse neurons delayed puberty and resulted in very low fertility. This reproductive phenotype was associated with a lower number of GnRH neurons in the hypothalamus of adult mice. Finally, Dmxl2 knockdown in an insulin-secreting cell line showed that rabconnectin-3α controlled the constitutive and glucose-induced secretion of insulin. In conclusion, this study shows that low levels of DMXL2 expression cause a

  3. Comparative analysis of the role of small G proteins in cell migration and cell death: Cytoprotective and promigratory effects of RalA

    International Nuclear Information System (INIS)

    Jeon, Hyejin; Zheng, Long Tai; Lee, Shinrye; Lee, Won-Ha; Park, Nammi; Park, Jae-Yong; Heo, Won Do; Lee, Myung-Shik; Suk, Kyoungho

    2011-01-01

    Small G protein superfamily consists of more than 150 members, and is classified into six families: the Ras, Rho, Rab, Arf, Ran, and RGK families. They regulate a wide variety of cell functions such as cell proliferation/differentiation, cytoskeletal reorganization, vesicle trafficking, nucleocytoplasmic transport and microtubule organization. The small G proteins have also been shown to regulate cell death/survival and cell shape. In this study, we compared the role of representative members of the six families of small G proteins in cell migration and cell death/survival, two cellular phenotypes that are associated with inflammation, tumorigenesis, and metastasis. Our results show that small G proteins of the six families differentially regulate cell death and cell cycle distribution. In particular, our results indicate that Rho family of small G proteins is antiapoptotic. Ras, Rho, and Ran families promoted cell migration. There was no significant correlation between the cell death- and cell migration-regulating activities of the small G proteins. Nevertheless, RalA was not only cytoprotective against multiple chemotherapeutic drugs, but also promigratory inducing stress fiber formation, which was accompanied by the activation of Akt and Erk pathways. Our study provides a framework for further systematic investigation of small G proteins in the perspectives of cell death/survival and motility in inflammation and cancer.

  4. The B7-1 cytoplasmic tail enhances intracellular transport and mammalian cell surface display of chimeric proteins in the absence of a linear ER export motif.

    Directory of Open Access Journals (Sweden)

    Yi-Chieh Lin

    Full Text Available Membrane-tethered proteins (mammalian surface display are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.

  5. Developmental shaping of dendritic arbors in Drosophila relies on tightly regulated intra-neuronal activity of protein kinase A (PKA).

    Science.gov (United States)

    Copf, Tijana

    2014-09-15

    Dendrites develop morphologies characterized by multiple levels of complexity that involve neuron type specific dendritic length and particular spatial distribution. How this is developmentally regulated and in particular which signaling molecules are crucial in the process is still not understood. Using Drosophila class IV dendritic arborization (da) neurons we test in vivo the effects of cell-autonomous dose-dependent changes in the activity levels of the cAMP-dependent Protein Kinase A (PKA) on the formation of complex dendritic arbors. We find that genetic manipulations of the PKA activity levels affect profoundly the arbor complexity with strongest impact on distal branches. Both decreasing and increasing PKA activity result in a reduced complexity of the arbors, as reflected in decreased dendritic length and number of branching points, suggesting an inverted U-shape response to PKA. The phenotypes are accompanied by changes in organelle distribution: Golgi outposts and early endosomes in distal dendritic branches are reduced in PKA mutants. By using Rab5 dominant negative we find that PKA interacts genetically with the early endosomal pathway. We test if the possible relationship between PKA and organelles may be the result of phosphorylation of the microtubule motor dynein components or Rab5. We find that Drosophila cytoplasmic dynein components are direct PKA phosphorylation targets in vitro, but not in vivo, thus pointing to a different putative in vivo target. Our data argue that tightly controlled dose-dependent intra-neuronal PKA activity levels are critical in determining the dendritic arbor complexity, one of the possible ways being through the regulation of organelle distribution. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Protein politics

    NARCIS (Netherlands)

    Vijver, Marike

    2005-01-01

    This study is part of the program of the interdisciplinary research group Profetas (protein foods, environment, technology and society). Profetas consists of technological, environmental and socio-economic research projects on protein food systems which result in the development of scenarios and

  7. Protein adhesives

    Science.gov (United States)

    Charles R. Frihart; Linda F. Lorenz

    2018-01-01

    Nature uses a wide variety of chemicals for providing adhesion internally (e.g., cell to cell) and externally (e.g., mussels to ships and piers). This adhesive bonding is chemically and mechanically complex, involving a variety of proteins, carbohydrates, and other compounds.Consequently,the effect of protein structures on adhesive properties is only partially...

  8. Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases.

    Science.gov (United States)

    Bahr, Ben A; Wisniewski, Meagan L; Butler, David

    2012-04-01

    Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ(1-38) peptide corresponded with decreased levels of Aβ(1-42), supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders to enhance

  9. Tau protein

    DEFF Research Database (Denmark)

    Frederiksen, Jette Lautrup Battistini; Kristensen, Kim; Bahl, Jmc

    2011-01-01

    Background: Tau protein has been proposed as biomarker of axonal damage leading to irreversible neurological impairment in MS. CSF concentrations may be useful when determining risk of progression from ON to MS. Objective: To investigate the association between tau protein concentration and 14......-3-3 protein in the cerebrospinal fluid (CSF) of patients with monosymptomatic optic neuritis (ON) versus patients with monosymptomatic onset who progressed to multiple sclerosis (MS). To evaluate results against data found in a complete literature review. Methods: A total of 66 patients with MS and/or ON from...... the Department of Neurology of Glostrup Hospital, University of Copenhagen, Denmark, were included. CSF samples were analysed for tau protein and 14-3-3 protein, and clinical and paraclinical information was obtained from medical records. Results: The study shows a significantly increased concentration of tau...

  10. The mechanism of lauric acid-modified protein nanocapsules escape from intercellular trafficking vesicles and its implication for drug delivery.

    Science.gov (United States)

    Jiang, Lijuan; Liang, Xin; Liu, Gan; Zhou, Yun; Ye, Xinyu; Chen, Xiuli; Miao, Qianwei; Gao, Li; Zhang, Xudong; Mei, Lin

    2018-11-01

    Protein nanocapsules have exhibited promising potential applications in the field of protein drug delivery. A major issue with various promising nano-sized biotherapeutics including protein nanocapsules is that owing to their particle size they are subject to cellular uptake via endocytosis, and become entrapped and then degraded within endolysosomes, which can significantly impair their therapeutic efficacy. In addition, many nano-sized biotherapeutics could be also sequestered by autophagosomes and degraded through the autolysosomal pathway. Thus, a limiting step in achieving an effective protein therapy is to facilitate the endosomal escape and auto-lysosomal escape to ensure cytosolic delivery of the protein drugs. Here, we prepared a protein nanocapsule based on BSA (nBSA) and the BSA nanocapsules modified with a bilayer of lauric acid (LA-nBSA) to investigate the escape effects from the endosome and autophagosome. The size distribution of nBSA and LA-nBSA analyzed using DLS presents a uniform diameter centered at 10 nm and 16 nm. The data also showed that FITC-labeled nBSA and LA-nBSA were taken up by the cells mainly through Arf-6-dependent endocytosis and Rab34-mediated macropinocytosis. In addition, LA-nBSA could efficiently escape from endosomal before the degradation in endo-lysosomes. Autophagy could also sequester the LA-nBSA through p62 autophagosome vesicles. These two types of nanocapsules underwent different intracellular destinies and lauric acid (LA) coating played a vital role in intracellular particle retention. In conclusion, the protein nanocapsules modified with LA could enhance the protein nanocapsules escape from intercellular trafficking vesicles, and protect the protein from degradation by the lysosomes.

  11. Biochemical characterization of native Usher protein complexes from a vesicular subfraction of tracheal epithelial cells.

    Science.gov (United States)

    Zallocchi, Marisa; Sisson, Joseph H; Cosgrove, Dominic

    2010-02-16

    Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in nondetergent buffer and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15, and VLGR-1 and a different one at the top of the gradient that included all of the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100-200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins cosediment into the gradient at a sedimentation coefficient of approximately 50 S, correlating with a predicted molecular mass of 2 x 10(6) Da. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher protein complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors.

  12. BIOCHEMICAL CHARACTERIZATION OF NATIVE USHER PROTEIN COMPLEXES FROM A VESICULAR SUBFRACTION OF TRACHEAL EPITHELIAL CELLS†

    Science.gov (United States)

    Zallocchi, Marisa; Sisson, Joseph H.; Cosgrove, Dominic

    2010-01-01

    Usher syndrome is the major cause of deaf/blindness in the world. It is a genetic heterogeneous disorder, with nine genes already identified as causative for the disease. We noted expression of all known Usher proteins in bovine tracheal epithelial cells, and exploited this system for large-scale biochemical analysis of Usher protein complexes. The dissected epithelia were homogenized in non-detergent buffer, and sedimented on sucrose gradients. At least two complexes were evident after the first gradient: one formed by specific isoforms of CDH23, PCDH15 and VLGR-1, and a different one at the top of the gradient that included all the Usher proteins and rab5, a transport vesicle marker. TEM analysis of these top fractions found them enriched in 100–200 nm vesicles, confirming a vesicular association of the Usher complex(es). Immunoisolation of these vesicles confirmed some of the associations already predicted and identified novel interactions. When the vesicles are lysed in the presence of phenylbutyrate, most of the Usher proteins co-sediment into the gradient at a sedimentation coefficient of approximately 50S, correlating with a predicted molecular mass of 2 × 106 Daltons. Although it is still unclear whether there is only one complex or several independent complexes that are trafficked within distinct vesicular pools, this work shows for the first time that native Usher proteins complexes occur in vivo. This complex(es) is present primarily in transport vesicles at the apical pole of tracheal epithelial cells, predicting that Usher proteins may be directionally transported as complexes in hair cells and photoreceptors. PMID:20058854

  13. Posttranslational modifications, localization, and protein interactions of optineurin, the product of a glaucoma gene.

    Directory of Open Access Journals (Sweden)

    Hongyu Ying

    Full Text Available BACKGROUND: Glaucoma is a major blinding disease. The most common form of this disease, primary open angle glaucoma (POAG, is genetically heterogeneous. One of the candidate genes, optineurin, is linked principally to normal tension glaucoma, a subtype of POAG. The present study was undertaken to illustrate the basic characteristics of optineurin. METHODOLOGY/PRINCIPAL FINDINGS: Lysates from rat retinal ganglion RGC5 cells were subjected to N- or O-deglycosylation or membrane protein extraction. The phosphorylation status was evaluated after immunoprecipitation. It was found that while phosphorylated, optineurin was neither N- nor O-glycosylated, and was by itself not a membrane protein. RGC5 and human retinal pigment epithelial cells were double stained with anti-optineurin and anti-GM130. The endogenous optineurin exhibited a diffuse, cytoplasmic distribution, but a population of the protein was associated with the Golgi apparatus. Turnover experiments showed that the endogenous optineurin was relatively short-lived, with a half-life of approximately 8 hours. Native blue gel electrophoresis revealed that the endogenous optineurin formed homohexamers. Optineurin also interacted with molecules including Rab8, myosin VI, and transferrin receptor to assemble into supermolecular complexes. When overexpressed, optineurin-green fluorescence protein (GFP fusion protein formed punctate structures termed "foci" in the perinuclear region. Treatment of nocadazole resulted in dispersion of the optineurin foci. In addition, tetracycline-regulated optineurin-GFPs expressing RGC5 stable cell lines were established for the first time. CONCLUSIONS/SIGNIFICANCE: The present study provides new information regarding basic characteristics of optineurin that are important for future efforts in defining precisely how optineurin functions normally and how mutations may result in pathology. The inducible optineurin-GFP-expressing cell lines are also anticipated to

  14. Dioscorea alata tuber proteome analysis shows over thirty dioscorin isoforms and novel tuber proteins.

    Science.gov (United States)

    Sharma, Shruti; Gupta, Ravi; Deswal, Renu

    2017-05-01

    In Dioscorea, dioscorin (31 kDa) is the major storage protein constituting 85% of the total tuber proteins. An integrated proteomic and biochemical approach was used to understand the physiological role of dioscorin in the two contrasting growth stages (germinating and mature tuber). HPLC analysis showed 3 fold reduction in mannitol and 12.88 and 1.24 fold increase in sucrose and maltose in the germinating tuber. A 1.8 and 3 fold increase in sucrose phosphate synthase and mannitol dehydrogenase activity respectively was observed in the germinating tuber while a 2 fold higher invertase probably lowers the sucrose accumulation in the mature tuber. SDS-PAGE and 2-D maps of the mature and germinating tubers confirmed depletion (more than 50%) of dioscorin on germination. Dioscorin was purified using ion exchange and gel filtration chromatography with 43.32 fold purification and 38.16 yield. Out of a trail of 35 spots at 31 kDa only 12 spots (identified as dioscorin isoforms) were present in the 2D gel of the purified fraction. To search for other tuber proteins besides dioscorin, the unbound fractions of DEAE column were analysed by 2DGE. DREB 1A, caffeic acid 3-O-methyltransferase and Rab-1 small GTP binding protein were identified perhaps for the first time in the Dioscorea proteome. The interactome analysis revealed these to be involved in oxidative stress, carotenoid synthesis and vesicular transport. This is perhaps the first attempt to identify tuber proteome (although limited) and to understand the physiological significance of these proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  15. In silico data analyses of the hotspot mutations of CHM gene in choroideremia disease

    Directory of Open Access Journals (Sweden)

    Saber Imani

    2018-06-01

    Full Text Available This data article provides compelling computational analysis of the hotspot CHM gene mutations that contribute to the progressive causativeness and susceptibility of Choroideremia in patients. We performed structural and molecular dynamics (MD simulation analysis on abnormal states of the CHM protein caused by deleterious and disease-causing hotspot mutant forms of CHM: S89C, E177K, and V529H. Within 40 ns, MD simulation time composed of the E177K mutant shows conformational alteration especially in several parts of the variant. Mathematically, we applied eigenvector analysis to determine the modes of flexibility and atomic positional fluctuations that contribute significantly to the overall motion of the CHM protein in terms of structural alteration, free energy landscapes (FEL, entropy, enthalpy, and principal component analysis (PCA.The data described here are related to the article entitled “Molecular Genetics Characterization and Homology Modeling of the CHM Gene Mutation: A study on Its Association with Choroideremia” (Imani et al., 2018 [1]. Keywords: In silico, Choroideremia, Rab escort protein 1, Molecular dynamic simulation

  16. Protein nanoparticles for therapeutic protein delivery.

    Science.gov (United States)

    Herrera Estrada, L P; Champion, J A

    2015-06-01

    Therapeutic proteins can face substantial challenges to their activity, requiring protein modification or use of a delivery vehicle. Nanoparticles can significantly enhance delivery of encapsulated cargo, but traditional small molecule carriers have some limitations in their use for protein delivery. Nanoparticles made from protein have been proposed as alternative carriers and have benefits specific to therapeutic protein delivery. This review describes protein nanoparticles made by self-assembly, including protein cages, protein polymers, and charged or amphipathic peptides, and by desolvation. It presents particle fabrication and delivery characterization for a variety of therapeutic and model proteins, as well as comparison of the features of different protein nanoparticles.

  17. Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

    Science.gov (United States)

    Wavre-Shapton, Silène T; Tolmachova, Tanya; Lopes da Silva, Mafalda; da Silva, Mafalda Lopes; Futter, Clare E; Seabra, Miguel C

    2013-01-01

    The retinal pigment epithelium (RPE) is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM) is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1). REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox), Tyr-Cre+). Transmission electron microscopy (TEM) was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox), Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD) suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

  18. Conditional ablation of the choroideremia gene causes age-related changes in mouse retinal pigment epithelium.

    Directory of Open Access Journals (Sweden)

    Silène T Wavre-Shapton

    Full Text Available The retinal pigment epithelium (RPE is a pigmented monolayer of cells lying between the photoreceptors and a layer of fenestrated capillaries, the choriocapillaris. Choroideremia (CHM is an X-linked progressive degeneration of these three layers caused by the loss of function of Rab Escort protein-1 (REP1. REP1 is involved in the prenylation of Rab proteins, key regulators of membrane trafficking. To study the pathological consequences of chronic disruption of membrane traffic in the RPE we used a cell type-specific knock-out mouse model of the disease, where the Chm/Rep1 gene is deleted only in pigmented cells (Chm(Flox, Tyr-Cre+. Transmission electron microscopy (TEM was used to quantitate the melanosome distribution in the RPE and immunofluorescent staining of rhodopsin was used to quantitate phagocytosed rod outer segments in retinal sections. The ultrastructure of the RPE and Bruch's membrane at different ages was characterised by TEM to analyse age-related changes occurring as a result of defects in membrane traffic pathways. Chm/Rep1 gene knockout in RPE cells resulted in reduced numbers of melanosomes in the apical processes and delayed phagosome degradation. In addition, the RPE accumulated pathological changes at 5-6 months of age similar to those observed in 2-year old controls. These included the intracellular accumulation of lipofuscin-containing deposits, disorganised basal infoldings and the extracellular accumulation of basal laminar and basal linear deposits. The phenotype of the Chm(Flox, Tyr-Cre+ mice suggests that loss of the Chm/Rep1 gene causes premature accumulation of features of aging in the RPE. Furthermore, the striking similarities between the present observations and some of the phenotypes reported in age-related macular degeneration (AMD suggest that membrane traffic defects may contribute to the pathogenesis of AMD.

  19. Molecular characterization of a novel X-linked syndrome involving developmental delay and deafness.

    Science.gov (United States)

    Hildebrand, Michael S; de Silva, Michelle G; Tan, Tiong Yang; Rose, Elizabeth; Nishimura, Carla; Tolmachova, Tanya; Hulett, Joanne M; White, Susan M; Silver, Jeremy; Bahlo, Melanie; Smith, Richard J H; Dahl, Hans-Henrik M

    2007-11-01

    X-linked syndromes associated with developmental delay and sensorineural hearing loss (SNHL) have been characterized at the molecular level, including Mohr-Tranebjaerg syndrome and Norrie disease. In this study we report on a novel X-linked recessive, congenital syndrome in a family with developmental delay and SNHL that maps to a locus associated with mental retardation (MR) for which no causative gene has been identified. The X-linked recessive inheritance and congenital nature of the syndrome was confirmed by detailed clinical investigation and the family history. Linkage mapping of the X-chromosome was conducted to ascertain the disease locus and candidate genes were screened by direct sequencing and STRP analysis. The recessive syndrome was mapped to Xp11.3-q21.32 and a deletion was identified in a regulatory region upstream of the POU3F4 gene in affected family members. Since mutations in POU3F4 cause deafness at the DFN3 locus, the deletion is the likely cause of the SNHL in this family. The choroideremia (CHM) gene was also screened and a novel missense change was identified. The alteration changes the serine residue at position 89 in the Rab escort 1 protein (REP-1) to a cysteine (S89C). Prenylation of Rab proteins was investigated in patients and the location of REP-1 expression in the brain determined. However, subsequent analysis revealed that this change in CHM was polymorphic having no effect on REP-1 function. Although the causative gene at the MR locus in this family has not been identified, there are a number of genes involved in syndromic and nonsyndromic forms of MR that are potential candidates. Copyright 2007 Wiley-Liss, Inc.

  20. Protein-Protein Interaction Databases

    DEFF Research Database (Denmark)

    Szklarczyk, Damian; Jensen, Lars Juhl

    2015-01-01

    Years of meticulous curation of scientific literature and increasingly reliable computational predictions have resulted in creation of vast databases of protein interaction data. Over the years, these repositories have become a basic framework in which experiments are analyzed and new directions...

  1. A unique deubiquitinase that deconjugates phosphoribosyl-linked protein ubiquitination

    Energy Technology Data Exchange (ETDEWEB)

    Qiu, Jiazhang; Yu, Kaiwen; Fei, Xiaowen; Liu, Yao; Nakayasu, Ernesto S.; Piehowski, Paul D.; Shaw, Jared B.; Puvar, Kedar; Das, Chittaranjan; Liu, Xiaoyun; Luo, Zhao-Qing

    2017-05-12

    Ubiquitination regulates many aspects of host immunity and thus is a common target for infectious agents. Recent studies revealed that members of the SidE effector family of the bacterial pathogen Legionella pneumophila attacked several small GTPases associated with the endoplasmic reticulum by a novel ubiquitination mechanism that does not require the E1 and E2 enzymes of the host ubiquitination machinery. Following ubiquitin activation by ADP- ribosylation via a mono-ADP-ribosylation motif, ADP-ribosylated ubiquitin is cleaved by a phosphodiesterasedomainwithinSdeA,whichisconcomitantwiththelinkof phosphoribosylated ubiquitin to serine residues in the substrate. Here we demonstrate that the activity of SidEs is regulated by SidJ, another effector encoded by a gene situated in the locus coding for three members of the SidE family (SdeC, SdeB and SdeA). SidJ functions to remove ubiquitin from SidEs-modified substrates by cleaving the phosphodiester bond that links phosphoribosylated ubiquitin to protein substrates. Further, the deubiquitinase activity of SidJ is essential for its role in L. pneumophila infection. Finally, the activity of SidJ is required for efficiently reducing the abundance of ubiquitinated Rab33b in infected cells within a few hours after bacterial uptake. Our results establish SidJ as a deubiquitinase that functions to impose temporal regulation of the activity of the SidE effectors. The identification of SidJ may shed light on future study of signaling cascades mediated by this unique ubiquitination that also potentially regulates cellular processes in eukaryotic cells.

  2. Numb endocytic adapter proteins regulate the transport and processing of the amyloid precursor protein in an isoform-dependent manner: implications for Alzheimer disease pathogenesis.

    Science.gov (United States)

    Kyriazis, George A; Wei, Zelan; Vandermey, Miriam; Jo, Dong-Gyu; Xin, Ouyang; Mattson, Mark P; Chan, Sic L

    2008-09-12

    Central to the pathogenesis of Alzheimer disease is the aberrant processing of the amyloid precursor protein (APP) to generate amyloid beta-peptide (Abeta), the principle component of amyloid plaques. The cell fate determinant Numb is a phosphotyrosine binding domain (PTB)-containing endocytic adapter protein that interacts with the carboxyl-terminal domain of APP. The physiological relevance of this interaction is unknown. Mammals produce four alternatively spliced variants of Numb that differ in the length of their PTB and proline-rich region. In the current study, we determined the influence of the four human Numb isoforms on the intracellular trafficking and processing of APP. Stable expression of Numb isoforms that differ in the PTB but not in the proline-rich region results in marked differences in the sorting of APP to the recycling and degradative pathways. Neural cells expressing Numb isoforms that lack the insert in the PTB (short PTB (SPTB)) exhibited marked accumulation of APP in Rab5A-labeled early endosomal and recycling compartments, whereas those expressing isoforms with the insertion in the PTB (long PTB (LPTB)) exhibited reduced amounts of cellular APP and its proteolytic derivatives relative to parental control cells. Neither the activities of the beta- and gamma-secretases nor the expression of APP mRNA were significantly different in the stably transfected cells, suggesting that the differential effects of the Numb proteins on APP metabolism is likely to be secondary to altered APP trafficking. In addition, the expression of SPTB-Numb increases at the expense of LPTB-Numb in neuronal cultures subjected to stress, suggesting a role for Numb in stress-induced Abeta production. Taken together, these results suggest distinct roles for the human Numb isoforms in APP metabolism and may provide a novel potential link between altered Numb isoform expression and increased Abeta generation.

  3. A new Em-like protein from Lactuca sativa, LsEm1, enhances drought and salt stress tolerance in Escherichia coli and rice.

    Science.gov (United States)

    Xiang, Dian-Jun; Man, Li-Li; Zhang, Chun-Lan; Peng-Liu; Li, Zhi-Gang; Zheng, Gen-Chang

    2018-02-07

    Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these

  4. Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells.

    Science.gov (United States)

    Panneerdoss, Subbarayalu; Viswanadhapalli, Suryavathi; Abdelfattah, Nourhan; Onyeagucha, Benjamin C; Timilsina, Santosh; Mohammad, Tabrez A; Chen, Yidong; Drake, Michael; Vuori, Kristiina; Kumar, T Rajendra; Rao, Manjeet K

    2017-09-19

    Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

  5. Genetics Home Reference: RAB18 deficiency

    Science.gov (United States)

    ... Morton J, Ainsworth JR, Horn D, Rosser E, Cole TR, Stolte-Dijkstra I, Fieggen K, Clayton-Smith ... medicine? What is newborn screening? New Pages Lyme disease Fibromyalgia White-Sutton syndrome All New & Updated Pages ...

  6. Aquaporin Protein-Protein Interactions

    Directory of Open Access Journals (Sweden)

    Jennifer Virginia Roche

    2017-10-01

    Full Text Available Aquaporins are tetrameric membrane-bound channels that facilitate transport of water and other small solutes across cell membranes. In eukaryotes, they are frequently regulated by gating or trafficking, allowing for the cell to control membrane permeability in a specific manner. Protein–protein interactions play crucial roles in both regulatory processes and also mediate alternative functions such as cell adhesion. In this review, we summarize recent knowledge about aquaporin protein–protein interactions; dividing the interactions into three types: (1 interactions between aquaporin tetramers; (2 interactions between aquaporin monomers within a tetramer (hetero-tetramerization; and (3 transient interactions with regulatory proteins. We particularly focus on the structural aspects of the interactions, discussing the small differences within a conserved overall fold that allow for aquaporins to be differentially regulated in an organism-, tissue- and trigger-specific manner. A deep knowledge about these differences is needed to fully understand aquaporin function and regulation in many physiological processes, and may enable design of compounds targeting specific aquaporins for treatment of human disease.

  7. Protein immobilization strategies for protein biochips

    NARCIS (Netherlands)

    Rusmini, F.; Rusmini, Federica; Zhong, Zhiyuan; Feijen, Jan

    2007-01-01

    In the past few years, protein biochips have emerged as promising proteomic and diagnostic tools for obtaining information about protein functions and interactions. Important technological innovations have been made. However, considerable development is still required, especially regarding protein

  8. AS160 associates with the Na+,K+-ATPase and mediates the adenosine monophosphate-stimulated protein kinase-dependent regulation of sodium pump surface expression.

    Science.gov (United States)

    Alves, Daiane S; Farr, Glen A; Seo-Mayer, Patricia; Caplan, Michael J

    2010-12-01

    The Na(+),K(+)-ATPase is the major active transport protein found in the plasma membranes of most epithelial cell types. The regulation of Na(+),K(+)-ATPase activity involves a variety of mechanisms, including regulated endocytosis and recycling. Our efforts to identify novel Na(+),K(+)-ATPase binding partners revealed a direct association between the Na(+),K(+)-ATPase and AS160, a Rab-GTPase-activating protein. In COS cells, coexpression of AS160 and Na(+),K(+)-ATPase led to the intracellular retention of the sodium pump. We find that AS160 interacts with the large cytoplasmic NP domain of the α-subunit of the Na(+),K(+)-ATPase. Inhibition of the activity of the adenosine monophosphate-stimulated protein kinase (AMPK) in Madin-Darby canine kidney cells through treatment with Compound C induces Na(+),K(+)-ATPase endocytosis. This effect of Compound C is prevented through the short hairpin RNA-mediated knockdown of AS160, demonstrating that AMPK and AS160 participate in a common pathway to modulate the cell surface expression of the Na(+),K(+)-ATPase.

  9. The E5 Proteins

    OpenAIRE

    DiMaio, Daniel; Petti, Lisa

    2013-01-01

    The E5 proteins are short transmembrane proteins encoded by many animal and human papillomaviruses. These proteins display transforming activity in cultured cells and animals, and they presumably also play a role in the productive virus life cycle. The E5 proteins are thought to act by modulating the activity of cellular proteins. Here, we describe the biological activities of the best-studied E5 proteins and discuss the evidence implicating specific protein targets and pathways in mediating ...

  10. EDITORIAL: Precision proteins Precision proteins

    Science.gov (United States)

    Demming, Anna

    2010-06-01

    Since the birth of modern day medicine, during the times of Hippocrates in ancient Greece, the profession has developed from the rudimentary classification of disease into a rigorous science with an inspiring capability to treat and cure. Scientific methodology has distilled clinical diagnostic tools from the early arts of prognosis, which used to rely as much on revelation and prophecy, as intuition and judgement [1]. Over the past decade, research into the interactions between proteins and nanosystems has provided some ingenious and apt techniques for delving into the intricacies of anatomical systems. In vivo biosensing has emerged as a vibrant field of research, as much of medical diagnosis relies on the detection of substances or an imbalance in the chemicals in the body. The inherent properties of nanoscale structures, such as cantilevers, make them well suited to biosensing applications that demand the detection of molecules at very low concentrations. Measurable deflections in cantilevers functionalised with antibodies provide quantitative indicators of the presence of specific antigens when the two react. Such developments have roused mounting interest in the interactions of proteins with nanostructures, such as carbon nanotubes [3], which have demonstrated great potential as generic biomarkers. Plasmonic properties are also being exploited in sensing applications, such as the molecular sentinel recently devised by researchers in the US. The device uses the plasmonic properties of a silver nanoparticle linked to a Raman labelled hairpin DNA probe to signal changes in the probe geometry resulting from interactions with substances in the environment. Success stories so far include the detection of two specific genes associated with breast cancer [4]. A greater understanding of how RNA interference regulates gene expression has highlighted the potential of using this natural process as another agent for combating disease in personalized medicine. However, the

  11. Protein docking prediction using predicted protein-protein interface

    Directory of Open Access Journals (Sweden)

    Li Bin

    2012-01-01

    Full Text Available Abstract Background Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. Results We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm, is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. Conclusion We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  12. Protein docking prediction using predicted protein-protein interface.

    Science.gov (United States)

    Li, Bin; Kihara, Daisuke

    2012-01-10

    Many important cellular processes are carried out by protein complexes. To provide physical pictures of interacting proteins, many computational protein-protein prediction methods have been developed in the past. However, it is still difficult to identify the correct docking complex structure within top ranks among alternative conformations. We present a novel protein docking algorithm that utilizes imperfect protein-protein binding interface prediction for guiding protein docking. Since the accuracy of protein binding site prediction varies depending on cases, the challenge is to develop a method which does not deteriorate but improves docking results by using a binding site prediction which may not be 100% accurate. The algorithm, named PI-LZerD (using Predicted Interface with Local 3D Zernike descriptor-based Docking algorithm), is based on a pair wise protein docking prediction algorithm, LZerD, which we have developed earlier. PI-LZerD starts from performing docking prediction using the provided protein-protein binding interface prediction as constraints, which is followed by the second round of docking with updated docking interface information to further improve docking conformation. Benchmark results on bound and unbound cases show that PI-LZerD consistently improves the docking prediction accuracy as compared with docking without using binding site prediction or using the binding site prediction as post-filtering. We have developed PI-LZerD, a pairwise docking algorithm, which uses imperfect protein-protein binding interface prediction to improve docking accuracy. PI-LZerD consistently showed better prediction accuracy over alternative methods in the series of benchmark experiments including docking using actual docking interface site predictions as well as unbound docking cases.

  13. Shotgun protein sequencing.

    Energy Technology Data Exchange (ETDEWEB)

    Faulon, Jean-Loup Michel; Heffelfinger, Grant S.

    2009-06-01

    A novel experimental and computational technique based on multiple enzymatic digestion of a protein or protein mixture that reconstructs protein sequences from sequences of overlapping peptides is described in this SAND report. This approach, analogous to shotgun sequencing of DNA, is to be used to sequence alternative spliced proteins, to identify post-translational modifications, and to sequence genetically engineered proteins.

  14. Introduction to protein blotting.

    Science.gov (United States)

    Kurien, Biji T; Scofield, R Hal

    2009-01-01

    Protein blotting is a powerful and important procedure for the immunodetection of proteins following electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer.

  15. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  16. Evolution of protein-protein interactions

    Indian Academy of Sciences (India)

    Evolution of protein-protein interactions · Our interests in protein-protein interactions · Slide 3 · Slide 4 · Slide 5 · Slide 6 · Slide 7 · Slide 8 · Slide 9 · Slide 10 · Slide 11 · Slide 12 · Slide 13 · Slide 14 · Slide 15 · Slide 16 · Slide 17 · Slide 18 · Slide 19 · Slide 20.

  17. Protein in diet

    Science.gov (United States)

    Diet - protein ... Protein foods are broken down into parts called amino acids during digestion. The human body needs a ... to eat animal products to get all the protein you need in your diet. Amino acids are ...

  18. Protein-losing enteropathy

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/007338.htm Protein-losing enteropathy To use the sharing features on this page, please enable JavaScript. Protein-losing enteropathy is an abnormal loss of protein ...

  19. Oligomeric protein structure networks: insights into protein-protein interactions

    Directory of Open Access Journals (Sweden)

    Brinda KV

    2005-12-01

    Full Text Available Abstract Background Protein-protein association is essential for a variety of cellular processes and hence a large number of investigations are being carried out to understand the principles of protein-protein interactions. In this study, oligomeric protein structures are viewed from a network perspective to obtain new insights into protein association. Structure graphs of proteins have been constructed from a non-redundant set of protein oligomer crystal structures by considering amino acid residues as nodes and the edges are based on the strength of the non-covalent interactions between the residues. The analysis of such networks has been carried out in terms of amino acid clusters and hubs (highly connected residues with special emphasis to protein interfaces. Results A variety of interactions such as hydrogen bond, salt bridges, aromatic and hydrophobic interactions, which occur at the interfaces are identified in a consolidated manner as amino acid clusters at the interface, from this study. Moreover, the characterization of the highly connected hub-forming residues at the interfaces and their comparison with the hubs from the non-interface regions and the non-hubs in the interface regions show that there is a predominance of charged interactions at the interfaces. Further, strong and weak interfaces are identified on the basis of the interaction strength between amino acid residues and the sizes of the interface clusters, which also show that many protein interfaces are stronger than their monomeric protein cores. The interface strengths evaluated based on the interface clusters and hubs also correlate well with experimentally determined dissociation constants for known complexes. Finally, the interface hubs identified using the present method correlate very well with experimentally determined hotspots in the interfaces of protein complexes obtained from the Alanine Scanning Energetics database (ASEdb. A few predictions of interface hot

  20. Regulation of B cell differentiation by intracellular membrane associated proteins and microRNAs: role in the antibody response

    Directory of Open Access Journals (Sweden)

    Zheng eLou

    2015-10-01

    Full Text Available B cells are central to adaptive immunity and their functions in antibody responses are exquisitely regulated. As suggested by recent findings, B cell differentiation is mediated by intracellular membrane structures (including endosomes, lysosomes and autophagosomes and protein factors specifically associated with these membranes, including Rab7, Atg5 and Atg7. These factors participate in vesicle formation/trafficking, signal transduction and induction of gene expression to promote antigen presentation, CSR/SHM, and generation/maintenance of plasma cells and memory B cells. Their expression is induced in B cells activated to differentiate and further fine-tuned by immune-modulating microRNAs, which coordinates CSR/SHM, plasma cell differentiation and memory B cell differentiation. These short non-coding RNAs would individually target multiple factors associated with the same intracellular membrane compartments and collaboratively target a single factor in addition to regulate AID and Blimp-1. These, together with regulation of microRNA biogenesis and activities by endosomes and autophagosomes, show that intracellular membranes and microRNAs, two broadly relevant cell constituents, play important roles in balancing gene expression to specify B cell differentiation processes for optimal antibody responses.

  1. Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes.

    Science.gov (United States)

    Chaudhary, Natasha; Gonzalez, Eva; Chang, Sung-Hee; Geng, Fuqiang; Rafii, Shahin; Altorki, Nasser K; McGraw, Timothy E

    2016-12-20

    Insulin activation of phosphatidylinositol 3-kinase (PI3K) regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin's effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

  2. Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes

    Directory of Open Access Journals (Sweden)

    Natasha Chaudhary

    2016-12-01

    Full Text Available Insulin activation of phosphatidylinositol 3-kinase (PI3K regulates metabolism, including the translocation of the Glut4 glucose transporter to the plasma membrane and inactivation of the FoxO1 transcription factor. Adenoviral protein E4-ORF1 stimulates cellular glucose metabolism by mimicking growth-factor activation of PI3K. We have used E4-ORF1 as a tool to dissect PI3K-mediated signaling in adipocytes. E4-ORF1 activation of PI3K in adipocytes recapitulates insulin regulation of FoxO1 but not regulation of Glut4. This uncoupling of PI3K effects occurs despite E4-ORF1 activating PI3K and downstream signaling to levels achieved by insulin. Although E4-ORF1 does not fully recapitulate insulin’s effects on Glut4, it enhances insulin-stimulated insertion of Glut4-containing vesicles to the plasma membrane independent of Rab10, a key regulator of Glut4 trafficking. E4-ORF1 also stimulates plasma membrane translocation of ubiquitously expressed Glut1 glucose transporter, an effect that is likely essential for E4-ORF1 to promote an anabolic metabolism in a broad range of cell types.

  3. NCBI nr-aa BLAST: CBRC-DMEL-03-0035 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-03-0035 ref|XP_001650651.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48197.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650651.1 2e-93 54% ...

  4. NCBI nr-aa BLAST: CBRC-DMEL-03-0035 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DMEL-03-0035 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 2e-93 54% ...

  5. NCBI nr-aa BLAST: CBRC-DSIM-03-0031 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DSIM-03-0031 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 2e-93 54% ...

  6. NCBI nr-aa BLAST: CBRC-DYAK-03-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-03-0017 ref|XP_001650651.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48197.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650651.1 2e-93 54% ...

  7. NCBI nr-aa BLAST: CBRC-AGAM-01-0094 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-01-0094 ref|XP_001650651.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48197.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650651.1 1e-172 69% ...

  8. NCBI nr-aa BLAST: CBRC-DYAK-03-0017 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-DYAK-03-0017 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 2e-93 54% ...

  9. NCBI nr-aa BLAST: CBRC-AGAM-01-0094 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-AGAM-01-0094 ref|XP_001650650.1| rab6 gtpase activating protein, gapcena (rabg...ap1 protein) [Aedes aegypti] gb|EAT48196.1| rab6 gtpase activating protein, gapcena (rabgap1 protein) [Aedes aegypti] XP_001650650.1 1e-172 69% ...

  10. Protein surface shielding agents in protein crystallization

    International Nuclear Information System (INIS)

    Hašek, J.

    2011-01-01

    The crystallization process can be controlled by protein surface shielding agents blocking undesirable competitive adhesion modes during non-equilibrium processes of deposition of protein molecules on the surface of growing crystalline blocks. The hypothesis is based on a number of experimental proofs from diffraction experiments and also retrieved from the Protein Data Bank. The molecules adhering temporarily on the surface of protein molecules change the propensity of protein molecules to deposit on the crystal surface in a definite position and orientation. The concepts of competitive adhesion modes and protein surface shielding agents acting on the surface of molecules in a non-equilibrium process of protein crystallization provide a useful platform for the control of crystallization. The desirable goal, i.e. a transient preference of a single dominating adhesion mode between protein molecules during crystallization, leads to uniform deposition of proteins in a crystal. This condition is the most important factor for diffraction quality and thus also for the accuracy of protein structure determination. The presented hypothesis is a generalization of the experimentally well proven behaviour of hydrophilic polymers on the surface of protein molecules of other compounds

  11. Role of ARF6 in internalization of metal-binding proteins, metallothionein and transferrin, and cadmium-metallothionein toxicity in kidney proximal tubule cells

    International Nuclear Information System (INIS)

    Wolff, Natascha A.; Lee, Wing-Kee; Abouhamed, Marouan; Thevenod, Frank

    2008-01-01

    Filtered metal-protein complexes, such as cadmium-metallothionein-1 (CdMT-1) or transferrin (Tf) are apically endocytosed partly via megalin/cubilin by kidney proximal tubule (PT) cells where CdMT-1 internalization causes apoptosis. Small GTPase ARF (ADP-ribosylation factor) proteins regulate endocytosis and vesicular trafficking. We investigated roles of ARF6, which has been shown to be involved in internalization of ligands and endocytic trafficking in PT cells, following MT-1/CdMT-1 and Tf uptake by PT cells. WKPT-0293 Cl.2 cells derived from rat PT S1 segment were transfected with hemagglutinin-tagged wild-type (ARF6-WT) or dominant negative (ARF6-T27N) forms of ARF6. Using immunofluorescence, endogenous ARF6 was associated with the plasma membrane (PM) as well as juxtanuclear and co-localized with Rab5a and Rab11 involved in early and recycling endosomal trafficking. Immunofluorescence staining of megalin showed reduced surface labelling in ARF6 dominant negative (ARF6-DN) cells. Intracellular Alexa Fluor 546-conjugated MT-1 uptake was reduced in ARF6-DN cells and CdMT-1 (14.8 μM for 24 h) toxicity was significantly attenuated from 27.3 ± 3.9% in ARF6-WT to 11.1 ± 4.0% in ARF6-DN cells (n = 6, P < 0.02). Moreover, reduced Alexa Fluor 546-conjugated Tf uptake was observed in ARF-DN cells (75.0 ± 4.6% versus 3.9 ± 3.9% of ARF6-WT cells, n = 3, P < 0.01) and/or remained near the PM (89.3 ± 5. 6% versus 45.2 ± 14.3% of ARF6-WT cells, n = 3, P < 0.05). In conclusion, the data support roles for ARF6 in receptor-mediated endocytosis and trafficking of MT-1/Tf to endosomes/lysosomes and CdMT-1 toxicity of PT cells

  12. Mossàrabs de València i "Llengua mossàrab"

    OpenAIRE

    Barceló Torres, María del Carmen

    1996-01-01

    Since the publication of Simonet's Glosario (1888) some scholars have been collecting data of sustain the hipothetical presence of Mozarab communities and the use of a Romance Language in the Valencian area under Islam. We show in this paper how they have commited abuses and arbitrarieties on the material and we interpret it in his true historical context.

  13. Protein sequence comparison and protein evolution

    Energy Technology Data Exchange (ETDEWEB)

    Pearson, W.R. [Univ. of Virginia, Charlottesville, VA (United States). Dept. of Biochemistry

    1995-12-31

    This tutorial was one of eight tutorials selected to be presented at the Third International Conference on Intelligent Systems for Molecular Biology which was held in the United Kingdom from July 16 to 19, 1995. This tutorial examines how the information conserved during the evolution of a protein molecule can be used to infer reliably homology, and thus a shared proteinfold and possibly a shared active site or function. The authors start by reviewing a geological/evolutionary time scale. Next they look at the evolution of several protein families. During the tutorial, these families will be used to demonstrate that homologous protein ancestry can be inferred with confidence. They also examine different modes of protein evolution and consider some hypotheses that have been presented to explain the very earliest events in protein evolution. The next part of the tutorial will examine the technical aspects of protein sequence comparison. Both optimal and heuristic algorithms and their associated parameters that are used to characterize protein sequence similarities are discussed. Perhaps more importantly, they survey the statistics of local similarity scores, and how these statistics can both be used to improve the selectivity of a search and to evaluate the significance of a match. They them examine distantly related members of three protein families, the serine proteases, the glutathione transferases, and the G-protein-coupled receptors (GCRs). Finally, the discuss how sequence similarity can be used to examine internal repeated or mosaic structures in proteins.

  14. Protein Structure Prediction by Protein Threading

    Science.gov (United States)

    Xu, Ying; Liu, Zhijie; Cai, Liming; Xu, Dong

    The seminal work of Bowie, Lüthy, and Eisenberg (Bowie et al., 1991) on "the inverse protein folding problem" laid the foundation of protein structure prediction by protein threading. By using simple measures for fitness of different amino acid types to local structural environments defined in terms of solvent accessibility and protein secondary structure, the authors derived a simple and yet profoundly novel approach to assessing if a protein sequence fits well with a given protein structural fold. Their follow-up work (Elofsson et al., 1996; Fischer and Eisenberg, 1996; Fischer et al., 1996a,b) and the work by Jones, Taylor, and Thornton (Jones et al., 1992) on protein fold recognition led to the development of a new brand of powerful tools for protein structure prediction, which we now term "protein threading." These computational tools have played a key role in extending the utility of all the experimentally solved structures by X-ray crystallography and nuclear magnetic resonance (NMR), providing structural models and functional predictions for many of the proteins encoded in the hundreds of genomes that have been sequenced up to now.

  15. Polymer Directed Protein Assemblies

    NARCIS (Netherlands)

    van Rijn, Patrick

    2013-01-01

    Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e. g., virus particles. Viruses are a multi-protein assembly of which the morphology is

  16. Amino acids and proteins

    Science.gov (United States)

    A balanced, safe diet with proteins is important to meet nutritional requirements. Proteins occur in animal as well as vegetable products in important quantities. In some countries, many people obtain much of their protein from animal products. In other regions, the major portion of dietary protein ...

  17. The Protein Model Portal

    OpenAIRE

    Arnold, Konstantin; Kiefer, Florian; Kopp, J?rgen; Battey, James N. D.; Podvinec, Michael; Westbrook, John D.; Berman, Helen M.; Bordoli, Lorenza; Schwede, Torsten

    2008-01-01

    Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploratio...

  18. Protein- protein interaction detection system using fluorescent protein microdomains

    Science.gov (United States)

    Waldo, Geoffrey S.; Cabantous, Stephanie

    2010-02-23

    The invention provides a protein labeling and interaction detection system based on engineered fragments of fluorescent and chromophoric proteins that require fused interacting polypeptides to drive the association of the fragments, and further are soluble and stable, and do not change the solubility of polypeptides to which they are fused. In one embodiment, a test protein X is fused to a sixteen amino acid fragment of GFP (.beta.-strand 10, amino acids 198-214), engineered to not perturb fusion protein solubility. A second test protein Y is fused to a sixteen amino acid fragment of GFP (.beta.-strand 11, amino acids 215-230), engineered to not perturb fusion protein solubility. When X and Y interact, they bring the GFP strands into proximity, and are detected by complementation with a third GFP fragment consisting of GFP amino acids 1-198 (strands 1-9). When GFP strands 10 and 11 are held together by interaction of protein X and Y, they spontaneous association with GFP strands 1-9, resulting in structural complementation, folding, and concomitant GFP fluorescence.

  19. Comparing side chain packing in soluble proteins, protein-protein interfaces, and transmembrane proteins.

    Science.gov (United States)

    Gaines, J C; Acebes, S; Virrueta, A; Butler, M; Regan, L; O'Hern, C S

    2018-05-01

    We compare side chain prediction and packing of core and non-core regions of soluble proteins, protein-protein interfaces, and transmembrane proteins. We first identified or created comparable databases of high-resolution crystal structures of these 3 protein classes. We show that the solvent-inaccessible cores of the 3 classes of proteins are equally densely packed. As a result, the side chains of core residues at protein-protein interfaces and in the membrane-exposed regions of transmembrane proteins can be predicted by the hard-sphere plus stereochemical constraint model with the same high prediction accuracies (>90%) as core residues in soluble proteins. We also find that for all 3 classes of proteins, as one moves away from the solvent-inaccessible core, the packing fraction decreases as the solvent accessibility increases. However, the side chain predictability remains high (80% within 30°) up to a relative solvent accessibility, rSASA≲0.3, for all 3 protein classes. Our results show that ≈40% of the interface regions in protein complexes are "core", that is, densely packed with side chain conformations that can be accurately predicted using the hard-sphere model. We propose packing fraction as a metric that can be used to distinguish real protein-protein interactions from designed, non-binding, decoys. Our results also show that cores of membrane proteins are the same as cores of soluble proteins. Thus, the computational methods we are developing for the analysis of the effect of hydrophobic core mutations in soluble proteins will be equally applicable to analyses of mutations in membrane proteins. © 2018 Wiley Periodicals, Inc.

  20. IGSF9 Family Proteins

    DEFF Research Database (Denmark)

    Hansen, Maria; Walmod, Peter Schledermann

    2013-01-01

    The Drosophila protein Turtle and the vertebrate proteins immunoglobulin superfamily (IgSF), member 9 (IGSF9/Dasm1) and IGSF9B are members of an evolutionarily ancient protein family. A bioinformatics analysis of the protein family revealed that invertebrates contain only a single IGSF9 family gene......, the longest isoforms of the proteins have the same general organization as the neural cell adhesion molecule family of cell adhesion molecule proteins, and like this family of proteins, IGSF9 family members are expressed in the nervous system. A review of the literature revealed that Drosophila Turtle...... facilitates homophilic cell adhesion. Moreover, IGSF9 family proteins have been implicated in the outgrowth and branching of neurites, axon guidance, synapse maturation, self-avoidance, and tiling. However, despite the few published studies on IGSF9 family proteins, reports on the functions of both Turtle...

  1. Personalizing Protein Nourishment

    Science.gov (United States)

    DALLAS, DAVID C.; SANCTUARY, MEGAN R.; QU, YUNYAO; KHAJAVI, SHABNAM HAGHIGHAT; VAN ZANDT, ALEXANDRIA E.; DYANDRA, MELISSA; FRESE, STEVEN A.; BARILE, DANIELA; GERMAN, J. BRUCE

    2016-01-01

    Proteins are not equally digestible—their proteolytic susceptibility varies by their source and processing method. Incomplete digestion increases colonic microbial protein fermentation (putrefaction), which produces toxic metabolites that can induce inflammation in vitro and have been associated with inflammation in vivo. Individual humans differ in protein digestive capacity based on phenotypes, particularly disease states. To avoid putrefaction-induced intestinal inflammation, protein sources and processing methods must be tailored to the consumer’s digestive capacity. This review explores how food processing techniques alter protein digestibility and examines how physiological conditions alter digestive capacity. Possible solutions to improving digestive function or matching low digestive capacity with more digestible protein sources are explored. Beyond the ileal digestibility measurements of protein digestibility, less invasive, quicker and cheaper techniques for monitoring the extent of protein digestion and fermentation are needed to personalize protein nourishment. Biomarkers of protein digestive capacity and efficiency can be identified with the toolsets of peptidomics, metabolomics, microbial sequencing and multiplexed protein analysis of fecal and urine samples. By monitoring individual protein digestive function, the protein component of diets can be tailored via protein source and processing selection to match individual needs to minimize colonic putrefaction and, thus, optimize gut health. PMID:26713355

  2. Prediction of Protein-Protein Interactions Related to Protein Complexes Based on Protein Interaction Networks

    Directory of Open Access Journals (Sweden)

    Peng Liu

    2015-01-01

    Full Text Available A method for predicting protein-protein interactions based on detected protein complexes is proposed to repair deficient interactions derived from high-throughput biological experiments. Protein complexes are pruned and decomposed into small parts based on the adaptive k-cores method to predict protein-protein interactions associated with the complexes. The proposed method is adaptive to protein complexes with different structure, number, and size of nodes in a protein-protein interaction network. Based on different complex sets detected by various algorithms, we can obtain different prediction sets of protein-protein interactions. The reliability of the predicted interaction sets is proved by using estimations with statistical tests and direct confirmation of the biological data. In comparison with the approaches which predict the interactions based on the cliques, the overlap of the predictions is small. Similarly, the overlaps among the predicted sets of interactions derived from various complex sets are also small. Thus, every predicted set of interactions may complement and improve the quality of the original network data. Meanwhile, the predictions from the proposed method replenish protein-protein interactions associated with protein complexes using only the network topology.

  3. Tissue-based quantitative proteome analysis of human hepatocellular carcinoma using tandem mass tags.

    Science.gov (United States)

    Megger, Dominik Andre; Rosowski, Kristin; Ahrens, Maike; Bracht, Thilo; Eisenacher, Martin; Schlaak, Jörg F; Weber, Frank; Hoffmann, Andreas-Claudius; Meyer, Helmut E; Baba, Hideo A; Sitek, Barbara

    2017-03-01

    Human hepatocellular carcinoma (HCC) is a severe malignant disease, and accurate and reliable diagnostic markers are still needed. This study was aimed for the discovery of novel marker candidates by quantitative proteomics. Proteomic differences between HCC and nontumorous liver tissue were studied by mass spectrometry. Among several significantly upregulated proteins, translocator protein 18 (TSPO) and Ras-related protein Rab-1A (RAB1A) were selected for verification by immunohistochemistry in an independent cohort. For RAB1A, a high accuracy for the discrimination of HCC and nontumorous liver tissue was observed. RAB1A was verified to be a potent biomarker candidate for HCC.

  4. Athoropometric measurements and plasma proteins in protein ...

    African Journals Online (AJOL)

    Athoropometric measurements and plasma proteins in protein energy malnutrition. MH Etukudo, EO Agbedana, OO Akinyinka, BOA Osifo. Abstract. No Abstract. Global Journal of Medical Sciences Vol. 5(1) 2006: 7-11. Full Text: EMAIL FREE FULL TEXT EMAIL FREE FULL TEXT · DOWNLOAD FULL TEXT DOWNLOAD ...

  5. Polymer Directed Protein Assemblies

    Directory of Open Access Journals (Sweden)

    Patrick van Rijn

    2013-05-01

    Full Text Available Protein aggregation and protein self-assembly is an important occurrence in natural systems, and is in some form or other dictated by biopolymers. Very obvious influences of biopolymers on protein assemblies are, e.g., virus particles. Viruses are a multi-protein assembly of which the morphology is dictated by poly-nucleotides namely RNA or DNA. This “biopolymer” directs the proteins and imposes limitations on the structure like the length or diameter of the particle. Not only do these bionanoparticles use polymer-directed self-assembly, also processes like amyloid formation are in a way a result of directed protein assembly by partial unfolded/misfolded biopolymers namely, polypeptides. The combination of proteins and synthetic polymers, inspired by the natural processes, are therefore regarded as a highly promising area of research. Directed protein assembly is versatile with respect to the possible interactions which brings together the protein and polymer, e.g., electrostatic, v.d. Waals forces or covalent conjugation, and possible combinations are numerous due to the large amounts of different polymers and proteins available. The protein-polymer interacting behavior and overall morphology is envisioned to aid in clarifying protein-protein interactions and are thought to entail some interesting new functions and properties which will ultimately lead to novel bio-hybrid materials.

  6. Protein and protein hydrolysates in sports nutrition.

    Science.gov (United States)

    van Loon, Luc J C; Kies, Arie K; Saris, Wim H M

    2007-08-01

    With the increasing knowledge about the role of nutrition in increasing exercise performance, it has become clear over the last 2 decades that amino acids, protein, and protein hydrolysates can play an important role. Most of the attention has been focused on their effects at a muscular level. As these nutrients are ingested, however, it also means that gastrointestinal digestibility and absorption can modulate their efficacy significantly. Therefore, discussing the role of amino acids, protein, and protein hydrolysates in sports nutrition entails holding a discussion on all levels of the metabolic route. On May 28-29, 2007, a small group of researchers active in the field of exercise science and protein metabolism presented an overview of the different aspects of the application of protein and protein hydrolysates in sports nutrition. In addition, they were asked to share their opinions on the future progress in their fields of research. In this overview, an introduction to the workshop and a short summary of its outcome is provided.

  7. Protein Data Bank (PDB)

    Data.gov (United States)

    U.S. Department of Health & Human Services — The Protein Data Bank (PDB) archive is the single worldwide repository of information about the 3D structures of large biological molecules, including proteins and...

  8. Learning about Proteins

    Science.gov (United States)

    ... Fitness Diseases & Conditions Infections Drugs & Alcohol School & Jobs Sports Expert Answers (Q&A) Staying Safe Videos for Educators Search English Español Learning About Proteins KidsHealth / For Kids / Learning About Proteins What's in ...

  9. Protein electrophoresis - serum

    Science.gov (United States)

    ... this page: //medlineplus.gov/ency/article/003540.htm Protein electrophoresis - serum To use the sharing features on ... JavaScript. This lab test measures the types of protein in the fluid (serum) part of a blood ...

  10. Polarizable protein packing

    KAUST Repository

    Ng, Albert H.; Snow, Christopher D.

    2011-01-01

    To incorporate protein polarization effects within a protein combinatorial optimization framework, we decompose the polarizable force field AMOEBA into low order terms. Including terms up to the third-order provides a fair approximation to the full

  11. Urine protein electrophoresis test

    Science.gov (United States)

    Urine protein electrophoresis; UPEP; Multiple myeloma - UPEP; Waldenström macroglobulinemia - UPEP; Amyloidosis - UPEP ... special paper and apply an electric current. The proteins move and form visible bands. These reveal the ...

  12. Allosteric Regulation of Proteins

    Indian Academy of Sciences (India)

    interactions with other proteins, or binding of small molecules. Covalent .... vealed through structural elucidation of the protein in free and oxygen-bound forms .... stance, molecular dynamic simulation of glutamine binding pro- tein shows that ...

  13. NMR of unfolded proteins

    Indian Academy of Sciences (India)

    Unknown

    2005-01-03

    Jan 3, 2005 ... covering all the systems, so far discovered.5,7,8,12. With the increasing ... Structural investigations on proteins by NMR are, currently ... rapid analysis of unfolded proteins. ...... and hence help in design of drugs against them.

  14. CSF total protein

    Science.gov (United States)

    CSF total protein is a test to determine the amount of protein in your spinal fluid, also called cerebrospinal fluid (CSF). ... The normal protein range varies from lab to lab, but is typically about 15 to 60 milligrams per deciliter (mg/dL) ...

  15. Protein - Which is Best?

    Science.gov (United States)

    Hoffman, Jay R; Falvo, Michael J

    2004-09-01

    Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids), whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function) are also reviewed. Key PointsHigher protein needs are seen in athletic populations.Animal proteins is an important source of protein, however potential health concerns do exist from a diet of protein

  16. Peptide segments in protein-protein interfaces

    Indian Academy of Sciences (India)

    Prakash

    2006-09-06

    Sep 6, 2006 ... contact surface from the rest of the protein surface have been used to identify ..... interfaces the contribution of the charged residues, such as. Lys, Asp and ..... Lawrence M C and Colman P M 1993 Shape complementarity at.

  17. Highly thermostable fluorescent proteins

    Science.gov (United States)

    Bradbury, Andrew M [Santa Fe, NM; Waldo, Geoffrey S [Santa Fe, NM; Kiss, Csaba [Los Alamos, NM

    2011-03-22

    Thermostable fluorescent proteins (TSFPs), methods for generating these and other stability-enhanced proteins, polynucleotides encoding such proteins, and assays and method for using the TSFPs and TSFP-encoding nucleic acid molecules are provided. The TSFPs of the invention show extremely enhanced levels of stability and thermotolerance. In one case, for example, a TSFP of the invention is so stable it can be heated to 99.degree. C. for short periods of time without denaturing, and retains 85% of its fluorescence when heated to 80.degree. C. for several minutes. The invention also provides a method for generating stability-enhanced variants of a protein, including but not limited to fluorescent proteins.

  18. Intracellular protein breakdown. 8

    International Nuclear Information System (INIS)

    Bohley, P.; Kirschke, H.; Langner, J.; Wiederanders, B.; Ansorge, S.

    1976-01-01

    Double-labelled proteins from rat liver cytosol ( 14 C in long-lived, 3 H in short-lived proteins after in-vivo-labelling) are used as substrates for unlabelled proteinases in vitro. Differences in the degradation rates of short-lived and long-lived proteins in vitro by different proteinases and after addition of different effectors allow conclusions concerning their importance for the in-vivo-turnover of substrate proteins. The main activity (>90%) of soluble lysosomal proteinases at pH 6.1 and pH 6.9 is caused by thiolproteinases, which degrade preferentially short-lived cytosol proteins. These proteinases are inhibited by leupeptin. Autolysis of double-labelled cell fractions shows a remarkably faster breakdown of short-lived substrate proteins only in the soluble part of lysosomes. Microsomal fractions degrade in vitro preferentially long-lived substrate proteins. (author)

  19. Protein carbonylation in plants

    DEFF Research Database (Denmark)

    Møller, Ian Max; Havelund, Jesper; Rogowska-Wrzesinska, Adelina

    2017-01-01

    This chapter provides an overview of the current knowledge on protein carbonylation in plants and its role in plant physiology. It starts with a brief outline of the turnover and production sites of reactive oxygen species (ROS) in plants and the causes of protein carbonylation. This is followed...... by a description of the methods used to study protein carbonylation in plants, which is also very brief as the methods are similar to those used in studies on animals. The chapter also focuses on protein carbonylation in plants in general and in mitochondria and in seeds in particular, as case stories where...... specific carbonylated proteins have been identified. Protein carbonylation appears to accumulate at all stages of seed development and germination investigated to date. In some cases, such as seed aging, it is probably simply an accumulation of oxidative damage. However, in other cases protein...

  20. Racemic protein crystallography.

    Science.gov (United States)

    Yeates, Todd O; Kent, Stephen B H

    2012-01-01

    Although natural proteins are chiral and are all of one "handedness," their mirror image forms can be prepared by chemical synthesis. This opens up new opportunities for protein crystallography. A racemic mixture of the enantiomeric forms of a protein molecule can crystallize in ways that natural proteins cannot. Recent experimental data support a theoretical prediction that this should make racemic protein mixtures highly amenable to crystallization. Crystals obtained from racemic mixtures also offer advantages in structure determination strategies. The relevance of these potential advantages is heightened by advances in synthetic methods, which are extending the size limit for proteins that can be prepared by chemical synthesis. Recent ideas and results in the area of racemic protein crystallography are reviewed.

  1. Texturized dairy proteins.

    Science.gov (United States)

    Onwulata, Charles I; Phillips, John G; Tunick, Michael H; Qi, Phoebi X; Cooke, Peter H

    2010-03-01

    Dairy proteins are amenable to structural modifications induced by high temperature, shear, and moisture; in particular, whey proteins can change conformation to new unfolded states. The change in protein state is a basis for creating new foods. The dairy products, nonfat dried milk (NDM), whey protein concentrate (WPC), and whey protein isolate (WPI) were modified using a twin-screw extruder at melt temperatures of 50, 75, and 100 degrees C, and moistures ranging from 20 to 70 wt%. Viscoelasticity and solubility measurements showed that extrusion temperature was a more significant (P extruded dairy protein ranged from rigid (2500 N) to soft (2.7 N). Extruding at or above 75 degrees C resulted in increased peak force for WPC (138 to 2500 N) and WPI (2.7 to 147.1 N). NDM was marginally texturized; the presence of lactose interfered with its texturization. WPI products extruded at 50 degrees C were not texturized; their solubility values ranged from 71.8% to 92.6%. A wide possibility exists for creating new foods with texturized dairy proteins due to the extensive range of states achievable. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, WPI, or WPC, or NDM were modified by extrusion processing. Extrusion temperature conditions were adjusted to 50, 75, or 100 degrees C, sufficient to change the structure of the dairy proteins, but not destroy them. Extrusion modified the structures of these dairy proteins for ease of use in starchy foods to boost nutrient levels. Dairy proteins can be used to boost the protein content in puffed snacks made from corn meal, but unmodified, they bind water and form doughy pastes with starch. To minimize the water binding property of dairy proteins, whey protein isolate, whey protein concentrate, or nonfat dried milk were modified by extrusion processing. Extrusion

  2. Protein kinesis: The dynamics of protein trafficking and stability

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1995-12-31

    The purpose of this conference is to provide a multidisciplinary forum for exchange of state-of-the-art information on protein kinesis. This volume contains abstracts of papers in the following areas: protein folding and modification in the endoplasmic reticulum; protein trafficking; protein translocation and folding; protein degradation; polarity; nuclear trafficking; membrane dynamics; and protein import into organelles.

  3. PROTEIN - WHICH IS BEST?

    Directory of Open Access Journals (Sweden)

    Michael J. Falvo

    2004-09-01

    Full Text Available Protein intake that exceeds the recommended daily allowance is widely accepted for both endurance and power athletes. However, considering the variety of proteins that are available much less is known concerning the benefits of consuming one protein versus another. The purpose of this paper is to identify and analyze key factors in order to make responsible recommendations to both the general and athletic populations. Evaluation of a protein is fundamental in determining its appropriateness in the human diet. Proteins that are of inferior content and digestibility are important to recognize and restrict or limit in the diet. Similarly, such knowledge will provide an ability to identify proteins that provide the greatest benefit and should be consumed. The various techniques utilized to rate protein will be discussed. Traditionally, sources of dietary protein are seen as either being of animal or vegetable origin. Animal sources provide a complete source of protein (i.e. containing all essential amino acids, whereas vegetable sources generally lack one or more of the essential amino acids. Animal sources of dietary protein, despite providing a complete protein and numerous vitamins and minerals, have some health professionals concerned about the amount of saturated fat common in these foods compared to vegetable sources. The advent of processing techniques has shifted some of this attention and ignited the sports supplement marketplace with derivative products such as whey, casein and soy. Individually, these products vary in quality and applicability to certain populations. The benefits that these particular proteins possess are discussed. In addition, the impact that elevated protein consumption has on health and safety issues (i.e. bone health, renal function are also reviewed

  4. The U.S. Navy, the Neutrality Patrol, and Atlantic Fleet Escort Operations, 1939-1941

    Science.gov (United States)

    1991-05-13

    Hereward in the Yucatan Channel, watching as the British ship refueled from a G- 3 class cruiser.3 3 Later that month, two American destroyers and the...4 0 On 14 December, the German freighter Arauca put out of Veracruz, and shaped a course for Hamburg. The ship’s cargo included " sisal , phosphate

  5. Factorization and escorting in the game-theoretical approach to non-extensive entropy measures

    DEFF Research Database (Denmark)

    Topsøe, Flemming

    2006-01-01

    A factorisation for a game between "nature" and "the physicist" is established, thereby searating the roles of the two parties.......A factorisation for a game between "nature" and "the physicist" is established, thereby searating the roles of the two parties....

  6. 76 FR 44880 - Security Zone; Escorted Vessels in Captain of the Port Ohio Valley Zone

    Science.gov (United States)

    2011-07-27

    ... potential terrorist acts and would enhance public and maritime safety and security. DATES: Comments and... coast of Yemen and the prior attack on the USS COLE demonstrate the maritime terrorism threat. These attacks manifest a continuing threat to U.S. maritime assets as described in the President's finding in...

  7. Fungal farmers or algal escorts: lichen adaptation from the algal perspective.

    Science.gov (United States)

    Piercey-Normore, Michele D; Deduke, Christopher

    2011-09-01

    Domestication of algae by lichen-forming fungi describes the symbiotic relationship between the photosynthetic (green alga or cyanobacterium; photobiont) and fungal (mycobiont) partnership in lichen associations (Goward 1992). The algal domestication implies that the mycobiont cultivates the alga as a monoculture within its thallus, analogous to a farmer cultivating a food crop. However, the initial photobiont 'selection' by the mycobiont may be predetermined by the habitat rather than by the farmer. When the mycobiont selects a photobiont from the available photobionts within a habitat, the mycobiont may influence photobiont growth and reproduction (Ahmadjian & Jacobs 1981) only after the interaction has been initiated. The theory of ecological guilds (Rikkinen et al. 2002) proposes that habitat limits the variety of photobionts available to the fungal partner. While some studies provide evidence to support the theory of ecological guilds in cyanobacterial lichens (Rikkinen et al. 2002), other studies propose models to explain variation in symbiont combinations in green algal lichens (Ohmura et al. 2006; Piercey-Normore 2006; Yahr et al. 2006) hypothesizing the existence of such guilds. In this issue of Molecular Ecology, Peksa & Škaloud (2011) test the theory of ecological guilds and suggest a relationship between algal habitat requirements and lichen adaptation in green algal lichens of the genus Lepraria. The environmental parameters examined in this study, exposure to rainfall, altitude and substratum type, are integral to lichen biology. Lichens have a poikilohydric nature, relying on the availability of atmospheric moisture for metabolic processes. Having no known active mechanism to preserve metabolic thallus moisture in times of drought, one would expect a strong influence of the environment on symbiont adaptation to specific habitats. Adaptation to changes in substrata and its properties would be expected with the intimate contact between crustose lichens in the genus Lepraria. Altitude has been suggested to influence species distributions in a wide range of taxonomic groups. This is one of the first studies to illustrate an ecological guild, mainly for exposure to rainfall (ombrophiles and ombrophobes), with green algal lichens. © 2011 Blackwell Publishing Ltd.

  8. 77 FR 4900 - Security Zone; Escorted Vessels in Captain of the Port Ohio Valley Zone

    Science.gov (United States)

    2012-02-01

    ... at The Brown Hotel, 335 West Broadway, Louisville, KY 40202, telephone 502-583-1234. You may submit... 1 p.m. to 4 p.m., at The Brown Hotel, 335 West Broadway, Louisville, KY 40202, telephone (502) 583...

  9. To serve and protect: Enzyme inhibitors as radiopeptide escorts promote tumor targeting

    NARCIS (Netherlands)

    B.A. Nock (Berthold); T. Maina (Theodosia); E.P. Krenning (Eric); M. de Jong (Marcel)

    2014-01-01

    textabstractRadiolabeled octreotide analogs are most successfully being applied today in clinical cancer imaging and treatment. Propagation of this paradigm to other radiopeptide families has been greatly hampered by the inherent poor metabolic stability of systemically administered peptide analogs.

  10. GGPP-Mediated Protein Geranylgeranylation in Oocyte Is Essential for the Establishment of Oocyte-Granulosa Cell Communication and Primary-Secondary Follicle Transition in Mouse Ovary.

    Directory of Open Access Journals (Sweden)

    Chen Jiang

    2017-01-01

    Full Text Available Folliculogenesis is a progressive and highly regulated process, which is essential to provide ova for later reproductive life, requires the bidirectional communication between the oocyte and granulosa cells. This physical connection-mediated communication conveys not only the signals from the oocyte to granulosa cells that regulate their proliferation but also metabolites from the granulosa cells to the oocyte for biosynthesis. However, the underlying mechanism of establishing this communication is largely unknown. Here, we report that oocyte geranylgeranyl diphosphate (GGPP, a metabolic intermediate involved in protein geranylgeranylation, is required to establish the oocyte-granulosa cell communication. GGPP and geranylgeranyl diphosphate synthase (Ggpps levels in oocytes increased during early follicular development. The selective depletion of GGPP in mouse oocytes impaired the proliferation of granulosa cells, primary-secondary follicle transition and female fertility. Mechanistically, GGPP depletion inhibited Rho GTPase geranylgeranylation and its GTPase activity, which was responsible for the accumulation of cell junction proteins in the oocyte cytoplasm and the failure to maintain physical connection between oocyte and granulosa cells. GGPP ablation also blocked Rab27a geranylgeranylation, which might account for the impaired secretion of oocyte materials such as Gdf9. Moreover, GGPP administration restored the defects in oocyte-granulosa cell contact, granulosa cell proliferation and primary-secondary follicle transition in Ggpps depletion mice. Our study provides the evidence that GGPP-mediated protein geranylgeranylation contributes to the establishment of oocyte-granulosa cell communication and then regulates the primary-secondary follicle transition, a key phase of folliculogenesis essential for female reproductive function.

  11. Specificity and affinity quantification of protein-protein interactions.

    Science.gov (United States)

    Yan, Zhiqiang; Guo, Liyong; Hu, Liang; Wang, Jin

    2013-05-01

    Most biological processes are mediated by the protein-protein interactions. Determination of the protein-protein structures and insight into their interactions are vital to understand the mechanisms of protein functions. Currently, compared with the isolated protein structures, only a small fraction of protein-protein structures are experimentally solved. Therefore, the computational docking methods play an increasing role in predicting the structures and interactions of protein-protein complexes. The scoring function of protein-protein interactions is the key responsible for the accuracy of the computational docking. Previous scoring functions were mostly developed by optimizing the binding affinity which determines the stability of the protein-protein complex, but they are often lack of the consideration of specificity which determines the discrimination of native protein-protein complex against competitive ones. We developed a scoring function (named as SPA-PP, specificity and affinity of the protein-protein interactions) by incorporating both the specificity and affinity into the optimization strategy. The testing results and comparisons with other scoring functions show that SPA-PP performs remarkably on both predictions of binding pose and binding affinity. Thus, SPA-PP is a promising quantification of protein-protein interactions, which can be implemented into the protein docking tools and applied for the predictions of protein-protein structure and affinity. The algorithm is implemented in C language, and the code can be downloaded from http://dl.dropbox.com/u/1865642/Optimization.cpp.

  12. General protein-protein cross-linking.

    Science.gov (United States)

    Alegria-Schaffer, Alice

    2014-01-01

    This protocol describes a general protein-to-protein cross-linking procedure using the water-soluble amine-reactive homobifunctional BS(3) (bis[sulfosuccinimidyl] suberate); however, the protocol can be easily adapted using other cross-linkers of similar properties. BS(3) is composed of two sulfo-NHS ester groups and an 11.4 Å linker. Sulfo-NHS ester groups react with primary amines in slightly alkaline conditions (pH 7.2-8.5) and yield stable amide bonds. The reaction releases N-hydroxysuccinimide (see an application of NHS esters on Labeling a protein with fluorophores using NHS ester derivitization). © 2014 Elsevier Inc. All rights reserved.

  13. Scoring functions for protein-protein interactions.

    Science.gov (United States)

    Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

    2013-12-01

    The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Computational Protein Design

    DEFF Research Database (Denmark)

    Johansson, Kristoffer Enøe

    Proteins are the major functional group of molecules in biology. The impact of protein science on medicine and chemical productions is rapidly increasing. However, the greatest potential remains to be realized. The fi eld of protein design has advanced computational modeling from a tool of support...... to a central method that enables new developments. For example, novel enzymes with functions not found in natural proteins have been de novo designed to give enough activity for experimental optimization. This thesis presents the current state-of-the-art within computational design methods together...... with a novel method based on probability theory. With the aim of assembling a complete pipeline for protein design, this work touches upon several aspects of protein design. The presented work is the computational half of a design project where the other half is dedicated to the experimental part...

  15. Dicty_cDB: VSE884 [Dicty_cDB

    Lifescience Database Archive (English)

    Full Text Available to Arabidopsis thaliana sequence At1g52280 GTP-binding protein RAB7D, putative see http://mips.gsf.de/proj/...ar to Arabidopsis thaliana sequence At1g52280 GTP-binding protein RAB7D, putative see http://mips.gsf.de/pro

  16. GenBank blastx search result: AK242967 [KOME

    Lifescience Database Archive (English)

    Full Text Available nsitive, 1, a gene for novel protein similar to human and rodent member RAS oncogene family RAB7 (RAB7), a gene for novel protein rem...otely similar to human host cell factor 1 (VP16-accessor

  17. Blue Emission in Proteins

    OpenAIRE

    Sarkar, Sohini; Sengupta, Abhigyan; Hazra, Partha; Mandal, Pankaj

    2014-01-01

    Recent literatures reported blue-green emission from amyloid fibril as exclusive signature of fibril formation. This unusual visible luminescence is regularly used to monitor fibril growth. Blue-green emission has also been observed in crystalline protein and in solution. However, the origin of this emission is not known exactly. Our spectroscopic study of serum proteins reveals that the blue-green emission is a property of protein monomer. Evidences suggest that semiconductor-like band struc...

  18. Pressure cryocooling protein crystals

    Science.gov (United States)

    Kim, Chae Un [Ithaca, NY; Gruner, Sol M [Ithaca, NY

    2011-10-04

    Preparation of cryocooled protein crystal is provided by use of helium pressurizing and cryocooling to obtain cryocooled protein crystal allowing collection of high resolution data and by heavier noble gas (krypton or xenon) binding followed by helium pressurizing and cryocooling to obtain cryocooled protein crystal for collection of high resolution data and SAD phasing simultaneously. The helium pressurizing is carried out on crystal coated to prevent dehydration or on crystal grown in aqueous solution in a capillary.

  19. Yeast ribosomal proteins

    International Nuclear Information System (INIS)

    Otaka, E.; Kobata, K.

    1978-01-01

    The cytoplasmic 80s ribosomal proteins from the cells of yeast Saccharomyces cerevisiae were analyzed by SDS two-dimensional polyacrylamide gel electrophoresis. Seventyfour proteins were identified and consecutively numbered from 1 to 74. Upon oxidation of the 80s proteins with performic acid, ten proteins (no. 15, 20, 35, 40, 44, 46, 49, 51, 54 and 55) were dislocated on the gel without change of the total number of protein spots. Five proteins (no. 8, 14, 16, 36 and 74) were phosphorylated in vivo as seen in 32 P-labelling experiments. The large and small subunits separated in low magnesium medium were analyzed by the above gel electrophoresis. At least forty-five and twenty-eight proteins were assumed to be in the large and small subunits, respectively. All proteins found in the 80s ribosomes, except for no. 3, were detected in either subunit without appearance of new spots. The acidic protein no. 3 seems to be lost during subunit dissociation. (orig.) [de

  20. Physics of protein folding

    Science.gov (United States)

    Finkelstein, A. V.; Galzitskaya, O. V.

    2004-04-01

    Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.

  1. Ultrafiltration of pegylated proteins

    Science.gov (United States)

    Molek, Jessica R.

    There is considerable clinical interest in the use of "second-generation" therapeutics produced by conjugation of a native protein with various polymers including polyethylene glycol (PEG). PEG--protein conjugates, so-called PEGylated proteins, can exhibit enhanced stability, half-life, and bioavailability. One of the challenges in the commercial production of PEGylated proteins is the purification required to remove unreacted polymer, native protein, and in many cases PEGylated proteins with nonoptimal degrees of conjugation. The overall objective of this thesis was to examine the use of ultrafiltration for the purification of PEGylated proteins. This included: (1) analysis of size-based separation of PEGylated proteins using conventional ultrafiltration membranes, (2) use of electrically-charged membranes to exploit differences in electrostatic interactions, and (3) examination of the effects of PEGylation on protein fouling. The experimental results were analyzed using appropriate theoretical models, with the underlying physical properties of the PEGylated proteins evaluated using size exclusion chromatography, capillary electrophoresis, dynamic light scattering, and reverse phase chromatography. PEGylated proteins were produced by covalent attachment of activated PEG to a protein via primary amines on the lysine residues. A simple model was developed for the reaction kinetics, which was used to explore the effect of reaction conditions and mode of operation on the distribution of PEGylated products. The effective size of the PEGylated proteins was evaluated using size exclusion chromatography, with appropriate correlations developed for the size in terms of the molecular weight of the native protein and attached PEG. The electrophoretic mobility of the PEGylated proteins were evaluated by capillary electrophoresis with the data in good agreement with a simple model accounting for the increase in protein size and the reduction in the number of protonated amine

  2. Advances in Protein Precipitation

    NARCIS (Netherlands)

    Golubovic, M.

    2009-01-01

    Proteins are biological macromolecules, which are among the key components of all living organisms. Proteins are nowadays present in all fields of biotech industry, such as food and feed, synthetic and pharmaceutical industry. They are isolated from their natural sources or produced in different

  3. Synthesis of Lipidated Proteins.

    Science.gov (United States)

    Mejuch, Tom; Waldmann, Herbert

    2016-08-17

    Protein lipidation is one of the major post-translational modifications (PTM) of proteins. The attachment of the lipid moiety frequently determines the localization and the function of the lipoproteins. Lipidated proteins participate in many essential biological processes in eukaryotic cells, including vesicular trafficking, signal transduction, and regulation of the immune response. Malfunction of these cellular processes usually leads to various diseases such as cancer. Understanding the mechanism of cellular signaling and identifying the protein-protein and protein-lipid interactions in which the lipoproteins are involved is a crucial task. To achieve these goals, fully functional lipidated proteins are required. However, access to lipoproteins by means of standard expression is often rather limited. Therefore, semisynthetic methods, involving the synthesis of lipidated peptides and their subsequent chemoselective ligation to yield full-length lipoproteins, were developed. In this Review we summarize the commonly used methods for lipoprotein synthesis and the development of the corresponding chemoselective ligation techniques. Several key studies involving full-length semisynthetic lipidated Ras, Rheb, and LC3 proteins are presented.

  4. Amino acids and proteins

    NARCIS (Netherlands)

    van Goudoever, Johannes B.; Vlaardingerbroek, Hester; van den Akker, Chris H.; de Groof, Femke; van der Schoor, Sophie R. D.

    2014-01-01

    Amino acids and protein are key factors for growth. The neonatal period requires the highest intake in life to meet the demands. Those demands include amino acids for growth, but proteins and amino acids also function as signalling molecules and function as neurotransmitters. Often the nutritional

  5. Protein Attachment on Nanodiamonds.

    Science.gov (United States)

    Lin, Chung-Lun; Lin, Cheng-Huang; Chang, Huan-Cheng; Su, Meng-Chih

    2015-07-16

    A recent advance in nanotechnology is the scale-up production of small and nonaggregated diamond nanoparticles suitable for biological applications. Using detonation nanodiamonds (NDs) with an average diameter of ∼4 nm as the adsorbents, we have studied the static attachment of three proteins (myoglobin, bovine serum albumin, and insulin) onto the nanoparticles by optical spectroscopy, mass spectrometry, and dynamic light scattering, and electrophoretic zeta potential measurements. Results show that the protein surface coverage is predominantly determined by the competition between protein-protein and protein-ND interactions, giving each protein a unique and characteristic structural configuration in its own complex. Specifically, both myoglobin and bovine serum albumin show a Langmuir-type adsorption behavior, forming 1:1 complexes at saturation, whereas insulin folds into a tightly bound multimer before adsorption. The markedly different adsorption patterns appear to be independent of the protein concentration and are closely related to the affinity of the individual proteins for the NDs. The present study provides a fundamental understanding for the use of NDs as a platform for nanomedical drug delivery.

  6. Poxviral Ankyrin Proteins

    Directory of Open Access Journals (Sweden)

    Michael H. Herbert

    2015-02-01

    Full Text Available Multiple repeats of the ankyrin motif (ANK are ubiquitous throughout the kingdoms of life but are absent from most viruses. The main exception to this is the poxvirus family, and specifically the chordopoxviruses, with ANK repeat proteins present in all but three species from separate genera. The poxviral ANK repeat proteins belong to distinct orthologue groups spread over different species, and align well with the phylogeny of their genera. This distribution throughout the chordopoxviruses indicates these proteins were present in an ancestral vertebrate poxvirus, and have since undergone numerous duplication events. Most poxviral ANK repeat proteins contain an unusual topology of multiple ANK motifs starting at the N-terminus with a C-terminal poxviral homologue of the cellular F-box enabling interaction with the cellular SCF ubiquitin ligase complex. The subtle variations between ANK repeat proteins of individual poxviruses suggest an array of different substrates may be bound by these protein-protein interaction domains and, via the F-box, potentially directed to cellular ubiquitination pathways and possible degradation. Known interaction partners of several of these proteins indicate that the NF-κB coordinated anti-viral response is a key target, whilst some poxviral ANK repeat domains also have an F-box independent affect on viral host-range.

  7. Protein oxidation and peroxidation

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan

    2016-01-01

    Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard...... to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners...... and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals...

  8. Protein restriction and cancer.

    Science.gov (United States)

    Yin, Jie; Ren, Wenkai; Huang, Xingguo; Li, Tiejun; Yin, Yulong

    2018-03-26

    Protein restriction without malnutrition is currently an effective nutritional intervention known to prevent diseases and promote health span from yeast to human. Recently, low protein diets are reported to be associated with lowered cancer incidence and mortality risk of cancers in human. In murine models, protein restriction inhibits tumor growth via mTOR signaling pathway. IGF-1, amino acid metabolic programing, FGF21, and autophagy may also serve as potential mechanisms of protein restriction mediated cancer prevention. Together, dietary intervention aimed at reducing protein intake can be beneficial and has the potential to be widely adopted and effective in preventing and treating cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

  9. Sensitizing properties of proteins

    DEFF Research Database (Denmark)

    Poulsen, Lars K.; Ladics, Gregory S; McClain, Scott

    2014-01-01

    The scope of allergy risk is diverse considering the myriad ways in which protein allergenicity is affected by physiochemical characteristics of proteins. The complexity created by the matrices of foods and the variability of the human immune system add additional challenges to understanding...... the relationship between sensitization potential and allergy disease. To address these and other issues, an April 2012 international symposium was held in Prague, Czech Republic, to review and discuss the state-of-the-science of sensitizing properties of protein allergens. The symposium, organized by the Protein...... Allergenicity Technical Committee of the International Life Sciences Institute's Health and Environmental Sciences Institute, featured presentations on current methods, test systems, research trends, and unanswered questions in the field of protein sensitization. A diverse group of over 70 interdisciplinary...

  10. Artificially Engineered Protein Polymers.

    Science.gov (United States)

    Yang, Yun Jung; Holmberg, Angela L; Olsen, Bradley D

    2017-06-07

    Modern polymer science increasingly requires precise control over macromolecular structure and properties for engineering advanced materials and biomedical systems. The application of biological processes to design and synthesize artificial protein polymers offers a means for furthering macromolecular tunability, enabling polymers with dispersities of ∼1.0 and monomer-level sequence control. Taking inspiration from materials evolved in nature, scientists have created modular building blocks with simplified monomer sequences that replicate the function of natural systems. The corresponding protein engineering toolbox has enabled the systematic development of complex functional polymeric materials across areas as diverse as adhesives, responsive polymers, and medical materials. This review discusses the natural proteins that have inspired the development of key building blocks for protein polymer engineering and the function of these elements in material design. The prospects and progress for scalable commercialization of protein polymers are reviewed, discussing both technology needs and opportunities.

  11. Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

    Science.gov (United States)

    Chellaiah, Meenakshi A; Schaller, Michael D

    2009-08-01

    PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

  12. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low-density-lipoprotein-receptor-related protein.

    Science.gov (United States)

    Andersen, O M; Petersen, H H; Jacobsen, C; Moestrup, S K; Etzerodt, M; Andreasen, P A; Thøgersen, H C

    2001-07-01

    The low-density-lipoprotein-receptor (LDLR)-related protein (LRP) is composed of several classes of domains, including complement-type repeats (CR), which occur in clusters that contain binding sites for a multitude of different ligands. Each approximately 40-residue CR domain contains three conserved disulphide linkages and an octahedral Ca(2+) cage. LRP is a scavenging receptor for ligands from extracellular fluids, e.g. alpha(2)-macroglobulin (alpha(2)M)-proteinase complexes, lipoprotein-containing particles and serine proteinase-inhibitor complexes, like the complex between urokinase-type plasminogen activator (uPA) and the plasminogen activator inhibitor-1 (PAI-1). In the present study we analysed the interaction of the uPA-PAI-1 complex with an ensemble of fragments representing a complete overlapping set of two-domain fragments accounting for the ligand-binding cluster II (CR3-CR10) of LRP. By ligand blotting, solid-state competition analysis and surface-plasmon-resonance analysis, we demonstrate binding to multiple CR domains, but show a preferential interaction between the uPA-PAI-1 complex and a two-domain fragment comprising CR domains 5 and 6 of LRP. We demonstrate that surface-exposed aspartic acid and tryptophan residues at identical positions in the two homologous domains, CR5 and CR6 (Asp(958,CR5), Asp(999,CR6), Trp(953,CR5) and Trp(994,CR6)), are critical for the binding of the complex as well as for the binding of the receptor-associated protein (RAP) - the folding chaperone/escort protein required for transport of LRP to the cell surface. Accordingly, the present work provides (1) an identification of a preferred binding site within LRP CR cluster II; (2) evidence that the uPA-PAI-1 binding site involves residues from two adjacent protein domains; and (3) direct evidence identifying specific residues as important for the binding of uPA-PAI-1 as well as for the binding of RAP.

  13. The Protein Model Portal.

    Science.gov (United States)

    Arnold, Konstantin; Kiefer, Florian; Kopp, Jürgen; Battey, James N D; Podvinec, Michael; Westbrook, John D; Berman, Helen M; Bordoli, Lorenza; Schwede, Torsten

    2009-03-01

    Structural Genomics has been successful in determining the structures of many unique proteins in a high throughput manner. Still, the number of known protein sequences is much larger than the number of experimentally solved protein structures. Homology (or comparative) modeling methods make use of experimental protein structures to build models for evolutionary related proteins. Thereby, experimental structure determination efforts and homology modeling complement each other in the exploration of the protein structure space. One of the challenges in using model information effectively has been to access all models available for a specific protein in heterogeneous formats at different sites using various incompatible accession code systems. Often, structure models for hundreds of proteins can be derived from a given experimentally determined structure, using a variety of established methods. This has been done by all of the PSI centers, and by various independent modeling groups. The goal of the Protein Model Portal (PMP) is to provide a single portal which gives access to the various models that can be leveraged from PSI targets and other experimental protein structures. A single interface allows all existing pre-computed models across these various sites to be queried simultaneously, and provides links to interactive services for template selection, target-template alignment, model building, and quality assessment. The current release of the portal consists of 7.6 million model structures provided by different partner resources (CSMP, JCSG, MCSG, NESG, NYSGXRC, JCMM, ModBase, SWISS-MODEL Repository). The PMP is available at http://www.proteinmodelportal.org and from the PSI Structural Genomics Knowledgebase.

  14. Coarse-grain modelling of protein-protein interactions

    NARCIS (Netherlands)

    Baaden, Marc; Marrink, Siewert J.

    2013-01-01

    Here, we review recent advances towards the modelling of protein-protein interactions (PPI) at the coarse-grained (CG) level, a technique that is now widely used to understand protein affinity, aggregation and self-assembly behaviour. PPI models of soluble proteins and membrane proteins are

  15. Protein-Protein Docking in Drug Design and Discovery.

    Science.gov (United States)

    Kaczor, Agnieszka A; Bartuzi, Damian; Stępniewski, Tomasz Maciej; Matosiuk, Dariusz; Selent, Jana

    2018-01-01

    Protein-protein interactions (PPIs) are responsible for a number of key physiological processes in the living cells and underlie the pathomechanism of many diseases. Nowadays, along with the concept of so-called "hot spots" in