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

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

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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

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

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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.

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

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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.

CXCR3 Directs Antigen-Specific Effector CD4+ T Cell Migration to the Lung During Parainfluenza Virus Infection

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Kohlmeier, Jacob E; Cookenham, Tres; Miller, Shannon C

2009-01-01

effector CD4(+) T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4......(+) T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4(+) T cell proliferation, phenotype......, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4(+) T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections....

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

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

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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

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

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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.

Rab5 regulates internalisation of P2X4 receptors and potentiation by ivermectin.

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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.

Rab7: roles in membrane trafficking and disease.

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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.

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

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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.

The GTPase Rab37 Participates in the Control of Insulin Exocytosis.

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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.

Essential role of RAB27A in determining constitutive human skin color.

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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 (�

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

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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.

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

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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.

Two Novel Rab2 Interactors Regulate Dense-core Vesicle Maturation

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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

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

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.

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

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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.

Cyclosporine-resistant, Rab27a-independent Mobilization of Intracellular Preformed CD40L Mediates Antigen-specific T Cell Help In Vitro

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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

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

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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.

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

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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.

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

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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.

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

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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

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

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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.

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

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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.

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

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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

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

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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

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

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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.

LRRK2 mediated Rab8a phosphorylation promotes lipid storage.

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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.

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

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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.

Legionella Effector AnkX Disrupts Host Cell Endocytic Recycling in a Phosphocholination-Dependent Manner

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Samual C. Allgood

2017-09-01

Full Text Available The facultative intracellular bacterium Legionella pneumophila proliferates within amoebae and human alveolar macrophages, and it is the causative agent of Legionnaires' disease, a life-threatening pneumonia. Within host cells, L. pneumophila establishes a replicative haven by delivering numerous effector proteins into the host cytosol, many of which target membrane trafficking by manipulating the function of Rab GTPases. The Legionella effector AnkX is a phosphocholine transferase that covalently modifies host Rab1 and Rab35. However, a detailed understanding of the biological consequence of Rab GTPase phosphocholination remains elusive. Here, we broaden the understanding of AnkX function by presenting three lines of evidence that it interferes with host endocytic recycling. First, using immunogold transmission electron microscopy, we determined that GFP-tagged AnkX ectopically produced in mammalian cells localizes at the plasma membrane and tubular membrane compartments, sites consistent with targeting the endocytic recycling pathway. Furthermore, the C-terminal region of AnkX was responsible for association with the plasma membrane, and we determined that this region was also able to bind the phosphoinositide lipids PI(3P and PI(4P in vitro. Second, we observed that mCherry-AnkX co-localized with Rab35, a regulator of recycling endocytosis and with major histocompatibility class I protein (MHC-I, a key immunoregulatory protein whose recycling from and back to the plasma membrane is Rab35-dependent. Third, we report that during infection of macrophages, AnkX is responsible for the disruption of endocytic recycling of transferrin, and AnkX's phosphocholination activity is critical for this function. These results support the hypothesis that AnkX targets endocytic recycling during host cell infection. Finally, we have demonstrated that the phosphocholination activity of AnkX is also critical for inhibiting fusion of the Legionella

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

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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.

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

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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.

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

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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.

Tissue specific heterogeneity in effector immune cell response

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Saba eTufail

2013-08-01

Full Text Available Post pathogen invasion, migration of effector T-cell subsets to specific tissue locations is of prime importance for generation of robust immune response. Effector T cells are imprinted with distinct ‘homing codes’ (adhesion molecules and chemokine receptors during activation which regulate their targeted trafficking to specific tissues. Internal cues in the lymph node microenvironment along with external stimuli from food (vitamin A and sunlight (vitamin D3 prime dendritic cells, imprinting them to play centrestage in the induction of tissue tropism in effector T cells. B cells as well, in a manner similar to effector T cells, exhibit tissue tropic migration. In this review, we have focused on the factors regulating the generation and migration of effector T cells to various tissues alongwith giving an overview of tissue tropism in B cells.

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

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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.

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

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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.

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

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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.

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

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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

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

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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.

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

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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

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

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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.

Peptide Nucleic Acid Knockdown and Intra-host Cell Complementation of Ehrlichia Type IV Secretion System Effector

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Pratibha Sharma

2017-06-01

Full Text Available Survival of Ehrlichia chaffeensis depends on obligatory intracellular infection. One of the barriers to E. chaffeensis research progress has been the inability, using conventional techniques, to generate knock-out mutants for genes essential for intracellular infection. This study examined the use of Peptide Nucleic Acids (PNAs technology to interrupt type IV secretion system (T4SS effector protein expression in E. chaffeensis followed by intracellular complementation of the effector to determine its requirement for infection. Successful E. chaffeensis infection depends on the E. chaffeensis-specific T4SS protein effector, ehrlichial translocated factor-1 (Etf-1, which induces Rab5-regulated autophagy to provide host cytosolic nutrients required for E. chaffeensis proliferation. Etf-1 is also imported by host cell mitochondria where it inhibits host cell apoptosis to prolong its infection. We designed a PNA specific to Etf-1 and showed that the PNA bound to the target region of single-stranded Etf-1 RNA using a competitive binding assay. Electroporation of E. chaffeensis with this PNA significantly reduced Etf-1 mRNA and protein, and the bacteria's ability to induce host cell autophagy and infect host cells. Etf-1 PNA-mediated inhibition of ehrlichial Etf-1 expression and E. chaffeensis infection could be intracellularly trans-complemented by ectopic expression of Etf-1-GFP in host cells. These data affirmed the critical role of bacterial T4SS effector in host cell autophagy and E. chaffeensis infection, and demonstrated the use of PNA to analyze the gene functions of obligate intracellular bacteria.

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

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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.

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

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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.

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

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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.

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

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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.

Comprehensive functional analysis of Rab GTPases in Drosophila nephrocytes.

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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.

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

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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.

A novel SIV gag-specific CD4(+)T-cell clone suppresses SIVmac239 replication in CD4(+)T cells revealing the interplay between antiviral effector cells and their infected targets.

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Ayala, Victor I; Trivett, Matthew T; Coren, Lori V; Jain, Sumiti; Bohn, Patrick S; Wiseman, Roger W; O'Connor, David H; Ohlen, Claes; Ott, David E

2016-06-01

To study CD4(+)T-cell suppression of AIDS virus replication, we isolated nine rhesus macaque SIVGag-specific CD4(+)T-cell clones. One responding clone, Gag68, produced a typical cytotoxic CD8(+)T-cell response: induction of intracellular IFN-γ, MIP-1α, MIP-1β, and CD107a degranulation. Gag68 effectively suppressed the spread of SIVmac239 in CD4(+)T cells with a corresponding reduction of infected Gag68 effector cells, suggesting that CD4(+)effectors need to suppress their own infection in addition to their targets to be effective. Gag68 TCR cloning and gene transfer into CD4(+)T cells enabled additional experiments with this unique specificity after the original clone senesced. Our data supports the idea that CD4(+)T cells can directly limit AIDS virus spread in T cells. Furthermore, Gag68 TCR transfer into CD4(+)T-cell clones with differing properties holds promise to better understand the suppressive effector mechanisms used by this important component of the antiviral response using the rhesus macaque model. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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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

Structural and enzymatic characterization of a host-specificity determinant from Salmonella

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Kohler, Amanda C. [Rockefeller University, New York, NY 10065 (United States); Spanò, Stefania; Galán, Jorge E. [Yale University School of Medicine, New Haven, CT 06536 (United States); Stebbins, C. Erec, E-mail: stebbins@rockefeller.edu [Rockefeller University, New York, NY 10065 (United States)

2014-02-01

The Salmonella effector protein GtgE functions as a cysteine protease to cleave a subset of the Rab-family GTPases and to prevent delivery of antimicrobial agents to the Salmonella-containing vacuole. GtgE is an effector protein from Salmonella Typhimurium that modulates trafficking of the Salmonella-containing vacuole. It exerts its function by cleaving the Rab-family GTPases Rab29, Rab32 and Rab38, thereby preventing the delivery of antimicrobial factors to the bacteria-containing vacuole. Here, the crystal structure of GtgE at 1.65 Å resolution is presented, and structure-based mutagenesis and in vivo infection assays are used to identify its catalytic triad. A panel of cysteine protease inhibitors were examined and it was determined that N-ethylmaleimide, antipain and chymostatin inhibit GtgE activity in vitro. These findings provide the basis for the development of novel therapeutic strategies to combat Salmonella infections.

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

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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.

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

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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.

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

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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.

Virus-specific regulatory T cells ameliorate encephalitis by repressing effector T cell functions from priming to effector stages.

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Jingxian Zhao

2014-08-01

Full Text Available Several studies have demonstrated the presence of pathogen-specific Foxp3+ CD4 regulatory T cells (Treg in infected animals, but little is known about where and how these cells affect the effector T cell responses and whether they are more suppressive than bulk Treg populations. We recently showed the presence of both epitope M133-specific Tregs (M133 Treg and conventional CD4 T cells (M133 Tconv in the brains of mice with coronavirus-induced encephalitis. Here, we provide new insights into the interactions between pathogenic Tconv and Tregs responding to the same epitope. M133 Tregs inhibited the proliferation but not initial activation of M133 Tconv in draining lymph nodes (DLN. Further, M133 Tregs inhibited migration of M133 Tconv from the DLN. In addition, M133 Tregs diminished microglia activation and decreased the number and function of Tconv in the infected brain. Thus, virus-specific Tregs inhibited pathogenic CD4 T cell responses during priming and effector stages, particularly those recognizing cognate antigen, and decreased mortality and morbidity without affecting virus clearance. These cells are more suppressive than bulk Tregs and provide a targeted approach to ameliorating immunopathological disease in infectious settings.

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

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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.

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

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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.

32 CFR 202.4 - Composition of a RAB.

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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...

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

[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.

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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

Rab32 is important for autophagy and lipid storage in Drosophila.

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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.

Rab7a modulates ER stress and ER morphology.

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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.

Proliferation requirements of cytomegalovirus-specific, effector-type human CD8+ T cells

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van Leeuwen, Ester M.; Gamadia, Laila E.; Baars, Paul A.; Remmerswaal, Ester B.; ten Berge, Ineke J.; van Lier, René A.

2002-01-01

Two prototypic types of virus-specific CD8(+) T cells can be found in latently infected individuals: CD45R0(+)CD27(+)CCR7(-) effector-memory, and CD45RA(+)CD27(-)CCR7(-) effector-type cells. It has recently been implied that CD45RA(+)CD27(-)CCR7(-) T cells are terminally differentiated effector

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

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

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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.

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

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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.

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

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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

Yersinia pestis Requires Host Rab1b for Survival in Macrophages.

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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.

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

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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.

The structural basis of Arf effector specificity: the crystal structure of ARF6 in a complex with JIP4.

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Isabet, Tatiana; Montagnac, Guillaume; Regazzoni, Karine; Raynal, Bertrand; El Khadali, Fatima; England, Patrick; Franco, Michel; Chavrier, Philippe; Houdusse, Anne; Ménétrey, Julie

2009-09-16

The JNK-interacting proteins, JIP3 and JIP4, are specific effectors of the small GTP-binding protein ARF6. The interaction of ARF6-GTP with the second leucine zipper (LZII) domains of JIP3/JIP4 regulates the binding of JIPs to kinesin-1 and dynactin. Here, we report the crystal structure of ARF6-GTP bound to the JIP4-LZII at 1.9 A resolution. The complex is a heterotetramer with dyad symmetry arranged in an ARF6-(JIP4)(2)-ARF6 configuration. Comparison of the ARF6-JIP4 interface with the equivalent region of ARF1 shows the structural basis of JIP4's specificity for ARF6. Using site-directed mutagenesis and surface plasmon resonance, we further show that non-conserved residues at the switch region borders are the key structural determinants of JIP4 specificity. A structure-derived model of the association of the ARF6-JIP3/JIP4 complex with membranes shows that the JIP4-LZII coiled-coil should lie along the membrane to prevent steric hindrances, resulting in only one ARF6 molecule bound. Such a heterotrimeric complex gives insights to better understand the ARF6-mediated motor switch regulatory function.

The primary immune response to Vaccinia virus vaccination includes cells with a distinct cytotoxic effector CD4 T-cell phenotype.

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Munier, C Mee Ling; van Bockel, David; Bailey, Michelle; Ip, Susanna; Xu, Yin; Alcantara, Sheilajen; Liu, Sue Min; Denyer, Gareth; Kaplan, Warren; Suzuki, Kazuo; Croft, Nathan; Purcell, Anthony; Tscharke, David; Cooper, David A; Kent, Stephen J; Zaunders, John J; Kelleher, Anthony D

2016-10-17

Smallpox was eradicated by a global program of inoculation with Vaccinia virus (VV). Robust VV-specific CD4 T-cell responses during primary infection are likely essential to controlling VV replication. Although there is increasing interest in cytolytic CD4 T-cells across many viral infections, the importance of these cells during acute VV infection is unclear. We undertook a detailed functional and genetic characterization of CD4 T-cells during acute VV-infection of humans. VV-specific T-cells were identified by up-regulation of activation markers directly ex vivo and through cytokine and co-stimulatory molecule expression. At day-13-post primary inoculation with VV, CD38highCD45RO+ CD4 T-cells were purified by cell sorting, RNA isolated and analysed by microarray. Differential expression of up-regulated genes in activated CD4 T-cells was confirmed at the mRNA and protein levels. We compared analyses of VV-specific CD4 T-cells to studies on 12 subjects with primary HIV infection (PHI). VV-specific T-cells lines were established from PBMCs collected post vaccination and checked for cytotoxicity potential. A median 11.9% CD4 T-cells were CD38highCD45RO+ at day-13 post-VV inoculation, compared to 3.0% prior and 10.4% during PHI. Activated CD4 T-cells had an up-regulation of genes related to cytolytic function, including granzymes K and A, perforin, granulysin, TIA-1, and Rab27a. No difference was seen between CD4 T-cell expression of perforin or TIA-1 to VV and PHI, however granzyme k was more dominant in the VV response. At 25:1 effector to target ratio, two VV-specific T-cell lines exhibited 62% and 30% cytotoxicity respectively and CD107a degranulation. We show for the first time that CD4 CTL are prominent in the early response to VV. Understanding the role of CD4 CTL in the generation of an effective anti-viral memory may help develop more effective vaccines for diseases such as HIV. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Recycling domains in plant cell morphogenesis: small GTPase effectors, plasma membrane signalling and the exocyst.

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Zárský, Viktor; Potocký, Martin

2010-04-01

The Rho/Rop small GTPase regulatory module is central for initiating exocytotically ACDs (active cortical domains) in plant cell cortex, and a growing array of Rop regulators and effectors are being discovered in plants. Structural membrane phospholipids are important constituents of cells as well as signals, and phospholipid-modifying enzymes are well known effectors of small GTPases. We have shown that PLDs (phospholipases D) and their product, PA (phosphatidic acid), belong to the regulators of the secretory pathway in plants. We have also shown that specific NOXs (NADPH oxidases) producing ROS (reactive oxygen species) are involved in cell growth as exemplified by pollen tubes and root hairs. Most plant cells exhibit several distinct plasma membrane domains (ACDs), established and maintained by endocytosis/exocytosis-driven membrane protein recycling. We proposed recently the concept of a 'recycling domain' (RD), uniting the ACD and the connected endosomal recycling compartment (endosome), as a dynamic spatiotemporal entity. We have described a putative GTPase-effector complex exocyst involved in exocytic vesicle tethering in plants. Owing to the multiplicity of its Exo70 subunits, this complex, along with many RabA GTPases (putative recycling endosome organizers), may belong to core regulators of RD organization in plants.

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

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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

Structural basis for sequence-specific recognition of DNA by TAL effectors

KAUST Repository

Deng, Dong

2012-01-05

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair by using two hypervariable residues [known as repeat variable diresidues (RVDs)] at positions 12 and 13. Here, we report the crystal structures of an 11.5-repeat TAL effector in both DNA-free and DNA-bound states. Each TAL repeat comprises two helices connected by a short RVD-containing loop. The 11.5 repeats form a right-handed, superhelical structure that tracks along the sense strand of DNA duplex, with RVDs contacting the major groove. The 12th residue stabilizes the RVD loop, whereas the 13th residue makes a base-specific contact. Understanding DNA recognition by TAL effectors may facilitate rational design of DNA-binding proteins with biotechnological applications.

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

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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.

Effector protein translocation by the Coxiella burnetii Dot/Icm type IV secretion system requires endocytic maturation of the pathogen-occupied vacuole.

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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

32 CFR 202.9 - Conducting RAB meetings.

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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...

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

Rab23 is a flagellar protein in Trypanosoma brucei

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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.

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

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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

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence.

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Rodriguez, Patricia A; Escudero-Martinez, Carmen; Bos, Jorunn I B

2017-03-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M persicae -host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. © 2017 American Society of Plant Biologists. All Rights Reserved.

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

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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.

Structural basis for sequence-specific recognition of DNA by TAL effectors

KAUST Repository

Deng, Dong; Yan, Chuangye; Pan, Xiaojing; Mahfouz, Magdy M.; Wang, Jiawei; Zhu, Jiankang; Shi, Yi Gong; Yan, Nieng

2012-01-01

TAL (transcription activator-like) effectors, secreted by phytopathogenic bacteria, recognize host DNA sequences through a central domain of tandem repeats. Each repeat comprises 33 to 35 conserved amino acids and targets a specific base pair

Membrane localization and dynamics of geranylgeranylated Rab5 hypervariable region.

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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.

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

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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.

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

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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.

Rice Rab11 is required for JA-mediated defense signaling

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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.

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.

Characterization and DNA-binding specificities of Ralstonia TAL-like effectors

KAUST Repository

Li, Lixin

2013-07-01

Transcription activator-like effectors (TALEs) from Xanthomonas sp. have been used as customizable DNA-binding modules for genome-engineering applications. Ralstonia solanacearum TALE-like proteins (RTLs) exhibit similar structural features to TALEs, including a central DNA-binding domain composed of 35 amino acid-long repeats. Here, we characterize the RTLs and show that they localize in the plant cell nucleus, mediate DNA binding, and might function as transcriptional activators. RTLs have a unique DNA-binding architecture and are enriched in repeat variable di-residues (RVDs), which determine repeat DNA-binding specificities. We determined the DNA-binding specificities for the RVD sequences ND, HN, NP, and NT. The RVD ND mediates highly specific interactions with C nucleotide, HN interacts specifically with A and G nucleotides, and NP binds to C, A, and G nucleotides. Moreover, we developed a highly efficient repeat assembly approach for engineering RTL effectors. Taken together, our data demonstrate that RTLs are unique DNA-targeting modules that are excellent alternatives to be tailored to bind to user-selected DNA sequences for targeted genomic and epigenomic modifications. These findings will facilitate research concerning RTL molecular biology and RTL roles in the pathogenicity of Ralstonia spp. © 2013 The Author.

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.

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

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.

Human reinforcement learning subdivides structured action spaces by learning effector-specific values.

Science.gov (United States)

Gershman, Samuel J; Pesaran, Bijan; Daw, Nathaniel D

2009-10-28

Humans and animals are endowed with a large number of effectors. Although this enables great behavioral flexibility, it presents an equally formidable reinforcement learning problem of discovering which actions are most valuable because of the high dimensionality of the action space. An unresolved question is how neural systems for reinforcement learning-such as prediction error signals for action valuation associated with dopamine and the striatum-can cope with this "curse of dimensionality." We propose a reinforcement learning framework that allows for learned action valuations to be decomposed into effector-specific components when appropriate to a task, and test it by studying to what extent human behavior and blood oxygen level-dependent (BOLD) activity can exploit such a decomposition in a multieffector choice task. Subjects made simultaneous decisions with their left and right hands and received separate reward feedback for each hand movement. We found that choice behavior was better described by a learning model that decomposed the values of bimanual movements into separate values for each effector, rather than a traditional model that treated the bimanual actions as unitary with a single value. A decomposition of value into effector-specific components was also observed in value-related BOLD signaling, in the form of lateralized biases in striatal correlates of prediction error and anticipatory value correlates in the intraparietal sulcus. These results suggest that the human brain can use decomposed value representations to "divide and conquer" reinforcement learning over high-dimensional action spaces.

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

An Aphid Effector Targets Trafficking Protein VPS52 in a Host-Specific Manner to Promote Virulence1[OPEN

Science.gov (United States)

2017-01-01

Plant- and animal-feeding insects secrete saliva inside their hosts, containing effectors, which may promote nutrient release and suppress immunity. Although for plant pathogenic microbes it is well established that effectors target host proteins to modulate host cell processes and promote disease, the host cell targets of herbivorous insects remain elusive. Here, we show that the existing plant pathogenic microbe effector paradigm can be extended to herbivorous insects in that effector-target interactions inside host cells modify critical host processes to promote plant susceptibility. We showed that the effector Mp1 from Myzus persicae associates with the host Vacuolar Protein Sorting Associated Protein52 (VPS52). Using natural variants, we provide a strong link between effector virulence activity and association with VPS52, and show that the association is highly specific to M. persicae-host interactions. Also, coexpression of Mp1, but not Mp1-like variants, specifically with host VPS52s resulted in effector relocalization to vesicle-like structures that associate with prevacuolar compartments. We show that high VPS52 levels negatively impact virulence, and that aphids are able to reduce VPS52 levels during infestation, indicating that VPS52 is an important virulence target. Our work is an important step forward in understanding, at the molecular level, how a major agricultural pest promotes susceptibility during infestation of crop plants. We give evidence that an herbivorous insect employs effectors that interact with host proteins as part of an effective virulence strategy, and that these effectors likely function in a species-specific manner. PMID:28100451

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

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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

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

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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

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

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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.

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

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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.

High Rab27A expression indicates favorable prognosis in CRC.

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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.

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

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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.

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

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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

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

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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.

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...

Autoreactive effector/memory CD4+ and CD8+ T cells infiltrating grafted and endogenous islets in diabetic NOD mice exhibit similar T cell receptor usage.

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Ramiro Diz

Full Text Available Islet transplantation provides a "cure" for type 1 diabetes but is limited in part by recurrent autoimmunity mediated by β cell-specific CD4(+ and CD8(+ T cells. Insight into the T cell receptor (TCR repertoire of effector T cells driving recurrent autoimmunity would aid the development of immunotherapies to prevent islet graft rejection. Accordingly, we used a multi-parameter flow cytometry strategy to assess the TCR variable β (Vβ chain repertoires of T cell subsets involved in autoimmune-mediated rejection of islet grafts in diabetic NOD mouse recipients. Naïve CD4(+ and CD8(+ T cells exhibited a diverse TCR repertoire, which was similar in all tissues examined in NOD recipients including the pancreas and islet grafts. On the other hand, the effector/memory CD8(+ T cell repertoire in the islet graft was dominated by one to four TCR Vβ chains, and specific TCR Vβ chain usage varied from recipient to recipient. Similarly, islet graft- infiltrating effector/memory CD4(+ T cells expressed a limited number of prevalent TCR Vβ chains, although generally TCR repertoire diversity was increased compared to effector/memory CD8(+ T cells. Strikingly, the majority of NOD recipients showed an increase in TCR Vβ12-bearing effector/memory CD4(+ T cells in the islet graft, most of which were proliferating, indicating clonal expansion. Importantly, TCR Vβ usage by effector/memory CD4(+ and CD8(+ T cells infiltrating the islet graft exhibited greater similarity to the repertoire found in the pancreas as opposed to the draining renal lymph node, pancreatic lymph node, or spleen. Together these results demonstrate that effector/memory CD4(+ and CD8(+ T cells mediating autoimmune rejection of islet grafts are characterized by restricted TCR Vβ chain usage, and are similar to T cells that drive destruction of the endogenous islets.

Rab GTPases in Immunity and Inflammation.

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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.

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).

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

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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.

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�

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

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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

Rab GTPases in Immunity and Inflammation

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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.

Effector-Triggered Immunity Determines Host Genotype-Specific Incompatibility in Legume-Rhizobium Symbiosis.

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Yasuda, Michiko; Miwa, Hiroki; Masuda, Sachiko; Takebayashi, Yumiko; Sakakibara, Hitoshi; Okazaki, Shin

2016-08-01

Symbiosis between legumes and rhizobia leads to the formation of N2-fixing root nodules. In soybean, several host genes, referred to as Rj genes, control nodulation. Soybean cultivars carrying the Rj4 gene restrict nodulation by specific rhizobia such as Bradyrhizobium elkanii We previously reported that the restriction of nodulation was caused by B. elkanii possessing a functional type III secretion system (T3SS), which is known for its delivery of virulence factors by pathogenic bacteria. In the present study, we investigated the molecular basis for the T3SS-dependent nodulation restriction in Rj4 soybean. Inoculation tests revealed that soybean cultivar BARC-2 (Rj4/Rj4) restricted nodulation by B. elkanii USDA61, whereas its nearly isogenic line BARC-3 (rj4/rj4) formed nitrogen-fixing nodules with the same strain. Root-hair curling and infection threads were not observed in the roots of BARC-2 inoculated with USDA61, indicating that Rj4 blocked B. elkanii infection in the early stages. Accumulation of H2O2 and salicylic acid (SA) was observed in the roots of BARC-2 inoculated with USDA61. Transcriptome analyses revealed that inoculation of USDA61, but not its T3SS mutant in BARC-2, induced defense-related genes, including those coding for hypersensitive-induced responsive protein, which act in effector-triggered immunity (ETI) in Arabidopsis. These findings suggest that B. elkanii T3SS triggers the SA-mediated ETI-type response in Rj4 soybean, which consequently blocks symbiotic interactions. This study revealed a common molecular mechanism underlying both plant-pathogen and plant-symbiont interactions, and suggests that establishment of a root nodule symbiosis requires the evasion or suppression of plant immune responses triggered by rhizobial effectors. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

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

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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.

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

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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.

Partial Diversity Generates Effector Immunity Specificity of the Bac41-Like Bacteriocins of Enterococcus faecalis Clinical Strains.

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Kurushima, Jun; Ike, Yasuyoshi; Tomita, Haruyoshi

2016-09-01

Bacteriocin 41 (Bac41) is the plasmid-encoded bacteriocin produced by the opportunistic pathogen Enterococcus faecalis Its genetic determinant consists of bacL1 (effector), bacL2 (regulator), bacA (effector), and bacI (immunity). The secreted effectors BacL1 and BacA coordinate to induce the lytic cell death of E. faecalis Meanwhile, the immunity factor BacI provides self-resistance to the Bac41 producer, E. faecalis, against the action of BacL1 and BacA. In this study, we demonstrated that more than half of the 327 clinical strains of E. faecalis screened had functional Bac41 genes. Analysis of the genetic structure of the Bac41 genes in the DNA sequences of the E. faecalis strains revealed that the Bac41-like genes consist of a relatively conserved region and a variable region located downstream from bacA Based on similarities in the variable region, the Bac41-like genes could be classified into type I, type IIa, and type IIb. Interestingly, the distinct Bac41 types had specific immunity factors for self-resistance, BacI1 or BacI2, and did not show cross-immunity to the other type of effector. We also demonstrated experimentally that the specificity of the immunity was determined by the combination of the C-terminal region of BacA and the presence of the unique BacI1 or BacI2 factor. These observations suggested that Bac41-like bacteriocin genes are extensively disseminated among E. faecalis strains in the clinical environment and can be grouped into at least three types. It was also indicated that the partial diversity results in specificity of self-resistance which may offer these strains a competitive advantage. Bacteriocins are antibacterial effectors produced by bacteria. In general, a bacteriocin-coding gene is accompanied by a cognate immunity gene that confers self-resistance on the bacteriocin-producing bacterium itself. We demonstrated that one of the bacteriocins, Bac41, is disseminated among E. faecalis clinical strains and the Bac41 subtypes with

Structural analyses of Legionella LepB reveal a new GAP fold that catalytically mimics eukaryotic RasGAP.

Science.gov (United States)

Yu, Qin; Hu, Liyan; Yao, Qing; Zhu, Yongqun; Dong, Na; Wang, Da-Cheng; Shao, Feng

2013-06-01

Rab GTPases are emerging targets of diverse bacterial pathogens. Here, we perform biochemical and structural analyses of LepB, a Rab GTPase-activating protein (GAP) effector from Legionella pneumophila. We map LepB GAP domain to residues 313-618 and show that the GAP domain is Rab1 specific with a catalytic activity higher than the canonical eukaryotic TBC GAP and the newly identified VirA/EspG family of bacterial RabGAP effectors. Exhaustive mutation analyses identify Arg444 as the arginine finger, but no catalytically essential glutamine residues. Crystal structures of LepB313-618 alone and the GAP domain of Legionella drancourtii LepB in complex with Rab1-GDP-AlF3 support the catalytic role of Arg444, and also further reveal a 3D architecture and a GTPase-binding mode distinct from all known GAPs. Glu449, structurally equivalent to TBC RabGAP glutamine finger in apo-LepB, undergoes a drastic movement upon Rab1 binding, which induces Rab1 Gln70 side-chain flipping towards GDP-AlF3 through a strong ionic interaction. This conformationally rearranged Gln70 acts as the catalytic cis-glutamine, therefore uncovering an unexpected RasGAP-like catalytic mechanism for LepB. Our studies highlight an extraordinary structural and catalytic diversity of RabGAPs, particularly those from bacterial pathogens.

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

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)

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.

TAL effectors specificity stems from negative discrimination.

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Basile I M Wicky

Full Text Available Transcription Activator-Like (TAL effectors are DNA-binding proteins secreted by phytopathogenic bacteria that interfere with native cellular functions by binding to plant DNA promoters. The key element of their architecture is a domain of tandem-repeats with almost identical sequences. Most of the polymorphism is located at two consecutive amino acids termed Repeat Variable Diresidue (RVD. The discovery of a direct link between the RVD composition and the targeted nucleotide allowed the design of TAL-derived DNA-binding tools with programmable specificities that revolutionized the field of genome engineering. Despite structural data, the molecular origins of this specificity as well as the recognition mechanism have remained unclear. Molecular simulations of the recent crystal structures suggest that most of the protein-DNA binding energy originates from non-specific interactions between the DNA backbone and non-variable residues, while RVDs contributions are negligible. Based on dynamical and energetic considerations we postulate that, while the first RVD residue promotes helix breaks--allowing folding of TAL as a DNA-wrapping super-helix--the second provides specificity through a negative discrimination of matches. Furthermore, we propose a simple pharmacophore-like model for the rationalization of RVD-DNA interactions and the interpretation of experimental findings concerning shared affinities and binding efficiencies. The explanatory paradigm presented herein provides a better comprehension of this elegant architecture and we hope will allow for improved designs of TAL-derived biotechnological tools.

α4β7+ CD4+ Effector/Effector Memory T Cells Differentiate into Productively and Latently Infected Central Memory T Cells by Transforming Growth Factor β1 during HIV-1 Infection.

Science.gov (United States)

Cheung, Ka-Wai; Wu, Tongjin; Ho, Sai Fan; Wong, Yik Chun; Liu, Li; Wang, Hui; Chen, Zhiwei

2018-04-15

HIV-1 transmission occurs mainly through mucosal tissues. During mucosal transmission, HIV-1 preferentially infects α 4 β 7 + gut-homing CCR7 - CD4 + effector/effector memory T cells (T EM ) and results in massive depletion of these cells and other subsets of T EM in gut-associated lymphoid tissues. However, besides being eliminated by HIV-1, the role of T EM during the early stage of infection remains inconclusive. Here, using in vitro -induced α 4 β 7 + gut-homing T EM (α 4 β 7 + T EM ), we found that α 4 β 7 + T EM differentiated into CCR7 + CD4 + central memory T cells (T CM ). This differentiation was HIV-1 independent but was inhibited by SB431542, a specific transforming growth factor β (TGF-β) receptor I kinase inhibitor. Consistently, T EM -to-T CM differentiation was observed in α 4 β 7 + T EM stimulated with TGF-β1 (TGF-β). The T CM properties of the TGF-β-induced T EM -derived T CM (α 4 β 7 + T CM ) were confirmed by their enhanced CCL19 chemotaxis and the downregulation of surface CCR7 upon T cell activation in vitro Importantly, the effect of TGF-β on T CM differentiation also held in T EM directly isolated from peripheral blood. To investigate the significance of the TGF-β-dependent T EM -to-T CM differentiation in HIV/AIDS pathogenesis, we observed that both productively and latently infected α 4 β 7 + T CM could differentiate from α 4 β 7 + T EM in the presence of TGF-β during HIV-1 infection. Collectively, this study not only provides a new insight for the plasticity of T EM but also suggests that the TGF-β-dependent T EM -to-T CM differentiation is a previously unrecognized mechanism for the formation of latently infected T CM after HIV-1 infection. IMPORTANCE HIV-1 is the causative agent of HIV/AIDS, which has led to millions of deaths in the past 30 years. Although the implementation of highly active antiretroviral therapy has remarkably reduced the HIV-1-related morbidity and mortality, HIV-1 is not eradicated in

Diverse Epitope Specificity, Immunodominance Hierarchy, and Functional Avidity of Effector CD4 T Cells Established During Priming Is Maintained in Lung After Influenza A Virus Infection.

Science.gov (United States)

Richards, Katherine A; DiPiazza, Anthony T; Rattan, Ajitanuj; Knowlden, Zackery A G; Yang, Hongmei; Sant, Andrea J

2018-01-01

One of the major contributions to protective immunity to influenza viruses that is provided by virus-specific CD4 T cells is delivery of effector function to the infected lung. However, there is little known about the selection and breadth of viral epitope-specific CD4 T cells that home to the lung after their initial priming. In this study, using a mouse model of influenza A infection and an unbiased method of epitope identification, the viral epitope-specific CD4 T cells elicited after infection were identified and quantified. We found that a very diverse specificity of CD4 T cells is primed by infection, including epitopes from hemagglutinin, neuraminidase, matrix protein, nucleoprotein, and non-structural protein-1. Using peptide-specific cytokine EliSpots, the diversity and immunodominance hierarchies established in the lung-draining lymph node were compared with specificities of CD4 T cells that home to the lung. Our studies revealed that CD4 T cells of all epitope specificities identified in peripheral lymphoid tissue home back to the lung and that most of these lung-homing cells are localized within the tissue rather than the pulmonary vasculature. There is a striking shift of CD4 T cell functionality that enriches for IFN-γ production as cells are primed in the lymph node, enter the lung vasculature, and finally establish residency in the tissue, but with no apparent shifts in their functional avidity. We conclude that CD4 T cells of broad viral epitope specificity are recruited into the lung after influenza infection, where they then have the opportunity to encounter infected or antigen-bearing antigen-presenting cells.

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.

Ganglioside-specific IgG and IgA recruit leukocyte effector functions in Guillain-Barre syndrome

NARCIS (Netherlands)

Sorge, N.M. van; Yuki, N.; Koga, M.; Susuki, K.; Jansen, M.D.; Kooten, C. van; Wokke, J.H.; Winkel, J.G.J. van de; Pol, W.L. van der; Berg, L.H. van den

2007-01-01

The capacity of ganglioside-specific autoantibodies to recruit leukocyte effector functions was studied. Serum samples from 87 patients with Guillain–Barré (GBS) or Miller Fisher syndrome (MFS), containing GM1-, GQ1b-, or GD1b-specific IgG or IgA, were tested for leukocyte activating capacity.

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

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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.

Comprehensive analysis of the specificity of transcription activator-like effector nucleases

DEFF Research Database (Denmark)

Juillerat, Alexandre; Dubois, Gwendoline; Valton, Julien

2014-01-01

A key issue when designing and using DNA-targeting nucleases is specificity. Ideally, an optimal DNA-targeting tool has only one recognition site within a genomic sequence. In practice, however, almost all designer nucleases available today can accommodate one to several mutations within...... their target site. The ability to predict the specificity of targeting is thus highly desirable. Here, we describe the first comprehensive experimental study focused on the specificity of the four commonly used repeat variable diresidues (RVDs; NI:A, HD:C, NN:G and NG:T) incorporated in transcription activator......-like effector nucleases (TALEN). The analysis of >15 500 unique TALEN/DNA cleavage profiles allowed us to monitor the specificity gradient of the RVDs along a TALEN/DNA binding array and to present a specificity scoring matrix for RVD/nucleotide association. Furthermore, we report that TALEN can only...

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

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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.

Effector CD4+ T cells recognize intravascular antigen presented by patrolling monocytes.

Science.gov (United States)

Westhorpe, Clare L V; Norman, M Ursula; Hall, Pam; Snelgrove, Sarah L; Finsterbusch, Michaela; Li, Anqi; Lo, Camden; Tan, Zhe Hao; Li, Songhui; Nilsson, Susan K; Kitching, A Richard; Hickey, Michael J

2018-02-21

Although effector CD4 + T cells readily respond to antigen outside the vasculature, how they respond to intravascular antigens is unknown. Here we show the process of intravascular antigen recognition using intravital multiphoton microscopy of glomeruli. CD4 + T cells undergo intravascular migration within uninflamed glomeruli. Similarly, while MHCII is not expressed by intrinsic glomerular cells, intravascular MHCII-expressing immune cells patrol glomerular capillaries, interacting with CD4 + T cells. Following intravascular deposition of antigen in glomeruli, effector CD4 + T-cell responses, including NFAT1 nuclear translocation and decreased migration, are consistent with antigen recognition. Of the MHCII + immune cells adherent in glomerular capillaries, only monocytes are retained for prolonged durations. These cells can also induce T-cell proliferation in vitro. Moreover, monocyte depletion reduces CD4 + T-cell-dependent glomerular inflammation. These findings indicate that MHCII + monocytes patrolling the glomerular microvasculature can present intravascular antigen to CD4 + T cells within glomerular capillaries, leading to antigen-dependent inflammation.

Identification of the Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the T3SS2-specific effector VopC.

Science.gov (United States)

Akeda, Yukihiro; Kodama, Toshio; Saito, Kazunobu; Iida, Tetsuya; Oishi, Kazunori; Honda, Takeshi

2011-11-01

The enteropathogen Vibrio parahaemolyticus possesses two sets of type III secretion systems, T3SS1 and T3SS2. Effector proteins secreted by these T3SSs are delivered into host cells, leading to cell death or diarrhea. However, it is not known how specific effectors are secreted through a specific T3SS when both T3SSs are expressed within bacteria. One molecule thought to determine secretion specificity is a T3SS-associated chaperone; however, no T3SS2-specific chaperone has been identified. Therefore, we screened T3SS2 chaperone candidates by a pull-down assay using T3SS2 effectors fused with glutathione-S-transferase. A secretion assay revealed that the newly identified cognate chaperone VocC for the T3SS2-specific effector VopC was required for the efficient secretion of the substrate through T3SS2. Further experiments determined the chaperone-binding domain and the amino-terminal secretion signal of the cognate effector. These findings, in addition to the previously identified T3SS1-specific chaperone, VecA, provide a strategy to clarify the specificity of effector secretion through T3SSs of V. parahaemolyticus. 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

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.

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.

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

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.

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.

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

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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.   

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

Science.gov (United States)

Richardson, Simone I; Chung, Amy W; Natarajan, Harini; Mabvakure, Batsirai; Mkhize, Nonhlanhla N; Garrett, Nigel; Abdool Karim, Salim; Moore, Penny L; Ackerman, Margaret E; Alter, Galit; Morris, Lynn

2018-04-01

While the induction of broadly neutralizing antibodies (bNAbs) is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP), complement deposition (ADCD), cellular cytotoxicity (ADCC) and cellular trogocytosis (ADCT) were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID) was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

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

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.

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

Profiling calcium signals of in vitro polarized human effector CD4+ T cells.

Science.gov (United States)

Kircher, Sarah; Merino-Wong, Maylin; Niemeyer, Barbara A; Alansary, Dalia

2018-06-01

Differentiation of naïve CD4 + T cells into effector subtypes with distinct cytokine profiles and physiological roles is a tightly regulated process, the imbalance of which can lead to an inadequate immune response or autoimmune disease. The crucial role of Ca 2+ signals, mainly mediated by the store operated Ca 2+ entry (SOCE) in shaping the immune response is well described. However, it is unclear if human effector CD4 + T cell subsets show differential Ca 2+ signatures in response to different stimulation methods. Herein, we provide optimized in vitro culture conditions for polarization of human CD4 + effector T cells and characterize their SOCE following both pharmacological store depletion and direct T-cell receptor (TCR) activation. Moreover, we measured whole cell Ca 2+ release activated Ca 2+ currents (I CRAC ) and investigated whether the observed differences correlate to the expression of CRAC genes. Our results show that Ca 2+ profiles of helper CD4 + Th1, Th2 and Th17 are distinct and in part shaped by the intensity of stimulation. Regulatory T cells (Treg) are unique being the subtype with the most prominent SOCE response. Analysis of in vivo differentiated Treg unraveled the role of differential expression of ORAI2 in fine-tuning signals in Treg vs. conventional CD4 + T cells. Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.

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

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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.

Signal transduction meets vesicle traffic: the software and hardware of GLUT4 translocation.

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Klip, Amira; Sun, Yi; Chiu, Tim Ting; Foley, Kevin P

2014-05-15

Skeletal muscle is the major tissue disposing of dietary glucose, a function regulated by insulin-elicited signals that impart mobilization of GLUT4 glucose transporters to the plasma membrane. This phenomenon, also central to adipocyte biology, has been the subject of intense and productive research for decades. We focus on muscle cell studies scrutinizing insulin signals and vesicle traffic in a spatiotemporal manner. Using the analogy of an integrated circuit to approach the intersection between signal transduction and vesicle mobilization, we identify signaling relays ("software") that engage structural/mechanical elements ("hardware") to enact the rapid mobilization and incorporation of GLUT4 into the cell surface. We emphasize how insulin signal transduction switches from tyrosine through lipid and serine phosphorylation down to activation of small G proteins of the Rab and Rho families, describe key negative regulation step of Rab GTPases through the GTPase-activating protein activity of the Akt substrate of 160 kDa (AS160), and focus on the mechanical effectors engaged by Rabs 8A and 10 (the molecular motor myosin Va), and the Rho GTPase Rac1 (actin filament branching and severing through Arp2/3 and cofilin). Finally, we illustrate how actin filaments interact with myosin 1c and α-Actinin4 to promote vesicle tethering as preamble to fusion with the membrane. Copyright © 2014 the American Physiological Society.

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.

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.

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

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

HIV-specific Fc effector function early in infection predicts the development of broadly neutralizing antibodies.

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Simone I Richardson

2018-04-01

Full Text Available While the induction of broadly neutralizing antibodies (bNAbs is a major goal of HIV vaccination strategies, there is mounting evidence to suggest that antibodies with Fc effector function also contribute to protection against HIV infection. Here we investigated Fc effector functionality of HIV-specific IgG plasma antibodies over 3 years of infection in 23 individuals, 13 of whom developed bNAbs. Antibody-dependent cellular phagocytosis (ADCP, complement deposition (ADCD, cellular cytotoxicity (ADCC and cellular trogocytosis (ADCT were detected in almost all individuals with levels of activity increasing over time. At 6 months post-infection, individuals with bNAbs had significantly higher levels of ADCD and ADCT that correlated with antibody binding to C1q and FcγRIIa respectively. In addition, antibodies from individuals with bNAbs showed more IgG subclass diversity to multiple HIV antigens which also correlated with Fc polyfunctionality. Germinal center activity represented by CXCL13 levels and expression of activation-induced cytidine deaminase (AID was found to be associated with neutralization breadth, Fc polyfunctionality and IgG subclass diversity. Overall, multivariate analysis by random forest classification was able to group bNAb individuals with 85% sensitivity and 80% specificity based on the properties of their antibody Fc early in HIV infection. Thus, the Fc effector function profile predicted the development of neutralization breadth in this cohort, suggesting that intrinsic immune factors within the germinal center provide a mechanistic link between the Fc and Fab of HIV-specific antibodies.

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

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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.

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.

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

CD4+CD25+ regulatory T cells control CD8+ T-cell effector differentiation by modulating IL-2 homeostasis

Science.gov (United States)

McNally, Alice; Hill, Geoffrey R.; Sparwasser, Tim; Thomas, Ranjeny; Steptoe, Raymond J.

2011-01-01

CD4+CD25+ regulatory T cells (Treg) play a crucial role in the regulation of immune responses. Although many mechanisms of Treg suppression in vitro have been described, the mechanisms by which Treg modulate CD8+ T cell differentiation and effector function in vivo are more poorly defined. It has been proposed, in many instances, that modulation of cytokine homeostasis could be an important mechanism by which Treg regulate adaptive immunity; however, direct experimental evidence is sparse. Here we demonstrate that CD4+CD25+ Treg, by critically regulating IL-2 homeostasis, modulate CD8+ T-cell effector differentiation. Expansion and effector differentiation of CD8+ T cells is promoted by autocrine IL-2 but, by competing for IL-2, Treg limit CD8+ effector differentiation. Furthermore, a regulatory loop exists between Treg and CD8+ effector T cells, where IL-2 produced during CD8+ T-cell effector differentiation promotes Treg expansion. PMID:21502514

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.

Functional differences between PD-1+ and PD-1- CD4+ effector T cells in healthy donors and patients with glioblastoma multiforme.

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Brittany A Goods

Full Text Available Immune checkpoint inhibitors targeting programmed cell death protein 1 (PD-1 have been highly successful in the treatment of cancer. While PD-1 expression has been widely investigated, its role in CD4+ effector T cells in the setting of health and cancer remains unclear, particularly in the setting of glioblastoma multiforme (GBM, the most aggressive and common form of brain cancer. We examined the functional and molecular features of PD-1+CD4+CD25-CD127+Foxp3-effector cells in healthy subjects and in patients with GBM. In healthy subjects, we found that PD-1+CD4+ effector cells are dysfunctional: they do not proliferate but can secrete large quantities of IFNγ. Strikingly, blocking antibodies against PD-1 did not rescue proliferation. RNA-sequencing revealed features of exhaustion in PD-1+ CD4 effectors. In the context of GBM, tumors were enriched in PD-1+ CD4+ effectors that were similarly dysfunctional and unable to proliferate. Furthermore, we found enrichment of PD-1+TIM-3+ CD4+ effectors in tumors, suggesting that co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial. RNA-sequencing of blood and tumors from GBM patients revealed distinct differences between CD4+ effectors from both compartments with enrichment in multiple gene sets from tumor infiltrating PD-1-CD4+ effectors cells. Enrichment of these gene sets in tumor suggests a more metabolically active cell state with signaling through other co-receptors. PD-1 expression on CD4 cells identifies a dysfunctional subset refractory to rescue with PD-1 blocking antibodies, suggesting that the influence of immune checkpoint inhibitors may involve recovery of function in the PD-1-CD4+ T cell compartment. Additionally, co-blockade of PD-1 and TIM-3 in GBM may be therapeutically beneficial.

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

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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.

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.

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

Graft rejection by cytolytic T cells. Specificity of the effector mechanism in the rejection of allogeneic marrow

International Nuclear Information System (INIS)

Nakamura, H.; Gress, R.E.

1990-01-01

Cellular effector mechanisms of allograft rejection remain incompletely described. Characterizing the rejection of foreign-marrow allografts rather than solid-organ grafts has the advantage that the cellular composition of the marrow graft, as a single cell suspension, can be altered to include cellular components with differing antigen expression. Rejection of marrow grafts is sensitive to lethal doses of radiation in the mouse but resistant to sublethal levels of radiation. In an effort to identify cells mediating host resistance, lymphocytes were isolated and cloned from spleens of mice 7 days after sublethal TBI (650 cGy) and inoculation with allogeneic marrow. All clones isolated were cytolytic with specificity for MHC encoded gene products of the allogeneic marrow donor. When cloned cells were transferred in vivo into lethally irradiated (1025 cGy) recipients unable to reject allogeneic marrow, results utilizing splenic 125IUdR uptake indicated that these MHC-specific cytotoxic clones could suppress marrow proliferation. In order to characterize the effector mechanism and the ability of the clones to affect final engraftment, double donor chimeras were constructed so that 2 target cell populations differing at the MHC from each other and from the host were present in the same marrow allograft. Results directly demonstrated an ability of CTL of host MHC type to mediate graft rejection and characterized the effector mechanism as one with specificity for MHC gene products

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

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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.

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

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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...

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

Multiple Avirulence Loci and Allele-Specific Effector Recognition Control the Pm3 Race-Specific Resistance of Wheat to Powdery Mildew[OPEN

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Roffler, Stefan; Stirnweis, Daniel; Treier, Georges; Herren, Gerhard; Korol, Abraham B.; Wicker, Thomas

2015-01-01

In cereals, several mildew resistance genes occur as large allelic series; for example, in wheat (Triticum aestivum and Triticum turgidum), 17 functional Pm3 alleles confer agronomically important race-specific resistance to powdery mildew (Blumeria graminis). The molecular basis of race specificity has been characterized in wheat, but little is known about the corresponding avirulence genes in powdery mildew. Here, we dissected the genetics of avirulence for six Pm3 alleles and found that three major Avr loci affect avirulence, with a common locus_1 involved in all AvrPm3-Pm3 interactions. We cloned the effector gene AvrPm3a2/f2 from locus_2, which is recognized by the Pm3a and Pm3f alleles. Induction of a Pm3 allele-dependent hypersensitive response in transient assays in Nicotiana benthamiana and in wheat demonstrated specificity. Gene expression analysis of Bcg1 (encoded by locus_1) and AvrPm3 a2/f2 revealed significant differences between isolates, indicating that in addition to protein polymorphisms, expression levels play a role in avirulence. We propose a model for race specificity involving three components: an allele-specific avirulence effector, a resistance gene allele, and a pathogen-encoded suppressor of avirulence. Thus, whereas a genetically simple allelic series controls specificity in the plant host, recognition on the pathogen side is more complex, allowing flexible evolutionary responses and adaptation to resistance genes. PMID:26452600

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

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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.

Functional heterogeneity of human effector CD8+ T cells.

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Takata, Hiroshi; Naruto, Takuya; Takiguchi, Masafumi

2012-02-09

Effector CD8(+) T cells are believed to be terminally differentiated cells having cytotoxic activity and the ability to produce effector cytokines such as INF-γ and TNF-α. We investigated the difference between CXCR1(+) and CXCR1(-) subsets of human effector CD27(-)CD28(-)CD8(+) T cells. The subsets expressed cytolytic molecules similarly and exerted substantial cytolytic activity, whereas only the CXCR1(-) subset had IL-2 productivity and self-proliferative activity and was more resistant to cell death than the CXCR1(+) subset. These differences were explained by the specific up-regulation of CAMK4, SPRY2, and IL-7R in the CXCR1(-) subset and that of pro-apoptotic death-associated protein kinase 1 (DAPK1) in the CXCR1(+) subset. The IL-2 producers were more frequently found in the IL-7R(+) subset of the CXCR1(-) effector CD8(+) T cells than in the IL-7R(-) subset. IL-7/IL-7R signaling promoted cell survival only in the CXCR1(-) subset. The present study has highlighted a novel subset of effector CD8(+) T cells producing IL-2 and suggests the importance of this subset in the homeostasis of effector CD8(+) T cells.

Identification of antibody glycosylation structures that predict monoclonal antibody Fc-effector function.

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Chung, Amy W; Crispin, Max; Pritchard, Laura; Robinson, Hannah; Gorny, Miroslaw K; Yu, Xiaojie; Bailey-Kellogg, Chris; Ackerman, Margaret E; Scanlan, Chris; Zolla-Pazner, Susan; Alter, Galit

2014-11-13

To determine monoclonal antibody (mAb) features that predict fragment crystalizable (Fc)-mediated effector functions against HIV. Monoclonal antibodies, derived from Chinese hamster ovary cells or Epstein-Barr virus-immortalized mouse heteromyelomas, with specificity to key regions of the HIV envelope including gp120-V2, gp120-V3 loop, gp120-CD4(+) binding site, and gp41-specific antibodies, were functionally profiled to determine the relative contribution of the variable and constant domain features of the antibodies in driving robust Fc-effector functions. Each mAb was assayed for antibody-binding affinity to gp140(SR162), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) and for the ability to bind to FcγRIIa, FcγRIIb and FcγRIIIa receptors. Antibody glycan profiles were determined by HPLC. Neither the specificity nor the affinity of the mAbs determined the potency of Fc-effector function. FcγRIIIa binding strongly predicted ADCC and decreased galactose content inversely correlated with ADCP, whereas N-glycolylneuraminic acid-containing structures exhibited enhanced ADCP. Additionally, the bi-antenary glycan arm onto which galactose was added predicted enhanced binding to FcγRIIIa and ADCC activity, independent of the specificity of the mAb. Our studies point to the specific Fc-glycan structures that can selectively promote Fc-effector functions independently of the antibody specificity. Furthermore, we demonstrated antibody glycan structures associated with enhanced ADCP activity, an emerging Fc-effector function that may aid in the control and clearance of HIV infection.

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

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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.

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

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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.

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

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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

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

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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.

GITR ligand-costimulation activates effector and regulatory functions of CD4+ T cells

International Nuclear Information System (INIS)

Igarashi, Hanna; Cao, Yujia; Iwai, Hideyuki; Piao, Jinhua; Kamimura, Yosuke; Hashiguchi, Masaaki; Amagasa, Teruo; Azuma, Miyuki

2008-01-01

Engagement of glucocorticoid-induced TNFR-related protein (GITR) enables the costimulation of both CD25 - CD4 + effector (Teff) and CD25 + CD4 + regulatory (Treg) cells; however, the effects of GITR-costimulation on Treg function remain controversial. In this study, we examined the effects of GITR ligand (GITRL) binding on the respective functions of CD4 + T cells. GITRL-P815 transfectants efficiently augmented anti-CD3-induced proliferation and cytokine production by Teff cells. Proliferation and IL-10 production in Treg were also enhanced by GITRL transfectants when exogenous IL-2 and stronger CD3 stimulation was provided. Concomitant GITRL-costimulation of Teff and Treg converted the anergic state of Treg into a proliferating state, maintaining and augmenting their function. Thus, GITRL-costimulation augments both effector and regulatory functions of CD4 + T cells. Our results suggest that highly activated and increased ratios of Treg reverse the immune-enhancing effects of GITRL-costimulation in Teff, which may be problematic for therapeutic applications using strong GITR agonists

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

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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.

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

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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.

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.

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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.

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

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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.

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

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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.

The Alpha-Melanocyte Stimulating Hormone Induces Conversion of Effector T Cells into Treg Cells

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Andrew W. Taylor

2011-01-01

Full Text Available The neuropeptide alpha-melanocyte stimulating hormone (α-MSH has an important role in modulating immunity and homeostasis. The production of IFN-γ by effector T cells is suppressed by α-MSH, while TGF-β production is promoted in the same cells. Such α-MSH-treated T cells have immune regulatory activity and suppress hypersensitivity, autoimmune diseases, and graft rejection. Previous characterizations of the α-MSH-induced Treg cells showed that the cells are CD4+ T cells expressing the same levels of CD25 as effector T cells. Therefore, we further analyzed the α-MSH-induced Treg cells for expression of effector and regulatory T-cell markers. Also, we examined the potential for α-MSH-induced Treg cells to be from the effector T-cell population. We found that the α-MSH-induced Treg cells are CD25+  CD4+ T cells that share similar surface markers as effector T cells, except that they express on their surface LAP. Also, the α-MSH treatment augments FoxP3 message in the effector T cells, and α-MSH induction of regulatory activity was limited to the effector CD25+ T-cell population. Therefore, α-MSH converts effector T cells into Treg cells, which suppress immunity targeting specific antigens and tissues.

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

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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.

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

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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.

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

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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.

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

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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.

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

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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.

The TAL effector PthA4 interacts with nuclear factors involved in RNA-dependent processes including a HMG protein that selectively binds poly(U RNA.

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Tiago Antonio de Souza

Full Text Available Plant pathogenic bacteria utilize an array of effector proteins to cause disease. Among them, transcriptional activator-like (TAL effectors are unusual in the sense that they modulate transcription in the host. Although target genes and DNA specificity of TAL effectors have been elucidated, how TAL proteins control host transcription is poorly understood. Previously, we showed that the Xanthomonas citri TAL effectors, PthAs 2 and 3, preferentially targeted a citrus protein complex associated with transcription control and DNA repair. To extend our knowledge on the mode of action of PthAs, we have identified new protein targets of the PthA4 variant, required to elicit canker on citrus. Here we show that all the PthA4-interacting proteins are DNA and/or RNA-binding factors implicated in chromatin remodeling and repair, gene regulation and mRNA stabilization/modification. The majority of these proteins, including a structural maintenance of chromosomes protein (CsSMC, a translin-associated factor X (CsTRAX, a VirE2-interacting protein (CsVIP2, a high mobility group (CsHMG and two poly(A-binding proteins (CsPABP1 and 2, interacted with each other, suggesting that they assemble into a multiprotein complex. CsHMG was shown to bind DNA and to interact with the invariable leucine-rich repeat region of PthAs. Surprisingly, both CsHMG and PthA4 interacted with PABP1 and 2 and showed selective binding to poly(U RNA, a property that is novel among HMGs and TAL effectors. Given that homologs of CsHMG, CsPABP1, CsPABP2, CsSMC and CsTRAX in other organisms assemble into protein complexes to regulate mRNA stability and translation, we suggest a novel role of TAL effectors in mRNA processing and translational control.

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

A splice variant of RILP induces lysosomal clustering independent of dynein recruitment

International Nuclear Information System (INIS)

Marsman, Marije; Jordens, Ingrid; Rocha, Nuno; Kuijl, Coenraad; Janssen, Lennert; Neefjes, Jacques

2006-01-01

The small GTPase Rab7 controls fusion and transport of late endocytic compartments. A critical mediator is the Rab7 effector RILP that recruits the minus-end dynein-dynactin motor complex to these compartments. We identified a natural occurring splice variant of RILP (RILPsv) lacking only 27 amino acids encoded by exon VII. Both variants bind Rab7, prolong its GTP-bound state, and induce clustering of late endocytic compartments. However, RILPsv does not recruit the dynein-dynactin complex, implicating exon VII in motor recruitment. Clustering might still occur via dimerization, since both RILP and RILPsv are able to form hetero- and homo-dimers. Moreover, both effectors compete for Rab7 binding but with different outcome for dynein-dynactin recruitment and transport. Hence, RILPsv provides an extra dimension to the control of vesicle fusion and transport by the small GTPase Rab7

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

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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.

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

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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.

Mutations of the catalytic subunit of RAB3GAP cause Warburg Micro syndrome

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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...

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

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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.

Temporal effects of Notch signaling and potential cooperation with multiple downstream effectors on adenohypophysis cell specification in zebrafish.

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Nakahara, Yoshinari; Muto, Akihiko; Hirabayashi, Ryo; Sakuma, Tetsushi; Yamamoto, Takashi; Kume, Shoen; Kikuchi, Yutaka

2016-05-01

The adenohypophysis (AH) consists of six distinct types of hormone-secreting cells. In zebrafish, although proper differentiation of all AH cell types has been shown to require Notch signaling within a period of 14-16 h postfertilization (hpf), the mechanisms underlying this process remain to be elucidated. Herein, we observed using the Notch inhibitor dibenzazepine (DBZ) that Notch signaling also contributed to AH cell specification beyond 16 hpf. Specification of distinct cell types was perturbed by DBZ treatment for different time frames, suggesting that AH cells are specified by Notch-dependent and cell-type-specific mechanisms. We also found that two hes-family genes, her4.1 and hey1, were expressed in the developing AH under the influence of Notch signaling. her4.1 knockdown reduced expression of proopiomelanocortin a (pomca), growth hormone (gh), and prolactin, whereas hey1 was responsible only for gh expression. Simultaneous loss of both Her4.1 and Hey1 produced milder phenotypes than that of DBZ-treated embryos. Moreover, DBZ treatment from 18 hpf led to a significant down-regulation of both gh and pomca genes only when combined with injection of a subthreshold level of her4.1-morpholino. These observations suggest that multiple downstream effectors, including Her4.1 and Hey1, mediate Notch signaling during AH cell specification. © 2016 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.

Effector-Triggered Self-Replication in Coupled Subsystems.

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Komáromy, Dávid; Tezcan, Meniz; Schaeffer, Gaël; Marić, Ivana; Otto, Sijbren

2017-11-13

In living systems processes like genome duplication and cell division are carefully synchronized through subsystem coupling. If we are to create life de novo, similar control over essential processes such as self-replication need to be developed. Here we report that coupling two dynamic combinatorial subsystems, featuring two separate building blocks, enables effector-mediated control over self-replication. The subsystem based on the first building block shows only self-replication, whereas that based on the second one is solely responsive toward a specific external effector molecule. Mixing the subsystems arrests replication until the effector molecule is added, resulting in the formation of a host-effector complex and the liberation of the building block that subsequently engages in self-replication. The onset, rate and extent of self-replication is controlled by the amount of effector present. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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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).

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...

Effector profiles distinguish formae speciales of Fusarium oxysporum.

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van Dam, Peter; Fokkens, Like; Schmidt, Sarah M; Linmans, Jasper H J; Kistler, H Corby; Ma, Li-Jun; Rep, Martijn

2016-11-01

Formae speciales (ff.spp.) of the fungus Fusarium oxysporum are often polyphyletic within the species complex, making it impossible to identify them on the basis of conserved genes. However, sequences that determine host-specific pathogenicity may be expected to be similar between strains within the same forma specialis. Whole genome sequencing was performed on strains from five different ff.spp. (cucumerinum, niveum, melonis, radicis-cucumerinum and lycopersici). In each genome, genes for putative effectors were identified based on small size, secretion signal, and vicinity to a "miniature impala" transposable element. The candidate effector genes of all genomes were collected and the presence/absence patterns in each individual genome were clustered. Members of the same forma specialis turned out to group together, with cucurbit-infecting strains forming a supercluster separate from other ff.spp. Moreover, strains from different clonal lineages within the same forma specialis harbour identical effector gene sequences, supporting horizontal transfer of genetic material. These data offer new insight into the genetic basis of host specificity in the F. oxysporum species complex and show that (putative) effectors can be used to predict host specificity in F. oxysporum. © 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

Formulation of the bivalent prostate cancer vaccine with surgifoam elicits antigen-specific effector T cells in PSA-transgenic mice.

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Karan, Dev

2017-10-13

We previously developed and characterized an adenoviral-based prostate cancer vaccine for simultaneous targeting of prostate-specific antigen (PSA) and prostate stem cell antigen (PSCA). We also demonstrated that immunization of mice with the bivalent vaccine (Ad 5 -PSA+PSCA) inhibited the growth of established prostate tumors. However, there are multiple challenges hindering the success of immunological therapies in the clinic. One of the prime concerns has been to overcome the immunological tolerance and maintenance of long-term effector T cells. In this study, we further characterized the use of the bivalent vaccine (Ad 5 -PSA+PSCA) in a transgenic mouse model expressing human PSA in the mouse prostate. We demonstrated the expression of PSA analyzed at the mRNA level (by RT-PCR) and protein level (by immunohistochemistry) in the prostate lobes harvested from the PSA-transgenic (PSA-Tg) mice. We established that the administration of the bivalent vaccine in surgifoam to the PSA-Tg mice induces strong PSA-specific effector CD8 + T cells as measured by IFN-γ secretion and in vitro cytotoxic T-cell assay. Furthermore, the use of surgifoam with Ad 5 -PSA+PSCA vaccine allows multiple boosting vaccinations with a significant increase in antigen-specific CD8 + T cells. These observations suggest that the formulation of the bivalent prostate cancer vaccine (Ad 5 -PSA+PSCA) with surgifoam bypasses the neutralizing antibody response, thus allowing multiple boosting. This formulation is also helpful for inducing an antigen-specific immune response in the presence of self-antigen, and maintains long-term effector CD8 + T cells. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

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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.

Epigenetic control of effectors in plant pathogens

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Mark eGijzen

2014-11-01

Full Text Available Plant pathogens display impressive versatility in adapting to host immune systems. Pathogen effector proteins facilitate disease but can become avirulence (Avr factors when the host acquires discrete recognition capabilities that trigger immunity. The mechanisms that lead to changes to pathogen Avr factors that enable escape from host immunity are diverse, and include epigenetic switches that allow for reuse or recycling of effectors. This perspective outlines possibilities of how epigenetic control of Avr effector gene expression may have arisen and persisted in plant pathogens, and how it presents special problems for diagnosis and detection of specific pathogen strains or pathotypes.

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

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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

Uncovering the Legionella genus effector repertoire - strength in diversity and numbers

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Burstein, David; Amaro, Francisco; Zusman, Tal; Lifshitz, Ziv; Cohen, Ofir; Gilbert, Jack A; Pupko, Tal; Shuman, Howard A; Segal, Gil

2016-01-01

Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution. PMID:26752266

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

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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.

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

Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.

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Abana, Chike O; Pilkinton, Mark A; Gaudieri, Silvana; Chopra, Abha; McDonnell, Wyatt J; Wanjalla, Celestine; Barnett, Louise; Gangula, Rama; Hager, Cindy; Jung, Dae K; Engelhardt, Brian G; Jagasia, Madan H; Klenerman, Paul; Phillips, Elizabeth J; Koelle, David M; Kalams, Spyros A; Mallal, Simon A

2017-11-01

Select CMV epitopes drive life-long CD8 + T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4 + T cells specific for human CMV (HCMV) are elevated in HIV + HCMV + subjects. To determine whether HCMV epitope-specific CD4 + T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4 + T cells in coinfected HLA-DR7 + long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4 + T cells were inflated among these HIV + subjects compared with those from an HIV - HCMV + HLA-DR7 + cohort or with HLA-DR7-restricted CD4 + T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4 + T cells consisted of effector memory or effector memory-RA + subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX 3 CR1, CD38, or HLA-DR but less often coexpressed CD38 + and HLA-DR + The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4 + T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease. Copyright © 2017 by The American Association of Immunologists, Inc.

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

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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.

Detailed analysis of Epstein–Barr virus-specific CD4+ and CD8+ T cell responses during infectious mononucleosis

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Scherrenburg, J; Piriou, E R W A N; Nanlohy, N M; van Baarle, D

2008-01-01

We studied simultaneously Epstein–Barr virus (EBV)-specific CD4+ and CD8+ T cell responses during and after infectious mononucleosis (IM), using a previously described 12-day stimulation protocol with EBNA1 or BZLF1 peptide pools. Effector function of EBV-specific T cells was determined after restimulation by measuring intracellular interferon-γ production. During IM, BZLF1-specifc CD4+ T cell responses were dominant compared with CD8+ T cell responses. EBNA1-specific CD4+ and CD8+ T cell responses were low and remained similar for 6 months. However, 6 months after IM, BZLF1-specific CD4+ T cell responses had declined, but CD8+ T cell responses had increased. At diagnosis, EBV-specific CD8+ T cells as studied by human leucocyte antigen class I tetramer staining comprised a tetramerbrightCD8bright population consisting mainly of CD27+ memory T cells and a tetramerdimCD8dim population consisting primarily of CD27- effector T cells. The remaining EBV-specific CD8+ T cell population 6 months after the diagnosis of IM consisted mainly of tetramerbrightCD8bright CD27+ T cells, suggesting preferential preservation of memory T cells after contraction of the EBV-specific T cell pool. PMID:18549439

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

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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).

CD4+ T cells are required to contain early extrathoracic TB dissemination and sustain multi-effector functions of CD8+ T and CD3− lymphocytes

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Yao, Shuyu; Huang, Dan; Chen, Crystal Y.; Halliday, Lisa; Wang, Richard C.; Chen, Zheng W.

2014-01-01

The possibility that CD4+ T cells can act as “innate-like” cells to contain very-early M. tuberculosis (Mtb) dissemination and function as master helpers to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes during development of adaptive immunity against primary tuberculosis(TB) has not been demonstrated. We showed that pulmonary Mtb infection of CD4-depleted macaques surprisingly led to very-early extrathoracic Mtb dissemination, whereas CD4 deficiency clearly resulted in rapid TB progression. CD4 depletion during Mtb infection revealed the ability of CD8+ T cells to compensate and rapidly differentiate to Th17-like/Th1-like, and cytotoxic-like effectors, but these effector functions were subsequently unsustainable due to CD4 deficiency. While CD3-negative non-T lymphocytes in presence of CD4+ T cells developed predominant Th22-like and NK-like (perforin production) responses to Mtb infection, CD4 depletion abrogated these Th22-/NK-like effector functions and favored IL-17 production by CD3-negative lymphocytes. CD4-depleted macaques exhibited no or few pulmonary T effector cells constitutively producing IFN-γ, TNFα, IL-17, IL-22, and perforin at the endpoint of more severe TB, but presented pulmonary IL-4+ T effectors. TB granulomas in CD4-depleted macaques contained fewer IL-22+ and perforin+ cells despite presence of IL-17+ and IL-4+ cells. These results implicate previously-unknown “innate-like” ability of CD4+ T cells to contain extrathoracic Mtb dissemination at very early stage. Data also suggest that CD4+ T cells are required to sustain multiple effector functions of CD8+ T cells and CD3-negative lymphocytes and to prevent rapid TB progression during Mtb infection of nonhuman primates. PMID:24489088

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

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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.

Effector-independent motor sequence representations exist in extrinsic and intrinsic reference frames.

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Wiestler, Tobias; Waters-Metenier, Sheena; Diedrichsen, Jörn

2014-04-02

Many daily activities rely on the ability to produce meaningful sequences of movements. Motor sequences can be learned in an effector-specific fashion (such that benefits of training are restricted to the trained hand) or an effector-independent manner (meaning that learning also facilitates performance with the untrained hand). Effector-independent knowledge can be represented in extrinsic/world-centered or in intrinsic/body-centered coordinates. Here, we used functional magnetic resonance imaging (fMRI) and multivoxel pattern analysis to determine the distribution of intrinsic and extrinsic finger sequence representations across the human neocortex. Participants practiced four sequences with one hand for 4 d, and then performed these sequences during fMRI with both left and right hand. Between hands, these sequences were equivalent in extrinsic or intrinsic space, or were unrelated. In dorsal premotor cortex (PMd), we found that sequence-specific activity patterns correlated higher for extrinsic than for unrelated pairs, providing evidence for an extrinsic sequence representation. In contrast, primary sensory and motor cortices showed effector-independent representations in intrinsic space, with considerable overlap of the two reference frames in caudal PMd. These results suggest that effector-independent representations exist not only in world-centered, but also in body-centered coordinates, and that PMd may be involved in transforming sequential knowledge between the two. Moreover, although effector-independent sequence representations were found bilaterally, they were stronger in the hemisphere contralateral to the trained hand. This indicates that intermanual transfer relies on motor memories that are laid down during training in both hemispheres, but preferentially draws upon sequential knowledge represented in the trained hemisphere.

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

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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.

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

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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.

Human Cytomegalovirus (HCMV)-Specific CD4+ T Cells Are Polyfunctional and Can Respond to HCMV-Infected Dendritic Cells In Vitro.

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Jackson, Sarah E; Sedikides, George X; Mason, Gavin M; Okecha, Georgina; Wills, Mark R

2017-03-15

Human cytomegalovirus (HCMV) infection and periodic reactivation are generally well controlled by the HCMV-specific T cell response in healthy people. While the CD8 + T cell response to HCMV has been extensively studied, the HCMV-specific CD4 + T cell effector response is not as well understood, especially in the context of direct interactions with HCMV-infected cells. We screened the gamma interferon (IFN-γ) and interleukin-10 (IL-10) responses to 6 HCMV peptide pools (pp65, pp71, IE1, IE2, gB, and US3, selected because they were the peptides most frequently responded to in our previous studies) in 84 donors aged 23 to 74 years. The HCMV-specific CD4 + T cell response to pp65, IE1, IE2, and gB was predominantly Th1 biased, with neither the loss nor the accumulation of these responses occurring with increasing age. A larger proportion of donors produced an IL-10 response to pp71 and US3, but the IFN-γ response was still dominant. CD4 + T cells specific to the HCMV proteins studied were predominantly effector memory cells and produced both cytotoxic (CD107a expression) and cytokine (macrophage inflammatory protein 1β secretion) effector responses. Importantly, when we measured the CD4 + T cell response to cytomegalovirus (CMV)-infected dendritic cells in vitro , we observed that the CD4 + T cells produced a range of cytotoxic and secretory effector functions, despite the presence of CMV-encoded immune evasion molecules. CD4 + T cell responses to HCMV-infected dendritic cells were sufficient to control the dissemination of virus in an in vitro assay. Together, the results show that HCMV-specific CD4 + T cell responses, even those from elderly individuals, are highly functional and are directly antiviral. IMPORTANCE Human cytomegalovirus (HCMV) infection is carried for a lifetime and in healthy people is kept under control by the immune system. HCMV has evolved many mechanisms to evade the immune response, possibly explaining why the virus is never eliminated

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

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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.

Life Stage-specific Proteomes of Legionella pneumophila Reveal a Highly Differential Abundance of Virulence-associated Dot/Icm effectors*

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Aurass, Philipp; Gerlach, Thomas; Becher, Dörte; Voigt, Birgit; Karste, Susanne; Bernhardt, Jörg; Riedel, Katharina; Hecker, Michael; Flieger, Antje

2016-01-01

Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests

Membranes linked by trans-SNARE complexes require lipids prone to non-bilayer structure for progression to fusion.

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Zick, Michael; Stroupe, Christopher; Orr, Amy; Douville, Deborah; Wickner, William T

2014-01-01

Like other intracellular fusion events, the homotypic fusion of yeast vacuoles requires a Rab GTPase, a large Rab effector complex, SNARE proteins which can form a 4-helical bundle, and the SNARE disassembly chaperones Sec17p and Sec18p. In addition to these proteins, specific vacuole lipids are required for efficient fusion in vivo and with the purified organelle. Reconstitution of vacuole fusion with all purified components reveals that high SNARE levels can mask the requirement for a complex mixture of vacuole lipids. At lower, more physiological SNARE levels, neutral lipids with small headgroups that tend to form non-bilayer structures (phosphatidylethanolamine, diacylglycerol, and ergosterol) are essential. Membranes without these three lipids can dock and complete trans-SNARE pairing but cannot rearrange their lipids for fusion. DOI: http://dx.doi.org/10.7554/eLife.01879.001.

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

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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

HIV-Infected Children Have Elevated Levels of PD-1+ Memory CD4 T Cells With Low Proliferative Capacity and High Inflammatory Cytokine Effector Functions.

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Foldi, Julia; Kozhaya, Lina; McCarty, Bret; Mwamzuka, Mussa; Marshed, Fatma; Ilmet, Tiina; Kilberg, Max; Kravietz, Adam; Ahmed, Aabid; Borkowsky, William; Unutmaz, Derya; Khaitan, Alka

2017-09-15

During human immunodeficiency virus (HIV) disease, chronic immune activation leads to T-cell exhaustion. PD-1 identifies "exhausted" CD8 T cells with impaired HIV-specific effector functions, but its role on CD4 T cells and in HIV-infected children is poorly understood. In a Kenyan cohort of vertically HIV-infected children, we measured PD-1+ CD4 T-cell frequencies and phenotype by flow cytometry and their correlation with HIV disease progression and immune activation. Second, in vitro CD4 T-cell proliferative and cytokine responses to HIV-specific and -nonspecific stimuli were assessed with and without PD-1 blockade. HIV-infected children have increased frequencies of PD-1+ memory CD4 T cells that fail to normalize with antiretroviral treatment. These cells are comprised of central and effector memory subsets and correlate with HIV disease progression, measured by viral load, CD4 percentage, CD4:CD8 T-cell ratio, and immune activation. Last, PD-1+ CD4 T cells predict impaired proliferative potential yet preferentially secrete the Th1 and Th17 cytokines interferon-γ and interleukin 17A, and are unresponsive to in vitro PD-1 blockade. This study highlights differences in PD-1+ CD4 T-cell memory phenotype and response to blockade between HIV-infected children and adults, with implications for potential immune checkpoint therapies. © The Author 2017. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.

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

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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.

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....

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

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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.

Identification of Anaplasma marginale type IV secretion system effector proteins.

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Svetlana Lockwood

Full Text Available Anaplasma marginale, an obligate intracellular alphaproteobacterium in the order Rickettsiales, is a tick-borne pathogen and the leading cause of anaplasmosis in cattle worldwide. Complete genome sequencing of A. marginale revealed that it has a type IV secretion system (T4SS. The T4SS is one of seven known types of secretion systems utilized by bacteria, with the type III and IV secretion systems particularly prevalent among pathogenic Gram-negative bacteria. The T4SS is predicted to play an important role in the invasion and pathogenesis of A. marginale by translocating effector proteins across its membrane into eukaryotic target cells. However, T4SS effector proteins have not been identified and tested in the laboratory until now.By combining computational methods with phylogenetic analysis and sequence identity searches, we identified a subset of potential T4SS effectors in A. marginale strain St. Maries and chose six for laboratory testing. Four (AM185, AM470, AM705 [AnkA], and AM1141 of these six proteins were translocated in a T4SS-dependent manner using Legionella pneumophila as a reporter system.The algorithm employed to find T4SS effector proteins in A. marginale identified four such proteins that were verified by laboratory testing. L. pneumophila was shown to work as a model system for A. marginale and thus can be used as a screening tool for A. marginale effector proteins. The first T4SS effector proteins for A. marginale have been identified in this work.

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

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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.

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

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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.

Identification and Characterisation CRN Effectors in Phytophthora capsici Shows Modularity and Functional Diversity.

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Remco Stam

Full Text Available Phytophthora species secrete a large array of effectors during infection of their host plants. The Crinkler (CRN gene family encodes a ubiquitous but understudied class of effectors with possible but as of yet unknown roles in infection. To appreciate CRN effector function in Phytophthora, we devised a simple Crn gene identification and annotation pipeline to improve effector prediction rates. We predicted 84 full-length CRN coding genes and assessed CRN effector domain diversity in sequenced Oomycete genomes. These analyses revealed evidence of CRN domain innovation in Phytophthora and expansion in the Peronosporales. We performed gene expression analyses to validate and define two classes of CRN effectors, each possibly contributing to infection at different stages. CRN localisation studies revealed that P. capsici CRN effector domains target the nucleus and accumulate in specific sub-nuclear compartments. Phenotypic analyses showed that few CRN domains induce necrosis when expressed in planta and that one cell death inducing effector, enhances P. capsici virulence on Nicotiana benthamiana. These results suggest that the CRN protein family form an important class of intracellular effectors that target the host nucleus during infection. These results combined with domain expansion in hemi-biotrophic and necrotrophic pathogens, suggests specific contributions to pathogen lifestyles. This work will bolster CRN identification efforts in other sequenced oomycete species and set the stage for future functional studies towards understanding CRN effector functions.

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.

TAL effectors: highly adaptable phytobacterial virulence factors and readily engineered DNA targeting proteins

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Doyle, Erin L.; Stoddard, Barry L.; Voytas, Daniel F.; Bogdanove, Adam J.

2013-01-01

Transcription activator-like (TAL) effectors are transcription factors injected into plant cells by pathogenic bacteria in the genus Xanthomonas. They function as virulence factors by activating host genes important for disease, or as avirulence factors by turning on genes that provide resistance. DNA binding specificity is encoded by polymorphic repeats in each protein that correspond one-to-one with different nucleotides. This code has facilitated target identification and opened new avenues for engineering disease resistance. It has also enabled TAL effector customization for targeted gene control, genome editing, and other applications. This article reviews the structural basis for TAL effector-DNA specificity, the impact of the TAL effector-DNA code on plant pathology and engineered resistance, and recent accomplishments and future challenges in TAL effector-based DNA targeting. PMID:23707478

Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots.

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Stergiopoulos, Ioannis; van den Burg, Harrold A; Okmen, Bilal; Beenen, Henriek G; van Liere, Sabine; Kema, Gert H J; de Wit, Pierre J G M

2010-04-20

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis, the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis, one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

Mtb-specific CD27low CD4 T cells as markers of lung tissue destruction during pulmonary tuberculosis in humans.

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Nikitina, Irina Yu; Kondratuk, Natalya A; Kosmiadi, George A; Amansahedov, Rasul B; Vasilyeva, Irina A; Ganusov, Vitaly V; Lyadova, Irina V

2012-01-01

Effector CD4 T cells represent a key component of the host's anti-tuberculosis immune defense. Successful differentiation and functioning of effector lymphocytes protects the host against severe M. tuberculosis (Mtb) infection. On the other hand, effector T cell differentiation depends on disease severity/activity, as T cell responses are driven by antigenic and inflammatory stimuli released during infection. Thus, tuberculosis (TB) progression and the degree of effector CD4 T cell differentiation are interrelated, but the relationships are complex and not well understood. We have analyzed an association between the degree of Mtb-specific CD4 T cell differentiation and severity/activity of pulmonary TB infection. The degree of CD4 T cell differentiation was assessed by measuring the percentages of highly differentiated CD27(low) cells within a population of Mtb- specific CD4 T lymphocytes ("CD27(low)IFN-γ(+)" cells). The percentages of CD27(low)IFN-γ+ cells were low in healthy donors (median, 33.1%) and TB contacts (21.8%) but increased in TB patients (47.3%, p76%), but varied in blood (12-92%). The major correlate for the accumulation of CD27(low)IFN-γ(+) cells in blood was lung destruction (r = 0.65, p = 2.7 × 10(-7)). A cutoff of 47% of CD27(low)IFN-γ(+) cells discriminated patients with high and low degree of lung destruction (sensitivity 89%, specificity 74%); a decline in CD27(low)IFN-γ(+)cells following TB therapy correlated with repair and/or reduction of lung destruction (ppulmonary TB. Accumulation of CD27(low)IFN-γ(+) cells in the blood is associated with lung destruction. The findings indicate that there is no deficiency in CD4 T cell differentiation during TB; evaluation of CD27(low)IFN-γ(+) cells provides a valuable means to assess TB activity, lung destruction, and tissue repair following TB therapy.

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

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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.

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

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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.

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

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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

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

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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.

Differential requirements of CD4(+) T-cell signals for effector cytotoxic T-lymphocyte (CTL) priming and functional memory CTL development at higher CD8(+) T-cell precursor frequency.

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Umeshappa, Channakeshava S; Nanjundappa, Roopa H; Xie, Yufeng; Freywald, Andrew; Xu, Qingyong; Xiang, Jim

2013-04-01

Increased CD8(+) T-cell precursor frequency (PF) precludes the requirement of CD4(+) helper T (Th) cells for primary CD8(+) cytotoxic T-lymphocyte (CTL) responses. However, the key questions of whether unhelped CTLs generated at higher PF are functional effectors, and whether unhelped CTLs can differentiate into functional memory cells at higher PF are unclear. In this study, ovalbumin (OVA) -pulsed dendritic cells (DC(OVA)) derived from C57BL/6, CD40 knockout (CD40(-/-)) or CD40 ligand knockout (CD40L(-/-)) mice were used to immunize C57BL/6, Ia(b-/-), CD40(-/-) or CD40L(-/-) mice, whose PF was previously increased with transfer of 1 × 10(6) CD8(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTI, OTI(CD40(-/-)) or OTI(CD40L(-/-)) mice. All the immunized mice were then assessed for effector and memory CTL responses. Following DC immunization, relatively comparable CTL priming occurred without CD4(+) T-cell help and Th-provided CD40/CD40L signalling. In addition, the unhelped CTLs were functional effectors capable of inducing therapeutic immunity against established OVA-expressing tumours. In contrast, the functional memory development of CTLs was severely impaired in the absence of CD4(+) T-cell help and CD40/CD40L signalling. Finally, unhelped memory CTLs failed to protect mice against lethal tumour challenge. Taken together, these results demonstrate that CD4(+) T-cell help at higher PF, is not required for effector CTL priming, but is required for functional memory CTL development against cancer. Our data may impact the development of novel preventive and therapeutic approaches in cancer patients with compromised CD4(+) T-cell functions. © 2012 Blackwell Publishing Ltd.

Small Molecules for Early Endosome-Specific Patch Clamping.

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Chen, Cheng-Chang; Butz, Elisabeth S; Chao, Yu-Kai; Grishchuk, Yulia; Becker, Lars; Heller, Stefan; Slaugenhaupt, Susan A; Biel, Martin; Wahl-Schott, Christian; Grimm, Christian

2017-07-20

To resolve the subcellular distribution of endolysosomal ion channels, we have established a novel experimental approach to selectively patch clamp Rab5 positive early endosomes (EE) versus Rab7/LAMP1-positive late endosomes/lysosomes (LE/LY). To functionally characterize ion channels in endolysosomal membranes with the patch-clamp technique, it is important to develop techniques to selectively enlarge the respective organelles. We found here that two small molecules, wortmannin and latrunculin B, enlarge Rab5-positive EE when combined but not Rab7-, LAMP1-, or Rab11 (RE)-positive vesicles. The two compounds act rapidly, specifically, and are readily applicable in contrast to genetic approaches or previously used compounds such as vacuolin, which enlarges EE, RE, and LE/LY. We apply this approach here to measure currents mediated by TRPML channels, in particular TRPML3, which we found to be functionally active in both EE and LE/LY in overexpressing cells as well as in endogenously expressing CD11b+ lung-tissue macrophages. Copyright © 2017 Elsevier Ltd. All rights reserved.

Development and testing of the cooling coil cleaning end effector

International Nuclear Information System (INIS)

Johnson, K.I.; Mullen, O.D.; Powell, M.R.; Daly, D.S.; Engel, D.W.

1997-01-01

The Retrieval Process Development and Enhancement (KPD ampersand E) program has developed and tested an end effector to support the waste retrieval mission at the Idaho National Engineering and Environmental Laboratory (INEEL). The end effector was developed specifically to remove a sticky waste material from the cooling coils in the High Level Liquid Waste (HLLW) tank, and to vacuum up a sediment layer that has settled beneath the cooling coils. An extensive testing program was conducted in the hydraulic test bed (HTB) at the Pacific Northwest National Laboratory (PNNL) to evaluate the performance of the end effector under simulated in-tank conditions. A mock up of the cooling coils was installed in the test bed tank, and simulated waste materials were included to represent the sticky waste on the tubes and the particulate waste settled beneath them. The testing program focused on assessing long-duration mining strategies for cleaning the cooling coils and removing the particulate waste forms. The report describes the results of the end effector testing program at PNNL. Section 2 describes the physical characteristics of the HLLW tanks, including the layout of the cooling coils, and it also describes what is known of the waste forms in the tanks. Section 3 describes the cleaning and retrieval strategy that was used in developing the end effector design. Section 4 describes the cooling coil mockup in the hydraulic test bed. Section 5 discusses the rationale used in selecting the simulants for the tarry waste and particulate waste forms. Section 6 describes the tests that were performed to evaluate cleaning of the cooling coils and retrieval of the particulate simulant. Section 7 summarizes the cleaning and retrieval tests, assesses the relative importance of cleaning the cooling coils and retrieving the particulate waste, and suggests modifications that would simplify the end effector design

Urinary CD4+ Effector Memory T Cells Reflect Renal Disease Activity in Antineutrophil Cytoplasmic Antibody-Associated Vasculitis

NARCIS (Netherlands)

Abdulahad, Wayel H.; Kallenberg, Cees G. M.; Limburg, Pieter C.; Stegeman, Coen A.

Objective. Numbers of circulating CD4+ effector memory T cells are proportionally increased in patients with proteinase 3 antineutrophil cytoplasmic antibody-associated vasculitis (AAV) whose disease is in remission and are decreased during active disease, which presumably reflects their migration

Shigella subverts the host recycling compartment to rupture its vacuole.

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Mellouk, Nora; Weiner, Allon; Aulner, Nathalie; Schmitt, Christine; Elbaum, Michael; Shorte, Spencer L; Danckaert, Anne; Enninga, Jost

2014-10-08

Shigella enters epithlial cells via internalization into a vacuole. Subsequent vacuolar membrane rupture allows bacterial escape into the cytosol for replication and cell-to-cell spread. Bacterial effectors such as IpgD, a PI(4,5)P2 phosphatase that generates PI(5)P and alters host actin, facilitate this internalization. Here, we identify host proteins involved in Shigella uptake and vacuolar membrane rupture by high-content siRNA screening and subsequently focus on Rab11, a constituent of the recycling compartment. Rab11-positive vesicles are recruited to the invasion site before vacuolar rupture, and Rab11 knockdown dramatically decreases vacuolar membrane rupture. Additionally, Rab11 recruitment is absent and vacuolar rupture is delayed in the ipgD mutant that does not dephosphorylate PI(4,5)P₂ into PI(5)P. Ultrastructural analyses of Rab11-positive vesicles further reveal that ipgD mutant-containing vacuoles become confined in actin structures that likely contribute to delayed vacular rupture. These findings provide insight into the underlying molecular mechanism of vacuole progression and rupture during Shigella invasion. Copyright © 2014 Elsevier Inc. All rights reserved.

Control of MarRAB Operon in Escherichia coli via Autoactivation and Autorepression

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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

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

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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

Hitting the Sweet Spot: Glycans as Targets of Fungal Defense Effector Proteins

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Markus Künzler

2015-05-01

Full Text Available Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparatively low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous fungi against microbial competitors and animal predators.

Space-based multifunctional end effector systems functional requirements and proposed designs

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Mishkin, A. H.; Jau, B. M.

1988-01-01

The end effector is an essential element of teleoperator and telerobot systems to be employed in space in the next decade. The report defines functional requirements for end effector systems to perform operations that are currently only feasible through Extra-Vehicular Activity (EVA). Specific tasks and functions that the end effectors must be capable of performing are delineated. Required capabilities for forces and torques, clearances, compliance, and sensing are described, using current EVA requirements as guidelines where feasible. The implications of these functional requirements on the elements of potential end effector systems are discussed. The systems issues that must be considered in the design of space-based manipulator systems are identified; including impacts on subsystems tightly coupled to the end effector, i.e., control station, information processing, manipulator arm, tool and equipment stowage. Possible end effector designs are divided into three categories: single degree-of-freedom end effectors, multiple degree of freedom end effectors, and anthropomorphic hands. Specific design alternatives are suggested and analyzed within the individual categories. Two evaluations are performed: the first considers how well the individual end effectors could substitute for EVA; the second compares how manipulator systems composed of the top performers from the first evaluation would improve the space shuttle Remote Manipulator System (RMS) capabilities. The analysis concludes that the anthropomorphic hand is best-suited for EVA tasks. A left- and right-handed anthropomorphic manipulator arm configuration is suggested as appropriate to be affixed to the RMS, but could also be used as part of the Smart Front End for the Orbital Maneuvering Vehicle (OMV). The technical feasibility of the anthropomorphic hand and its control are demonstrated. An evolutionary development approach is proposed and approximate scheduling provided for implementing the suggested

Transcriptional Reprogramming during Effector-to-Memory Transition Renders CD4+ T Cells Permissive for Latent HIV-1 Infection.

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Shan, Liang; Deng, Kai; Gao, Hongbo; Xing, Sifei; Capoferri, Adam A; Durand, Christine M; Rabi, S Alireza; Laird, Gregory M; Kim, Michelle; Hosmane, Nina N; Yang, Hung-Chih; Zhang, Hao; Margolick, Joseph B; Li, Linghua; Cai, Weiping; Ke, Ruian; Flavell, Richard A; Siliciano, Janet D; Siliciano, Robert F

2017-10-17

The latent reservoir for HIV-1 in resting memory CD4 + T cells is the major barrier to curing HIV-1 infection. Studies of HIV-1 latency have focused on regulation of viral gene expression in cells in which latent infection is established. However, it remains unclear how infection initially becomes latent. Here we described a unique set of properties of CD4 + T cells undergoing effector-to-memory transition including temporary upregulation of CCR5 expression and rapid downregulation of cellular gene transcription. These cells allowed completion of steps in the HIV-1 life cycle through integration but suppressed HIV-1 gene transcription, thus allowing the establishment of latency. CD4 + T cells in this stage were substantially more permissive for HIV-1 latent infection than other CD4 + T cells. Establishment of latent HIV-1 infection in CD4 + T could be inhibited by viral-specific CD8 + T cells, a result with implications for elimination of latent HIV-1 infection by T cell-based vaccines. Copyright © 2017. Published by Elsevier Inc.

Timing of in utero malaria exposure influences fetal CD4 T cell regulatory versus effector differentiation

Directory of Open Access Journals (Sweden)

Mary Prahl

2016-10-01

Full Text Available Abstract Background In malaria-endemic areas, the first exposure to malaria antigens often occurs in utero when the fetal immune system is poised towards the development of tolerance. Children exposed to placental malaria have an increased risk of clinical malaria in the first few years of life compared to unexposed children. Recent work has suggested the potential of pregnancy-associated malaria to induce immune tolerance in children living in malaria-endemic areas. A study was completed to evaluate the effect of malaria exposure during pregnancy on fetal immune tolerance and effector responses. Methods Using cord blood samples from a cohort of mother-infant pairs followed from early in pregnancy until delivery, flow cytometry analysis was completed to assess the relationship between pregnancy-associated malaria and fetal cord blood CD4 and dendritic cell phenotypes. Results Cord blood FoxP3+ Treg counts were higher in infants born to mothers with Plasmodium parasitaemia early in pregnancy (12–20 weeks of gestation; p = 0.048, but there was no association between Treg counts and the presence of parasites in the placenta at the time of delivery (by loop-mediated isothermal amplification (LAMP; p = 0.810. In contrast, higher frequencies of activated CD4 T cells (CD25+FoxP3−CD127+ were observed in the cord blood of neonates with active placental Plasmodium infection at the time of delivery (p = 0.035. This population exhibited evidence of effector memory differentiation, suggesting priming of effector T cells in utero. Lastly, myeloid dendritic cells were higher in the cord blood of infants with histopathologic evidence of placental malaria (p < 0.0001. Conclusion Together, these data indicate that in utero exposure to malaria drives expansion of both regulatory and effector T cells in the fetus, and that the timing of this exposure has a pivotal role in determining the polarization of the fetal immune response.

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.

Effector proteins of rust fungi.

Science.gov (United States)

Petre, Benjamin; Joly, David L; Duplessis, Sébastien

2014-01-01

Rust fungi include many species that are devastating crop pathogens. To develop resistant plants, a better understanding of rust virulence factors, or effector proteins, is needed. Thus far, only six rust effector proteins have been described: AvrP123, AvrP4, AvrL567, AvrM, RTP1, and PGTAUSPE-10-1. Although some are well established model proteins used to investigate mechanisms of immune receptor activation (avirulence activities) or entry into plant cells, how they work inside host tissues to promote fungal growth remains unknown. The genome sequences of four rust fungi (two Melampsoraceae and two Pucciniaceae) have been analyzed so far. Genome-wide analyses of these species, as well as transcriptomics performed on a broader range of rust fungi, revealed hundreds of small secreted proteins considered as rust candidate secreted effector proteins (CSEPs). The rust community now needs high-throughput approaches (effectoromics) to accelerate effector discovery/characterization and to better understand how they function in planta. However, this task is challenging due to the non-amenability of rust pathosystems (obligate biotrophs infecting crop plants) to traditional molecular genetic approaches mainly due to difficulties in culturing these species in vitro. The use of heterologous approaches should be promoted in the future.

Type IV secretion system of Brucella spp. and its effectors.

Science.gov (United States)

Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

2015-01-01

Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

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.

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

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

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

Genome-scale identification of Legionella pneumophila effectors using a machine learning approach.

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David Burstein

2009-07-01

Full Text Available A large number of highly pathogenic bacteria utilize secretion systems to translocate effector proteins into host cells. Using these effectors, the bacteria subvert host cell processes during infection. Legionella pneumophila translocates effectors via the Icm/Dot type-IV secretion system and to date, approximately 100 effectors have been identified by various experimental and computational techniques. Effector identification is a critical first step towards the understanding of the pathogenesis system in L. pneumophila as well as in other bacterial pathogens. Here, we formulate the task of effector identification as a classification problem: each L. pneumophila open reading frame (ORF was classified as either effector or not. We computationally defined a set of features that best distinguish effectors from non-effectors. These features cover a wide range of characteristics including taxonomical dispersion, regulatory data, genomic organization, similarity to eukaryotic proteomes and more. Machine learning algorithms utilizing these features were then applied to classify all the ORFs within the L. pneumophila genome. Using this approach we were able to predict and experimentally validate 40 new effectors, reaching a success rate of above 90%. Increasing the number of validated effectors to around 140, we were able to gain novel insights into their characteristics. Effectors were found to have low G+C content, supporting the hypothesis that a large number of effectors originate via horizontal gene transfer, probably from their protozoan host. In addition, effectors were found to cluster in specific genomic regions. Finally, we were able to provide a novel description of the C-terminal translocation signal required for effector translocation by the Icm/Dot secretion system. To conclude, we have discovered 40 novel L. pneumophila effectors, predicted over a hundred additional highly probable effectors, and shown the applicability of machine

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...

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

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

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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.

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.

Type IV Secretion System of Brucella spp. and its Effectors

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Yuehua eKe

2015-10-01

Full Text Available Brucella spp. cause brucellosis in domestic and wild animals. They are intracellular bacterial pathogens and used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we will discuss roles of Brucella VirB T4SS and in more detail of all 15 identified effectors, which may be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells, suggesting that it plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. So, we listed some key molecular events in the intracellular life cycle of Brucella potentially targeted by the VirB T4SS effectors. Elucidating functions of the effectors secreted will be crucial to clarifying mechanism of T4SS during infection. Studying the effectors secreted by Brucella spp. might provide insights into the mechanisms by which the bacteria hijack the host signaling pathways, which help us to develop better vaccines and therapies against brucellosis.

Primary murine CD4+ T cells fail to acquire the ability to produce effector cytokines when active Ras is present during Th1/Th2 differentiation.

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Sujit V Janardhan

Full Text Available Constitutive Ras signaling has been shown to augment IL-2 production, reverse anergy, and functionally replace many aspects of CD28 co-stimulation in CD4+ T cells. These data raise the possibility that introduction of active Ras into primary T cells might result in improved functionality in pathologic situations of T cell dysfunction, such as cancer or chronic viral infection. To test the biologic effects of active Ras in primary T cells, CD4+ T cells from Coxsackie-Adenovirus Receptor Transgenic mice were transduced with an adenovirus encoding active Ras. As expected, active Ras augmented IL-2 production in naive CD4+ T cells. However, when cells were cultured for 4 days under conditions to promote effector cell differentiation, active Ras inhibited the ability of CD4+ T cells to acquire a Th1 or Th2 effector cytokine profile. This differentiation defect was not due to deficient STAT4 or STAT6 activation by IL-12 or IL-4, respectively, nor was it associated with deficient induction of T-bet and GATA-3 expression. Impaired effector cytokine production in active Ras-transduced cells was associated with deficient demethylation of the IL-4 gene locus. Our results indicate that, despite augmenting acute activation of naïve T cells, constitutive Ras signaling inhibits the ability of CD4+ T cells to properly differentiate into Th1/Th2 effector cytokine-producing cells, in part by interfering with epigenetic modification of effector gene loci. Alternative strategies to potentiate Ras pathway signaling in T cells in a more regulated fashion should be considered as a therapeutic approach to improve immune responses in vivo.

Hypoxia Enhances Immunosuppression by Inhibiting CD4+ Effector T Cell Function and Promoting Treg Activity

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Astrid M. Westendorf

2017-03-01

Full Text Available Background/Aims: Hypoxia occurs in many pathological conditions, including inflammation and cancer. Within this context, hypoxia was shown to inhibit but also to promote T cell responses. Due to this controversial function, we aimed to explore whether an insufficient anti-tumour response during colitis-associated colon cancer could be ascribed to a hypoxic microenvironment. Methods: Colitis-associated colon cancer was induced in wildtype mice, and hypoxia as well as T cell immunity were analysed in the colonic tumour tissues. In addition, CD4+ effector T cells and regulatory T cells were cultured under normoxic and hypoxic conditions and examined regarding their phenotype and function. Results: We observed severe hypoxia in the colon of mice suffering from colitis-associated colon cancer that was accompanied by a reduced differentiation of CD4+ effector T cells and an enhanced number and suppressive activity of regulatory T cells. Complementary ex vivo and in vitro studies revealed that T cell stimulation under hypoxic conditions inhibited the differentiation, proliferation and IFN-γ production of TH1 cells and enhanced the suppressive capacity of regulatory T cells. Moreover, we identified an active role for HIF-1α in the modulation of CD4+ T cell functions under hypoxic conditions. Conclusion: Our data indicate that oxygen availability can function as a local modulator of CD4+ T cell responses and thus influences tumour immune surveillance in inflammation-associated colon cancer.

Autoreactive T effector memory differentiation mirrors β-cell function in type 1 diabetes.

Science.gov (United States)

Yeo, Lorraine; Woodwyk, Alyssa; Sood, Sanjana; Lorenc, Anna; Eichmann, Martin; Pujol-Autonell, Irma; Melchiotti, Rossella; Skowera, Ania; Fidanis, Efthymios; Dolton, Garry M; Tungatt, Katie; Sewell, Andrew K; Heck, Susanne; Saxena, Alka; Beam, Craig A; Peakman, Mark

2018-05-31

In type 1 diabetes, cytotoxic CD8 T cells with specificity for β-cell autoantigens are found in the pancreatic islets where they are implicated in the destruction of insulin-secreting β cells. In contrast, the disease relevance of β-cell-reactive CD8 T cells that are detectable in the circulation, and their relationship to β-cell function, are not known. Here, we tracked multiple, circulating β-cell-reactive CD8 T cell subsets and measured β-cell function longitudinally for two years, starting immediately after diagnosis of type 1 diabetes. We found that change in β-cell-specific effector memory CD8 T cells expressing CD57 was positively correlated with C-peptide change in subjects below 12 years of age. Autoreactive CD57+ effector memory CD8 T cells bore the signature of enhanced effector function (higher expression of granzyme B, killer specific protein 37 and CD16, and reduced expression of CD28) compared with their CD57-negative counterparts, and network association modelling indicated that the dynamics of β-cell-reactive CD57+ effector memory CD8 T cell subsets were strongly linked. Thus, coordinated changes in circulating β-cell-specific CD8 T cells within the CD57+ effector memory subset calibrate to functional insulin reserve in type 1 diabetes, providing a tool for immune monitoring and a mechanism-based target for immunotherapy.

A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay.

Science.gov (United States)

Kim, G G; Donnenberg, V S; Donnenberg, A D; Gooding, W; Whiteside, T L

2007-08-31

Natural killer (NK) cell-or T cell-mediated cytotoxicity traditionally is measured in 4-16 h (51)Cr-release assays (CRA). A new four-color flow cytometry-based cytotoxicity assay (FCC) was developed to simultaneously measure NK cell cytotoxicity and NK cell phenotype (CD3(-)CD16(+)CD56(+)). Target cells, K562 or Daudi, were labeled with Cell Tracker Orange (CTO) prior to the addition of effector cells. Following co-incubation, 7 amino-actinomycin D (7-AAD) was added to measure death of target cells. The phenotype of effectors, viability of targets, the formation of tumor-effector cell conjugates and absolute numbers of all cells were measured based on light scatter (FSC/SSC), double discrimination of the fluorescence peak integral and height, and fluorescence intensity. Kinetic studies (0.5 and 1 to 4 h) at different effector to target (E:T) cell ratios (50, 25, 12, and 6) confirmed that the 3 h incubation was optimal. The FCC assay is more sensitive than the CRA, has a coefficient of variation (CV) 8-13% and reliably measures NK cell-or lymphokine-activated killer (LAK) cell-mediated killing of target cells in normal controls and subjects with cancer. The FCC assay can be used to study a range of phenotypic attributes, in addition to lytic activity of various subsets of effector cells, without radioactive tracers and thus, it is relatively inexpensive. The FCC assay has a potential for providing information about molecular interactions underlying target cell lysis and thus becoming a major tool for studies of disease pathogenesis as well as development of novel immune therapies.

TAL effectors and the executor R genes.

Science.gov (United States)

Zhang, Junli; Yin, Zhongchao; White, Frank

2015-01-01

Transcription activator-like (TAL) effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R) genes have been characterized-recessive, dominant non-transcriptional, and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

TAL effectors and the executor R genes

Directory of Open Access Journals (Sweden)

Junli eZhang

2015-08-01

Full Text Available Transcription activation-like (TAL effectors are bacterial type III secretion proteins that function as transcription factors in plants during Xanthomonas/plant interactions, conditioning either host susceptibility and/or host resistance. Three types of TAL effector associated resistance (R genes have been characterized - recessive, dominant non-transcriptional and dominant TAL effector-dependent transcriptional based resistance. Here, we discuss the last type of R genes, whose functions are dependent on direct TAL effector binding to discrete effector binding elements in the promoters. Only five of the so-called executor R genes have been cloned, and commonalities are not clear. We have placed the protein products in two groups for conceptual purposes. Group 1 consists solely of the protein from pepper, BS3, which is predicted to have catalytic function on the basis of homology to a large conserved protein family. Group 2 consists of BS4C-R, XA27, XA10, and XA23, all of which are relatively short proteins from pepper or rice with multiple potential transmembrane domains. Group 2 members have low sequence similarity to proteins of unknown function in closely related species. Firm predictions await further experimentation on these interesting new members to the R gene repertoire, which have potential broad application in new strategies for disease resistance.

A translocator-specific export signal establishes the translocator-effector secretion hierarchy that is important for type III secretion system function

Science.gov (United States)

Tomalka, Amanda G.; Stopford, Charles M.; Lee, Pei-Chung; Rietsch, Arne

2012-01-01

Summary Type III secretion systems are used by many Gram-negative pathogens to directly deliver effector proteins into the cytoplasm of host cells. To accomplish this, bacteria secrete translocator proteins that form a pore in the host-cell membrane through which the effector proteins are then introduced into the host cell. Evidence from multiple systems indicates that the pore-forming translocator proteins are exported before effectors, but how this secretion hierarchy is established is unclear. Here we used the P. aeruginosa translocator protein PopD as a model to identify its export signals. The amino-terminal secretion signal and chaperone, PcrH, are required for export under all conditions. Two novel signals in PopD, one proximal to the chaperone-binding site and one at the very C-terminus of the protein, are required for export of PopD before effector proteins. These novel export signals establish the translocator-effector secretion hierarchy, which in turn, is critical for the delivery of effectors into host cells. PMID:23121689

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 ...

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.

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

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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.

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

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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

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. �

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

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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

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.

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

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.

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

Science.gov (United States)

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Oomycetes, effectors, and all that jazz.

Science.gov (United States)

Bozkurt, Tolga O; Schornack, Sebastian; Banfield, Mark J; Kamoun, Sophien

2012-08-01

Plant pathogenic oomycetes secrete a diverse repertoire of effector proteins that modulate host innate immunity and enable parasitic infection. Understanding how effectors evolve, translocate and traffic inside host cells, and perturb host processes are major themes in the study of oomycete-plant interactions. The last year has seen important progress in the study of oomycete effectors with, notably, the elucidation of the 3D structures of five RXLR effectors, and novel insights into how cytoplasmic effectors subvert host cells. In this review, we discuss these and other recent advances and highlight the most important open questions in oomycete effector biology. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

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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.

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.

Tumor-specific CD4+ T cells develop cytotoxic activity and eliminate virus-induced tumor cells in the absence of regulatory T cells.

Science.gov (United States)

Akhmetzyanova, Ilseyar; Zelinskyy, Gennadiy; Schimmer, Simone; Brandau, Sven; Altenhoff, Petra; Sparwasser, Tim; Dittmer, Ulf

2013-02-01

The important role of tumor-specific cytotoxic CD8(+) T cells is well defined in the immune control of the tumors, but the role of effector CD4(+) T cells is poorly understood. In the current research, we have used a murine retrovirus-induced tumor cell line of C57BL/6 mouse origin, namely FBL-3 cells, as a model to study basic mechanisms of immunological control and escape during tumor formation. This study shows that tumor-specific CD4(+) T cells are able to protect against virus-induced tumor cells. We show here that there is an expansion of tumor-specific CD4(+) T cells producing cytokines and cytotoxic molecule granzyme B (GzmB) in the early phase of tumor growth. Importantly, we demonstrate that in vivo depletion of regulatory T cells (Tregs) and CD8(+) T cells in FBL-3-bearing DEREG transgenic mice augments IL-2 and GzmB production by CD4(+) T cells and increases FV-specific CD4(+) T-cell effector and cytotoxic responses leading to the complete tumor regression. Therefore, the capacity to reject tumor acquired by tumor-reactive CD4(+) T cells largely depends on the direct suppressive activity of Tregs. We suggest that a cytotoxic CD4(+) T-cell immune response may be induced to enhance resistance against oncovirus-associated tumors.

Generation of specific antitumor cytotoxic T-lymphocytes in the monoculture

International Nuclear Information System (INIS)

Lupatov, A.Yu.; Brondz, B.D.

1992-01-01

A new model for the generation of specific antitumor cytotoxic T-lymphocytes (CTL) was proposed. In contrast to other models, it allows to generate effector CTL without immunization in vitro. For estimation of cytotoxic activity, chromium-51 release assay was used. It has been shown that effector CTL were absent in the lymph nodes in 1-fold as well as 2-fold immunization. Specific CTL were detected only after secondary immunization and subsequent cultivation in vitro. Effector cells had Thy1.2 + , Lyt2 + , L3T4 - phenotypes. Presence in vitro of exogenous IL-2 was needed for the generation of CTL against MX-11 sarcoma but not against EL4 lymphoma. The release of IL-2 from lymphomas cells could stimulate generation of the effector cells through activation of the endogenous production of IL-2, or due to some other factors

Investigation of a bio-inspired lift-enhancing effector on a 2D airfoil.

Science.gov (United States)

Johnston, Joe; Gopalarathnam, Ashok

2012-09-01

A flap mounted on the upper surface of an airfoil, called a 'lift-enhancing effector', has been shown in wind tunnel tests to have a similar function to a bird's covert feathers, which rise off the wing's surface in response to separated flows. The effector, fabricated from a thin Mylar sheet, is allowed to rotate freely about its leading edge. The tests were performed in the NCSU subsonic wind tunnel at a chord Reynolds number of 4 × 10(5). The maximum lift coefficient with the effector was the same as that for the clean airfoil, but was maintained over an angle-of-attack range from 12° to almost 20°, resulting in a very gentle stall behavior. To better understand the aerodynamics and to estimate the deployment angle of the free-moving effector, fixed-angle effectors fabricated out of stiff wood were also tested. A progressive increase in the stall angle of attack with increasing effector angle was observed, with diminishing returns beyond the effector angle of 60°. Drag tests on both the free-moving and fixed effectors showed a marked improvement in drag at high angles of attack. Oil flow visualization on the airfoil with and without the fixed-angle effectors proved that the effector causes the separation point to move aft on the airfoil, as compared to the clean airfoil. This is thought to be the main mechanism by which an effector improves both lift and drag. A comparison of the fixed-effector results with those from the free-effector tests shows that the free effector's deployment angle is between 30° and 45°. When operating at and beyond the clean airfoil's stall angle, the free effector automatically deploys to progressively higher angles with increasing angles of attack. This slows down the rapid upstream movement of the separation point and avoids the severe reduction in the lift coefficient and an increase in the drag coefficient that are seen on the clean airfoil at the onset of stall. Thus, the effector postpones the stall by 4-8° and makes the

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

Evaluation of secretion prediction highlights differing approaches needed for oomycete and fungal effectors

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Jana eSperschneider

2015-12-01

Full Text Available The steadily increasing number of sequenced fungal and oomycete genomes has enabled detailed studies of how these eukaryotic microbes infect plants and cause devastating losses in food crops. During infection, fungal and oomycete pathogens secrete effector molecules which manipulate host plant cell processes to the pathogen’s advantage. Proteinaceous effectors are synthesised intracellularly and must be externalised to interact with host cells. Computational prediction of secreted proteins from genomic sequences is an important technique to narrow down the candidate effector repertoire for subsequent experimental validation. In this study, we benchmark secretion prediction tools on experimentally validated fungal and oomycete effectors. We observe that for a set of fungal SwissProt protein sequences, SignalP 4 and the neural network predictors of SignalP 3 (D-score and SignalP 2 perform best. For effector prediction in particular, the use of a sensitive method can be desirable to obtain the most complete candidate effector set. We show that the neural network predictors of SignalP 2 and 3, as well as TargetP were the most sensitive tools for fungal effector secretion prediction, whereas the hidden Markov model predictors of SignalP 2 and 3 were the most sensitive tools for oomycete effectors. Thus, previous versions of SignalP retain value for oomycete effector prediction, as the current version, SignalP 4, was unable to reliably predict the signal peptide of the oomycete Crinkler effectors in the test set. Our assessment of subcellular localisation predictors shows that cytoplasmic effectors are often predicted as not extracellular. This limits the reliability of secretion predictions that depend on these tools. We present our assessment with a view to informing future pathogenomics studies and suggest revised pipelines for secretion prediction to obtain optimal effector predictions in fungi and oomycetes.

Identification and Initial Characterization of the Effectors of an Anther Smut Fungus and Potential Host Target Proteins

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Venkata S. Kuppireddy

2017-11-01

Full Text Available (1 Background: Plant pathogenic fungi often display high levels of host specificity and biotrophic fungi; in particular, they must manipulate their hosts to avoid detection and to complete their obligate pathogenic lifecycles. One important strategy of such fungi is the secretion of small proteins that serve as effectors in this process. Microbotryum violaceum is a species complex whose members infect members of the Caryophyllaceae; M. lychnidis-dioicae, a parasite on Silene latifolia, is one of the best studied interactions. We are interested in identifying and characterizing effectors of the fungus and possible corresponding host targets; (2 Methods: In silico analysis of the M. lychnidis-dioicae genome and transcriptomes allowed us to predict a pool of small secreted proteins (SSPs with the hallmarks of effectors, including a lack of conserved protein family (PFAM domains and also localized regions of disorder. Putative SSPs were tested for secretion using a yeast secretion trap method. We then used yeast two-hybrid analyses for candidate-secreted effectors to probe a cDNA library from a range of growth conditions of the fungus, including infected plants; (3 Results: Roughly 50 SSPs were identified by in silico analysis. Of these, 4 were studied further and shown to be secreted, as well as examined for potential host interactors. One of the putative effectors, MVLG_01732, was found to interact with Arabidopsis thaliana calcium-dependent lipid binding protein (AtCLB and with cellulose synthase interactive protein 1 orthologues; and (4 Conclusions: The identification of a pool of putative effectors provides a resource for functional characterization of fungal proteins that mediate the delicate interaction between pathogen and host. The candidate targets of effectors, e.g., AtCLB, involved in pollen germination suggest tantalizing insights that could drive future studies.

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

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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.

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.

A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

Science.gov (United States)

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.

Intraspecies Competition in Serratia marcescens Is Mediated by Type VI-Secreted Rhs Effectors and a Conserved Effector-Associated Accessory Protein.

Science.gov (United States)

Alcoforado Diniz, Juliana; Coulthurst, Sarah J

2015-07-01

The type VI secretion system (T6SS) is widespread in Gram-negative bacteria and can deliver toxic effector proteins into eukaryotic cells or competitor bacteria. Antibacterial T6SSs are increasingly recognized as key mediators of interbacterial competition and may contribute to the outcome of many polymicrobial infections. Multiple antibacterial effectors can be delivered by these systems, with diverse activities against target cells and distinct modes of secretion. Polymorphic toxins containing Rhs repeat domains represent a recently identified and as-yet poorly characterized class of T6SS-dependent effectors. Previous work had revealed that the potent antibacterial T6SS of the opportunistic pathogen Serratia marcescens promotes intraspecies as well as interspecies competition (S. L. Murdoch, K. Trunk, G. English, M. J. Fritsch, E. Pourkarimi, and S. J. Coulthurst, J Bacteriol 193:6057-6069, 2011, http://dx.doi.org/10.1128/JB.05671-11). In this study, two new Rhs family antibacterial effectors delivered by this T6SS have been identified. One of these was shown to act as a DNase toxin, while the other contains a novel, cytoplasmic-acting toxin domain. Importantly, using S. marcescens, it has been demonstrated for the first time that Rhs proteins, rather than other T6SS-secreted effectors, can be the primary determinant of intraspecies competition. Furthermore, a new family of accessory proteins associated with T6SS effectors has been identified, exemplified by S. marcescens EagR1, which is specifically required for deployment of its associated Rhs effector. Together, these findings provide new insight into how bacteria can use the T6SS to deploy Rhs-family effectors and mediate different types of interbacterial interactions. Infectious diseases caused by bacterial pathogens represent a continuing threat to health and economic prosperity. To counter this threat, we must understand how such organisms survive and prosper. The type VI secretion system is a weapon that

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

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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.

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

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors.

Science.gov (United States)

Lopez, David; Ribeiro, Sébastien; Label, Philippe; Fumanal, Boris; Venisse, Jean-Stéphane; Kohler, Annegret; de Oliveira, Ricardo R; Labutti, Kurt; Lipzen, Anna; Lail, Kathleen; Bauer, Diane; Ohm, Robin A; Barry, Kerrie W; Spatafora, Joseph; Grigoriev, Igor V; Martin, Francis M; Pujade-Renaud, Valérie

2018-01-01

-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors

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David Lopez

2018-03-01

-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

The Legionella pneumophila IcmSW complex interacts with multiple Dot/Icm effectors to facilitate type IV translocation.

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Eric D Cambronne

2007-12-01

Full Text Available Many gram-negative pathogens use a type IV secretion system (T4SS to deliver effector proteins into eukaryotic host cells. The fidelity of protein translocation depends on the efficient recognition of effector proteins by the T4SS. Legionella pneumophila delivers a large number of effector proteins into eukaryotic cells using the Dot/Icm T4SS. How the Dot/Icm system is able to recognize and control the delivery of effectors is poorly understood. Recent studies suggest that the IcmS and IcmW proteins interact to form a stable complex that facilitates translocation of effector proteins by the Dot/Icm system by an unknown mechanism. Here we demonstrate that the IcmSW complex is necessary for the productive translocation of multiple Dot/Icm effector proteins. Effector proteins that were able to bind IcmSW in vitro required icmS and icmW for efficient translocation into eukaryotic cells during L. pneumophila infection. We identified regions in the effector protein SidG involved in icmSW-dependent translocation. Although the full-length SidG protein was translocated by an icmSW-dependent mechanism, deletion of amino terminal regions in the SidG protein resulted in icmSW-independent translocation, indicating that the IcmSW complex is not contributing directly to recognition of effector proteins by the Dot/Icm system. Biochemical and genetic studies showed that the IcmSW complex interacts with a central region of the SidG protein. The IcmSW interaction resulted in a conformational change in the SidG protein as determined by differences in protease sensitivity in vitro. These data suggest that IcmSW binding to effectors could enhance effector protein delivery by mediating a conformational change that facilitates T4SS recognition of a translocation domain located in the carboxyl region of the effector protein.

Characterization of the largest effector gene cluster of Ustilago maydis.

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Thomas Brefort

2014-07-01

Full Text Available In the genome of the biotrophic plant pathogen Ustilago maydis, many of the genes coding for secreted protein effectors modulating virulence are arranged in gene clusters. The vast majority of these genes encode novel proteins whose expression is coupled to plant colonization. The largest of these gene clusters, cluster 19A, encodes 24 secreted effectors. Deletion of the entire cluster results in severe attenuation of virulence. Here we present the functional analysis of this genomic region. We show that a 19A deletion mutant behaves like an endophyte, i.e. is still able to colonize plants and complete the infection cycle. However, tumors, the most conspicuous symptoms of maize smut disease, are only rarely formed and fungal biomass in infected tissue is significantly reduced. The generation and analysis of strains carrying sub-deletions identified several genes significantly contributing to tumor formation after seedling infection. Another of the effectors could be linked specifically to anthocyanin induction in the infected tissue. As the individual contributions of these genes to tumor formation were small, we studied the response of maize plants to the whole cluster mutant as well as to several individual mutants by array analysis. This revealed distinct plant responses, demonstrating that the respective effectors have discrete plant targets. We propose that the analysis of plant responses to effector mutant strains that lack a strong virulence phenotype may be a general way to visualize differences in effector function.

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

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Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

2013-01-01

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants.

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Jwa, Nam-Soo; Hwang, Byung Kook

2017-01-01

Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

Convergent Evolution of Pathogen Effectors toward Reactive Oxygen Species Signaling Networks in Plants

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Nam-Soo Jwa

2017-09-01

Full Text Available Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP-triggered immunity (PTI and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.

Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

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Hu, Gui-Qiu; Song, Pei-Xuan; Chen, Wei; Qi, Shuai; Yu, Shui-Xing; Du, Chong-Tao; Deng, Xu-Ming; Ouyang, Hong-Sheng; Yang, Yong-Jun

2017-10-01

Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1β secretion, cleaved caspase-1 production and ASC speckle formation were detected. We found that SopB could inhibit host IL-1β secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1β secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB. Copyright © 2017. Published by Elsevier Ltd.

Human reinforcement learning subdivides structured action spaces by learning effector-specific values

OpenAIRE

Gershman, Samuel J.; Pesaran, Bijan; Daw, Nathaniel D.

2009-01-01

Humans and animals are endowed with a large number of effectors. Although this enables great behavioral flexibility, it presents an equally formidable reinforcement learning problem of discovering which actions are most valuable, due to the high dimensionality of the action space. An unresolved question is how neural systems for reinforcement learning – such as prediction error signals for action valuation associated with dopamine and the striatum – can cope with this “curse of dimensionality...

The immune checkpoint regulator PD-L1 is a specific target for naturally occurring CD4(+) T cells

DEFF Research Database (Denmark)

Munir, Shamaila; Andersen, Gitte Holmen; Svane, Inge Marie

2013-01-01

Programmed cell death 1 ligand 1 (PD-L1) is an important regulator of T-cell responses and may consequently limit anticancer immunity. We have recently identified PD-L1-specific, cytotoxic CD8(+) T cells. In the present study, we develop these findings and report that CD4(+) helper T cells...... spontaneously recognize PD-L1. We examined the locality of a previously identified HLA-A*0201-restricted PD-L1-epitope for the presence of possible CD4(+) T-cell epitopes. Thus, we identified naturally occurring PD-L1-specific CD4(+) T cells among the peripheral blood lymphocytes of cancer patients...... and - to lesser extents - healthy donors, by means of ELISPOT assays. PD-L1-specific CD4(+) T cells appeared to be TH17 cells exhibiting an effector T-cell cytokine profile. Hence, PD-L1-specific CD4(+) T cells released interferon γ (IFNγ), tumor necrosis factor α (TNFα) and interleukin-17 (IL-17) in response...

Expansion of PD-1-positive effector CD4 T cells in an experimental model of SLE: contribution to the self-organized criticality theory.

Science.gov (United States)

Miyazaki, Yumi; Tsumiyama, Ken; Yamane, Takashi; Ito, Mitsuhiro; Shiozawa, Shunichi

2013-04-18

We have developed a systems biology concept to explain the origin of systemic autoimmunity. From our studies of systemic lupus erythematosus (SLE) we have concluded that this disease is the inevitable consequence of over-stimulating the host's immune system by repeated exposure to antigen to levels that surpass a critical threshold, which we term the system's "self-organized criticality". We observed that overstimulation of CD4 T cells in mice led to the development of autoantibody-inducing CD4 T cells (aiCD4 T) capable of generating various autoantibodies and pathological lesions identical to those observed in SLE. We show here that this is accompanied by the significant expansion of a novel population of effector T cells characterized by expression of programmed death-1 (PD-1)-positive, CD27(low), CD127(low), CCR7(low) and CD44(high)CD62L(low) markers, as well as increased production of IL-2 and IL-6. In addition, repeated immunization caused the expansion of CD8 T cells into fully-matured cytotoxic T lymphocytes (CTL) that express Ly6C(high)CD122(high) effector and memory markers. Thus, overstimulation with antigen leads to the expansion of a novel effector CD4 T cell population that expresses an unusual memory marker, PD-1, and that may contribute to the pathogenesis of SLE.

Target selection biases from recent experience transfer across effectors.

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Moher, Jeff; Song, Joo-Hyun

2016-02-01

Target selection is often biased by an observer's recent experiences. However, not much is known about whether these selection biases influence behavior across different effectors. For example, does looking at a red object make it easier to subsequently reach towards another red object? In the current study, we asked observers to find the uniquely colored target object on each trial. Randomly intermixed pre-trial cues indicated the mode of action: either an eye movement or a visually guided reach movement to the target. In Experiment 1, we found that priming of popout, reflected in faster responses following repetition of the target color on consecutive trials, occurred regardless of whether the effector was repeated from the previous trial or not. In Experiment 2, we examined whether an inhibitory selection bias away from a feature could transfer across effectors. While priming of popout reflects both enhancement of the repeated target features and suppression of the repeated distractor features, the distractor previewing effect isolates a purely inhibitory component of target selection in which a previewed color is presented in a homogenous display and subsequently inhibited. Much like priming of popout, intertrial suppression biases in the distractor previewing effect transferred across effectors. Together, these results suggest that biases for target selection driven by recent trial history transfer across effectors. This indicates that representations in memory that bias attention towards or away from specific features are largely independent from their associated actions.

The N-terminal domains of Vps3 and Vps8 are critical for localization and function of the CORVET tethering complex on endosomes.

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Nadine Epp

Full Text Available Endosomal biogenesis depends on multiple fusion and fission events. For fusion, the heterohexameric CORVET complex as an effector of the endosomal Rab5/Vps21 GTPase has a central function in the initial tethering event. Here, we show that the CORVET-specific Vps3 and Vps8 subunits, which interact with Rab5/Vps21, require their N-terminal domains for localization and function. Surprisingly, CORVET may lack either one of the two N-terminal domains, but not both, to promote protein sorting via the endosome. The dually truncated complex mislocalizes to the cytosol and is impaired in endocytic protein sorting, but not in assembly. Furthermore, the endosomal localization can be rescued by overexpression of Vps21 or one of the truncated CORVET subunits, even though CORVET assembly is not impaired by loss of the N-terminal domains or in strains lacking all endosomal Rab5s and Ypt7. We thus conclude that CORVET requires only its C-terminal domains for assembly and has beyond its putative β-propeller domains additional binding sites for endosomes, which could be important to bind Vps21 and other endosome-specific factors for efficient endosome tethering.

Repeat-containing protein effectors of plant-associated organisms

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Carl H. Mesarich

2015-10-01

Full Text Available Many plant-associated organisms, including microbes, nematodes, and insects, deliver effector proteins into the apoplast, vascular tissue, or cell cytoplasm of their prospective hosts. These effectors function to promote colonization, typically by altering host physiology or by modulating host immune responses. The same effectors however, can also trigger host immunity in the presence of cognate host immune receptor proteins, and thus prevent colonization. To circumvent effector-triggered immunity, or to further enhance host colonization, plant-associated organisms often rely on adaptive effector evolution. In recent years, it has become increasingly apparent that several effectors of plant-associated organisms are repeat-containing proteins (RCPs that carry tandem or non-tandem arrays of an amino acid sequence or structural motif. In this review, we highlight the diverse roles that these repeat domains play in RCP effector function. We also draw attention to the potential role of these repeat domains in adaptive evolution with regards to RCP effector function and the evasion of effector-triggered immunity. The aim of this review is to increase the profile of RCP effectors from plant-associated organisms.

Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes.

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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.

Adenovirus Protein E4-ORF1 Activation of PI3 Kinase Reveals Differential Regulation of Downstream Effector Pathways in Adipocytes

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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.

Persistent expansion of CD4(+) effector memory T cells in Wegener's granulomatosis

NARCIS (Netherlands)

Abdulahad, W. H.; van der Geld, Y. M.; Stegeman, C. A.; Kallenberg, C. G. M.

In order to test the hypothesis that Wegener's granulomatosis (WG) is associated with an ongoing immune effector response, even in remission, we examined the distribution of peripheral naive and memory T-lymphocytes in this disease, and analyzed the function-related phenotypes of the memory T-cell

Global study of holistic morphological effectors in the budding yeast Saccharomyces cerevisiae.

Science.gov (United States)

Suzuki, Godai; Wang, Yang; Kubo, Karen; Hirata, Eri; Ohnuki, Shinsuke; Ohya, Yoshikazu

2018-02-20

The size of the phenotypic effect of a gene has been thoroughly investigated in terms of fitness and specific morphological traits in the budding yeast Saccharomyces cerevisiae, but little is known about gross morphological abnormalities. We identified 1126 holistic morphological effectors that cause severe gross morphological abnormality when deleted, and 2241 specific morphological effectors with weak holistic effects but distinctive effects on yeast morphology. Holistic effectors fell into many gene function categories and acted as network hubs, affecting a large number of morphological traits, interacting with a large number of genes, and facilitating high protein expression. Holistic morphological abnormality was useful for estimating the importance of a gene to morphology. The contribution of gene importance to fitness and morphology could be used to efficiently classify genes into functional groups. Holistic morphological abnormality can be used as a reproducible and reliable gene feature for high-dimensional morphological phenotyping. It can be used in many functional genomic applications.

Class IA phosphatidylinositol 3-kinase p110α regulates phagosome maturation.

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Emily P Thi

Full Text Available Of the various phosphatidylinositol 3- kinases (PI3Ks, only the class III enzyme Vps34 has been shown to regulate phagosome maturation. During studies of phagosome maturation in THP-1 cells deficient in class IA PI3K p110α, we discovered that this PI3K isoform is required for vacuole maturation to progress beyond acquisition of Rab7 leading to delivery of lysosomal markers. Bead phagosomes from THP-1 cells acquired p110α and contained PI3P and PI(3,4,5P3; however, p110α and PI(3,4,5P3 levels in phagosomes from p110α knockdown cells were decreased. Phagosomes from p110α knock down cells showed normal acquisition of both Rab5 and EEA-1, but were markedly deficient in the lysosomal markers LAMP-1 and LAMP-2, and the lysosomal hydrolase, β-galactosidase. Phagosomes from p110α deficient cells also displayed impaired fusion with Texas Red dextran-loaded lysosomes. Despite lacking lysosomal components, phagosomes from p110α deficient cells recruited normal levels of Rab7, Rab-interacting lysosomal protein (RILP and homotypic vacuole fusion and protein sorting (HOPs components Vps41 and Vps16. The latter observations demonstrated that phagosomal Rab7 was active and capable of recruiting effectors involved in membrane fusion. Nevertheless, active Rab7 was not sufficient to bring about the delivery of lysosomal proteins to the maturing vacuole, which is shown for the first time to be dependent on a class I PI3K.

Type VI secretion system MIX-effectors carry both antibacterial and anti-eukaryotic activities.

Science.gov (United States)

Ray, Ann; Schwartz, Nika; de Souza Santos, Marcela; Zhang, Junmei; Orth, Kim; Salomon, Dor

2017-11-01

Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti-eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine-like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX-effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti-eukaryotic effectors. One of these is a MIX-effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX-effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells.

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Joanne M Lumsden

Full Text Available A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP-specific CD4(+ T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM and/or central memory (T(CM CD4(+ T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both T(E/EM and T(CM cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4(+ T(E/EM cells and of CD4(+ T(CM cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4(+ T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4(+ T(E/EM cells and of CD4(+ T(E/EM cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both T(E/EM and T(CM cells are major producers of IL-2.

The genome sequence and effector complement of the flax rust pathogen Melampsora lini

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Adnane eNemri

2014-03-01

Full Text Available Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp. Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimise parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analysed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

Science.gov (United States)

Nemri, Adnane; Saunders, Diane G O; Anderson, Claire; Upadhyaya, Narayana M; Win, Joe; Lawrence, Gregory J; Jones, David A; Kamoun, Sophien; Ellis, Jeffrey G; Dodds, Peter N

2014-01-01

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their

Diverse Secreted Effectors Are Required for Salmonella Persistence in a Mouse Infection Model

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Kidwai, Afshan S.; Mushamiri, Ivy T.; Niemann, George; Brown, Roslyn N.; Adkins, Joshua N.; Heffron, Fred

2013-08-12

Salmonella enterica serovar Typhimurium causes typhoid-like disease in mice and is a model of typhoid fever in humans. One of the hallmarks of typhoid is persistence, the ability of the bacteria to survive in the host weeks after infection. Virulence factors called effectors facilitate this process by direct transfer to the cytoplasm of infected cells thereby subverting cellular processes. Secretion of effectors to the cell cytoplasm takes place through multiple routes, including two separate type III secretion (T3SS) apparati as well as outer membrane vesicles. The two T3SS are encoded on separate pathogenicity islands, SPI-1 and -2, with SPI-1 more strongly associated with the intestinal phase of infection, and SPI-2 with the systemic phase. Both T3SS are required for persistence, but the effectors required have not been systematically evaluated. In this study, mutations in 48 described effectors were tested for persistence. We replaced each effector with a specific DNA barcode sequence by allelic exchange and co-infected with a wild-type reference to calculate the ratio of wild-type parent to mutant at different times after infection. The competitive index (CI) was determined by quantitative PCR in which primers that correspond to the barcode were used for amplification. Mutations in all but seven effectors reduced persistence demonstrating that most effectors were required. One exception was CigR, a recently discovered effector that is widely conserved throughout enteric bacteria. Deletion of cigR increased lethality, suggesting that it may be an anti-virulence factor. The fact that almost all Salmonella effectors are required for persistence argues against redundant functions. This is different from effector repertoires in other intracellular pathogens such as Legionella.

USP2-45 Is a Circadian Clock Output Effector Regulating Calcium Absorption at the Post-Translational Level.

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Daniel Pouly

Full Text Available The mammalian circadian clock influences most aspects of physiology and behavior through the transcriptional control of a wide variety of genes, mostly in a tissue-specific manner. About 20 clock-controlled genes (CCGs oscillate in virtually all mammalian tissues and are generally considered as core clock components. One of them is Ubiquitin-Specific Protease 2 (Usp2, whose status remains controversial, as it may be a cogwheel regulating the stability or activity of core cogwheels or an output effector. We report here that Usp2 is a clock output effector related to bodily Ca2+ homeostasis, a feature that is conserved across evolution. Drosophila with a whole-body knockdown of the orthologue of Usp2, CG14619 (dUsp2-kd, predominantly die during pupation but are rescued by dietary Ca2+ supplementation. Usp2-KO mice show hyperabsorption of dietary Ca2+ in small intestine, likely due to strong overexpression of the membrane scaffold protein NHERF4, a regulator of the Ca2+ channel TRPV6 mediating dietary Ca2+ uptake. In this tissue, USP2-45 is found in membrane fractions and negatively regulates NHERF4 protein abundance in a rhythmic manner at the protein level. In clock mutant animals (Cry1/Cry2-dKO, rhythmic USP2-45 expression is lost, as well as the one of NHERF4, confirming the inverse relationship between USP2-45 and NHERF4 protein levels. Finally, USP2-45 interacts in vitro with NHERF4 and endogenous Clathrin Heavy Chain. Taken together these data prompt us to define USP2-45 as the first clock output effector acting at the post-translational level at cell membranes and possibly regulating membrane permeability of Ca2+.

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.

A Role for EHD4 in the Regulation of Early Endosomal Transport

Science.gov (United States)

Sharma, Mahak; Naslavsky, Naava; Caplan, Steve

2009-01-01

All four of the C-terminal Eps15 homology domain (EHD) proteins have been implicated in the regulation of endocytic trafficking. However, the high level of amino acid sequence identity among these proteins has made it challenging to elucidate the precise function of individual EHD proteins. We demonstrate here with specific peptide antibodies that endogenous EHD4 localizes to Rab5-, early embryonic antigen 1 (EEA1)- and Arf6-containing endosomes and colocalizes with internalized transferrin in the cell periphery. Knock-down of EHD4 expression by both small interfering RNA and short hairpin RNA leads to the generation of enlarged early endosomal structures that contain Rab5 and EEA1 as well as internalized transferrin or major histocompatibility complex class I molecules. In addition, cargo destined for degradation, such as internalized low-density lipoprotein, also accumulates in the enlarged early endosomes in EHD4-depleted cells. Moreover, we have demonstrated that these enlarged early endosomes are enriched in levels of the activated GTP-bound Rab5. Finally, we show that endogenous EHD4 and EHD1 interact in cells, suggesting coordinated involvement in the regulation of receptor transport along the early endosome to endocytic recycling compartment axis. The results presented herein provide evidence that EHD4 is involved in the control of trafficking at the early endosome and regulates exit of cargo toward both the recycling compartment and the late endocytic pathway. PMID:18331452

Differential sensitivity of regulatory and effector T cells to cell death: a prerequisite for transplant tolerance

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Sylvaine eYou

2015-05-01

Full Text Available Despite significant progress achieved in transplantation, immunosuppressive therapies currently used to prevent graft rejection are still endowed with severe side effects impairing their efficiency over the long term. Thus, the development of graft-specific, non toxic innovative therapeutic strategies has become a major challenge, the goal being to selectively target alloreactive effector T cells while sparing CD4+Foxp3+ regulatory T cells (Tregs to promote operational tolerance. Various approaches, notably the one based on monoclonal antibodies or fusion proteins directed against the TCR/CD3 complex, TCR coreceptors, or costimulatory molecules, have been proposed to reduce the alloreactive T cell pool which is an essential prerequisite to create a therapeutic window allowing Tregs to induce and maintain allograft tolerance. In this minireview, we focus on the differential sensitivity of Tregs and effector T cells to the depleting and inhibitory effect of these immunotherapies, with a particular emphasis on CD3-specific antibodies that beyond their immunosuppressive effect, also express potent tolerogenic capacities.

Specific Hypersensitive Response–Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato

NARCIS (Netherlands)

Mesarich, Carl H.; Ӧkmen, Bilal; Rovenich, Hanna; Griffiths, Scott A.; Wang, Changchun; Karimi Jashni, Mansoor; Mihajlovski, Aleksandar; Collemare, Jérôme; Hunziker, Lukas; Deng, Cecilia H.; Burgt, Van Der Ate; Beenen, Henriek G.; Templeton, Matthew D.; Bradshaw, Rosie E.; Wit, De Pierre J.G.M.

2018-01-01

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes

Allelic barley MLA immune receptors recognize sequence-unrelated avirulence effectors of the powdery mildew pathogen.

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Lu, Xunli; Kracher, Barbara; Saur, Isabel M L; Bauer, Saskia; Ellwood, Simon R; Wise, Roger; Yaeno, Takashi; Maekawa, Takaki; Schulze-Lefert, Paul

2016-10-18

Disease-resistance genes encoding intracellular nucleotide-binding domain and leucine-rich repeat proteins (NLRs) are key components of the plant innate immune system and typically detect the presence of isolate-specific avirulence (AVR) effectors from pathogens. NLR genes define the fastest-evolving gene family of flowering plants and are often arranged in gene clusters containing multiple paralogs, contributing to copy number and allele-specific NLR variation within a host species. Barley mildew resistance locus a (Mla) has been subject to extensive functional diversification, resulting in allelic resistance specificities each recognizing a cognate, but largely unidentified, AVR a gene of the powdery mildew fungus, Blumeria graminis f. sp. hordei (Bgh). We applied a transcriptome-wide association study among 17 Bgh isolates containing different AVR a genes and identified AVR a1 and AVR a13 , encoding candidate-secreted effectors recognized by Mla1 and Mla13 alleles, respectively. Transient expression of the effector genes in barley leaves or protoplasts was sufficient to trigger Mla1 or Mla13 allele-specific cell death, a hallmark of NLR receptor-mediated immunity. AVR a1 and AVR a13 are phylogenetically unrelated, demonstrating that certain allelic MLA receptors evolved to recognize sequence-unrelated effectors. They are ancient effectors because corresponding loci are present in wheat powdery mildew. AVR A1 recognition by barley MLA1 is retained in transgenic Arabidopsis, indicating that AVR A1 directly binds MLA1 or that its recognition involves an evolutionarily conserved host target of AVR A1 Furthermore, analysis of transcriptome-wide sequence variation among the Bgh isolates provides evidence for Bgh population structure that is partially linked to geographic isolation.

A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

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Harms, Alexander; Liesch, Marius; Körner, Jonas; Québatte, Maxime; Engel, Philipp; Dehio, Christoph

2017-10-01

Host-targeting type IV secretion systems (T4SS) evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP) domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery) domain-similar to the Bartonella effector proteins (Beps) that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping stone in the

A bacterial toxin-antitoxin module is the origin of inter-bacterial and inter-kingdom effectors of Bartonella.

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Alexander Harms

2017-10-01

Full Text Available Host-targeting type IV secretion systems (T4SS evolved from conjugative T4SS machineries that mediate interbacterial plasmid transfer. However, the origins of effectors secreted by these virulence devices have remained largely elusive. Previous work showed that some effectors exhibit homology to toxins of bacterial toxin-antitoxin modules, but the evolutionary trajectories underlying these ties had not been resolved. We previously reported that FicT toxins of FicTA toxin-antitoxin modules disrupt cellular DNA topology via their enzymatic FIC (filamentation induced by cAMP domain. Intriguingly, the FIC domain of the FicT toxin VbhT of Bartonella schoenbuchensis is fused to a type IV secretion signal-the BID (Bep intracellular delivery domain-similar to the Bartonella effector proteins (Beps that are secreted into eukaryotic host cells via the host-targeting VirB T4SS. In this study, we show that the VbhT toxin is an interbacterial effector protein secreted via the conjugative Vbh T4SS that is closely related to the VirB T4SS and encoded by plasmid pVbh of B. schoenbuchensis. We therefore propose that the Vbh T4SS together with its effector VbhT represent an evolutionary missing link on a path that leads from a regular conjugation system and FicTA toxin-antitoxin modules to the VirB T4SS and the Beps. Intriguingly, phylogenetic analyses revealed that the fusion of FIC and BID domains has probably occurred independently in VbhT and the common ancestor of the Beps, suggesting parallel evolutionary paths. Moreover, several other examples of TA module toxins that are bona fide substrates of conjugative T4SS indicate that their recruitment as interbacterial effectors is prevalent and serves yet unknown biological functions in the context of bacterial conjugation. We propose that the adaptation for interbacterial transfer favors the exaptation of FicT and other TA module toxins as inter-kingdom effectors and may thus constitute an important stepping

In Planta Functional Analysis and Subcellular Localization of the Oomycete Pathogen Plasmopara viticola Candidate RXLR Effector Repertoire

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Yunxiao Liu

2018-04-01

Full Text Available Downy mildew is one of the most destructive diseases of grapevine, causing tremendous economic loss in the grape and wine industry. The disease agent Plasmopara viticola is an obligate biotrophic oomycete, from which over 100 candidate RXLR effectors have been identified. In this study, 83 candidate RXLR effector genes (PvRXLRs were cloned from the P. viticola isolate “JL-7-2” genome. The results of the yeast signal sequence trap assay indicated that most of the candidate effectors are secretory proteins. The biological activities and subcellular localizations of all the 83 effectors were analyzed via a heterologous Agrobacterium-mediated Nicotiana benthamiana expression system. Results showed that 52 effectors could completely suppress cell death triggered by elicitin, 10 effectors could partially suppress cell death, 11 effectors were unable to suppress cell death, and 10 effectors themselves triggered cell death. Live-cell imaging showed that the majority of the effectors (76 of 83 could be observed with informative fluorescence signals in plant cells, among which 34 effectors were found to be targeted to both the nucleus and cytosol, 29 effectors were specifically localized in the nucleus, and 9 effectors were targeted to plant membrane system. Interestingly, three effectors PvRXLR61, 86 and 161 were targeted to chloroplasts, and one effector PvRXLR54 was dually targeted to chloroplasts and mitochondria. However, western blot analysis suggested that only PvRXLR86 carried a cleavable N-terminal transit peptide and underwent processing in planta. Many effectors have previously been predicted to target organelles, however, to the best of our knowledge, this is the first study to provide experimental evidence of oomycete effectors targeted to chloroplasts and mitochondria.

Identification and characterization of LysM effectors in Penicillium expansum.

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Levin, Elena; Ballester, Ana Rosa; Raphael, Ginat; Feigenberg, Oleg; Liu, Yongsheng; Norelli, John; Gonzalez-Candelas, Luis; Ma, Jing; Dardick, Christopher; Wisniewski, Michael; Droby, Samir

2017-01-01

P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4) were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

Identification and characterization of LysM effectors in Penicillium expansum.

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Elena Levin

Full Text Available P. expansum is regarded as one of the most important postharvest rots of apple fruit and is also of great concern to fruit processing industries. Elucidating the pathogenicity mechanism of this pathogen is of utmost importance for the development of effective and safe management strategies. Although, many studies on modification of the host environment by the pathogen were done, its interactions with fruit during the early stages of infection and the virulence factors that mediate pathogenicity have not been fully defined. Effectors carrying LysM domain have been identified in numerous pathogenic fungi and their role in the first stages of infection has been established. In this study, we identified 18 LysM genes in the P. expansum genome. Amino acid sequence analysis indicated that P. expansum LysM proteins belong to a clade of fungal-specific LysM. Eleven of the discovered LysM genes were found to have secretory pathway signal peptide, among them, 4 (PeLysM1 PeLysM2, PeLysM3 and PeLysM4 were found to be highly expressed during the infection and development of decay of apple fruit. Effect of targeted deletion of the four putative PeLysM effectors on the growth and pathogenicity was studied. Possible interactions of PeLysM with host proteins was investigated using the yeast-two-hybrid system.

Abstract and Effector-Selective Decision Signals Exhibit Qualitatively Distinct Dynamics before Delayed Perceptual Reports.

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Twomey, Deirdre M; Kelly, Simon P; O'Connell, Redmond G

2016-07-13

Electrophysiological research has isolated neural signatures of decision formation in a variety of brain regions. Studies in rodents and monkeys have focused primarily on effector-selective signals that translate the emerging decision into a specific motor plan, but, more recently, research on the human brain has identified an abstract signature of evidence accumulation that does not appear to play any direct role in action preparation. The functional dissociations between these distinct signal types have only begun to be characterized, and their dynamics during decisions with deferred actions with or without foreknowledge of stimulus-effector mapping, a commonly studied task scenario in single-unit and functional imaging investigations, have not been established. Here we traced the dynamics of distinct abstract and effector-selective decision signals in the form of the broad-band centro-parietal positivity (CPP) and limb-selective β-band (8-16 and 18-30 Hz) EEG activity, respectively, during delayed-reported motion direction decisions with and without foreknowledge of direction-response mapping. With foreknowledge, the CPP and β-band signals exhibited a similar gradual build-up following evidence onset, but whereas choice-predictive β-band activity persisted up until the delayed response, the CPP dropped toward baseline after peaking. Without foreknowledge, the CPP exhibited identical dynamics, whereas choice-selective β-band activity was eliminated. These findings highlight qualitative functional distinctions between effector-selective and abstract decision signals and are of relevance to the assumptions founding functional neuroimaging investigations of decision-making. Neural signatures of evidence accumulation have been isolated in numerous brain regions. Although animal neurophysiology has largely concentrated on effector-selective decision signals that translate the emerging decision into a specific motor plan, recent research on the human brain has

Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on diots and monocots

NARCIS (Netherlands)

Stergiopoulos, I.; Burg, van den H.A.; Ökmen, B.; Beenen, H.G.; Liere, van S.; Kema, G.H.J.; Wit, de P.J.G.M.

2010-01-01

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce

Altered T cell memory and effector cell development in chronic lymphatic filarial infection that is independent of persistent parasite antigen.

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Cathy Steel

2011-04-01

Full Text Available Chronic lymphatic filarial (LF infection is associated with suppression of parasite-specific T cell responses that persist even following elimination of infection. While several mechanisms have been implicated in mediating this T cell specific downregulation, a role for alterations in the homeostasis of T effector and memory cell populations has not been explored. Using multiparameter flow cytometry, we investigated the role of persistent filarial infection on the maintenance of T cell memory in patients from the filarial-endemic Cook Islands. Compared to filarial-uninfected endemic normals (EN, microfilaria (mf positive infected patients (Inf had a reduced CD4 central memory (T(CM compartment. In addition, Inf patients tended to have more effector memory cells (T(EM and fewer effector cells (T(EFF than did ENs giving significantly smaller T(EFF:T(EM ratios. These contracted T(CM and T(EFF populations were still evident in patients previously mf+ who had cleared their infection (CLInf. Moreover, the density of IL-7Rα, necessary for T memory cell maintenance (but decreased in T effector cells, was significantly higher on memory cells of Inf and CLInf patients, although there was no evidence for decreased IL-7 or increased soluble IL7-Rα, both possible mechanisms for signaling defects in memory cells. However, effector cells that were present in Inf and CLInf patients had lower percentages of HLA-DR suggesting impaired function. These changes in T cell populations appear to reflect chronicity of infection, as filarial-infected children, despite the presence of active infection, did not show alterations in the frequencies of these T cell phenotypes. These data indicate that filarial-infected patients have contracted T(CM compartments and a defect in effector cell development, defects that persist even following clearance of infection. The fact that these global changes in memory and effector cell compartments do not yet occur in infected children

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.

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

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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.

De novo-engineered transcription activator-like effector (TALE) hybrid nuclease with novel DNA binding specificity creates double-strand breaks

KAUST Repository

Mahfouz, Magdy M.

2011-01-24

Site-specific and rare cutting nucleases are valuable tools for genome engineering. The generation of double-strand DNA breaks (DSBs) promotes homologous recombination in eukaryotes and can facilitate gene targeting, additions, deletions, and inactivation. Zinc finger nucleases have been used to generate DSBs and subsequently, for genome editing but with low efficiency and reproducibility. The transcription activator-like family of type III effectors (TALEs) contains a central domain of tandem repeats that could be engineered to bind specific DNA targets. Here, we report the generation of a Hax3-based hybrid TALE nuclease with a user-selected DNA binding specificity. We show that the engineered TALE nuclease can bind to its target sequence in vitro and that the homodimeric TALE nuclease can cleave double-stranded DNA in vitro if the DNA binding sites have the proper spacing and orientation. Transient expression assays in tobacco leaves suggest that the hybrid nuclease creates DSB in its target sequence, which is subsequently repaired by nonhomologous end-joining repair. Taken together, our data show the feasibility of engineering TALE-based hybrid nucleases capable of generating site-specific DSBs and the great potential for site-specific genome modification in plants and eukaryotes in general.

Effectors of Th1 and Th17 cells act on astrocytes and augment their neuroinflammatory properties.

Science.gov (United States)

Prajeeth, Chittappen K; Kronisch, Julius; Khorooshi, Reza; Knier, Benjamin; Toft-Hansen, Henrik; Gudi, Viktoria; Floess, Stefan; Huehn, Jochen; Owens, Trevor; Korn, Thomas; Stangel, Martin

2017-10-16

Autoreactive Th1 and Th17 cells are believed to mediate the pathology of multiple sclerosis in the central nervous system (CNS). Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of the neuroinflammation. Previously, we have shown that only Th1 but not Th17 effectors activate microglia. However, it is not clear which cells are targets of Th17 effectors in the CNS. To understand the effects driven by Th17 cells in the CNS, we induced experimental autoimmune encephalomyelitis in wild-type mice and CD4 + T cell-specific integrin α4-deficient mice where trafficking of Th1 cells into the CNS was affected. We compared microglial and astrocyte response in the brain and spinal cord of these mice. We further treated astrocytes with supernatants from highly pure Th1 and Th17 cultures and assessed the messenger RNA expression of neurotrophic factors, cytokines and chemokines, using real-time PCR. Data obtained was analyzed using the Kruskal-Wallis test. We observed in α4-deficient mice weak microglial activation but comparable astrogliosis to that of wild-type mice in the regions of the brain populated with Th17 infiltrates, suggesting that Th17 cells target astrocytes and not microglia. In vitro, in response to supernatants from Th1 and Th17 cultures, astrocytes showed altered expression of neurotrophic factors, pro-inflammatory cytokines and chemokines. Furthermore, increased expression of chemokines in Th1- and Th17-treated astrocytes enhanced recruitment of microglia and transendothelial migration of Th17 cells in vitro. Our results demonstrate the delicate interaction between T cell subsets and glial cells and how they communicate to mediate their effects. Effectors of Th1 act on both microglia and astrocytes whereas Th17 effectors preferentially target astrocytes to promote neuroinflammation.

Cytosolic Access of Intracellular Bacterial Pathogens: The Shigella Paradigm.

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Mellouk, Nora; Enninga, Jost

2016-01-01

Shigella is a Gram-negative bacterial pathogen, which causes bacillary dysentery in humans. A crucial step of Shigella infection is its invasion of epithelial cells. Using a type III secretion system, Shigella injects several bacterial effectors ultimately leading to bacterial internalization within a vacuole. Then, Shigella escapes rapidly from the vacuole, it replicates within the cytosol and spreads from cell-to-cell. The molecular mechanism of vacuolar rupture used by Shigella has been studied in some detail during the recent years and new paradigms are emerging about the underlying molecular events. For decades, bacterial effector proteins were portrayed as main actors inducing vacuolar rupture. This includes the effector/translocators IpaB and IpaC. More recently, this has been challenged and an implication of the host cell in the process of vacuolar rupture has been put forward. This includes the bacterial subversion of host trafficking regulators, such as the Rab GTPase Rab11. The involvement of the host in determining bacterial vacuolar integrity has also been found for other bacterial pathogens, particularly for Salmonella. Here, we will discuss our current view of host factor and pathogen effector implications during Shigella vacuolar rupture and the steps leading to it.

Systematic Identification of Intracellular-Translocated Candidate Effectors in Edwardsiella piscicida

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Lingzhi Zhang

2018-02-01

Full Text Available Many bacterial pathogens inject effectors directly into host cells to target a variety of host cellular processes and promote bacterial dissemination and survival. Identifying the bacterial effectors and elucidating their functions are central to understanding the molecular pathogenesis of these pathogens. Edwardsiella piscicida is a pathogen with a wide host range, and very few of its effectors have been identified to date. Here, based on the genes significantly regulated by macrophage infection, we identified 25 intracellular translocation-positive candidate effectors, including all five previously reported effectors, namely EseG, EseJ, EseH, EseK, and EvpP. A subsequent secretion analysis revealed diverse secretion patterns for the 25 effector candidates, suggesting that multiple transport pathways were involved in the internalization of these candidate effectors. Further, we identified two novel type VI secretion system (T6SS putative effectors and three outer membrane vesicles (OMV-dependent putative effectors among the candidate effectors described above, and further analyzed their contribution to bacterial virulence in a zebrafish model. This work demonstrates an effective approach for screening bacterial effectors and expands the effectors repertoire in E. piscicida.

A resistance locus in the American heirloom rice variety Carolina Gold Select is triggered by TAL effectors with diverse predicted targets and is effective against African strains of Xanthomonas oryzae pv. oryzicola.

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Triplett, Lindsay R; Cohen, Stephen P; Heffelfinger, Christopher; Schmidt, Clarice L; Huerta, Alejandra I; Tekete, Cheick; Verdier, Valerie; Bogdanove, Adam J; Leach, Jan E

2016-09-01

The rice pathogens Xanthomonas oryzae pathovar (pv.) oryzae and pv. oryzicola produce numerous transcription activator-like (TAL) effectors that increase bacterial virulence by activating expression of host susceptibility genes. Rice resistance mechanisms against TAL effectors include polymorphisms that prevent effector binding to susceptibility gene promoters, or that allow effector activation of resistance genes. This study identifies, in the heirloom variety Carolina Gold Select, a third mechanism of rice resistance involving TAL effectors. This resistance manifests through strong suppression of disease development in response to diverse TAL effectors from both X. oryzae pathovars. The resistance can be triggered by an effector with only 3.5 central repeats, is independent of the composition of the repeat variable di-residues that determine TAL effector binding specificity, and is independent of the transcriptional activation domain. We determined that the resistance is conferred by a single dominant locus, designated Xo1, that maps to a 1.09 Mbp fragment on chromosome 4. The Xo1 interval also confers complete resistance to the strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The strong phenotypic similarity between the TAL effector-triggered resistance conferred by Xo1 and that conferred by the tomato resistance gene Bs4 suggests that monocots and dicots share an ancient or convergently evolved mechanism to recognize analogous TAL effector epitopes. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

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

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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.

GTP Binding and Oncogenic Mutations May Attenuate Hypervariable Region (HVR)-Catalytic Domain Interactions in Small GTPase K-Ras4B, Exposing the Effector Binding Site*

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Lu, Shaoyong; Banerjee, Avik; Jang, Hyunbum; Zhang, Jian; Gaponenko, Vadim; Nussinov, Ruth

2015-01-01

K-Ras4B, a frequently mutated oncogene in cancer, plays an essential role in cell growth, differentiation, and survival. Its C-terminal membrane-associated hypervariable region (HVR) is required for full biological activity. In the active GTP-bound state, the HVR interacts with acidic plasma membrane (PM) headgroups, whereas the farnesyl anchors in the membrane; in the inactive GDP-bound state, the HVR may interact with both the PM and the catalytic domain at the effector binding region, obstructing signaling and nucleotide exchange. Here, using molecular dynamics simulations and NMR, we aim to figure out the effects of nucleotides (GTP and GDP) and frequent (G12C, G12D, G12V, G13D, and Q61H) and infrequent (E37K and R164Q) oncogenic mutations on full-length K-Ras4B. The mutations are away from or directly at the HVR switch I/effector binding site. Our results suggest that full-length wild-type GDP-bound K-Ras4B (K-Ras4BWT-GDP) is in an intrinsically autoinhibited state via tight HVR-catalytic domain interactions. The looser association in K-Ras4BWT-GTP may release the HVR. Some of the oncogenic mutations weaken the HVR-catalytic domain association in the K-Ras4B-GDP/-GTP bound states, which may facilitate the HVR disassociation in a nucleotide-independent manner, thereby up-regulating oncogenic Ras signaling. Thus, our results suggest that mutations can exert their effects in more than one way, abolishing GTP hydrolysis and facilitating effector binding. PMID:26453300

ULtiMATE system for rapid assembly of customized TAL effectors.

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Junjiao Yang

Full Text Available Engineered TAL-effector nucleases (TALENs and TALE-based constructs have become powerful tools for eukaryotic genome editing. Although many methods have been reported, it remains a challenge for the assembly of designer-based TALE repeats in a fast, precise and cost-effective manner. We present an ULtiMATE (USER-based Ligation Mediated Assembly of TAL Effector system for speedy and accurate assembly of customized TALE constructs. This method takes advantage of uracil-specific excision reagent (USER to create multiple distinct sticky ends between any neighboring DNA fragments for specific ligation. With pre-assembled templates, multiple TALE DNA-binding domains could be efficiently assembled in order within hours with minimal manual operation. This system has been demonstrated to produce both functional TALENs for effective gene knockout and TALE-mediated gene-specific transcription activation (TALE-TA. The feature of both ease-of-operation and high efficiency of ULtiMATE system makes it not only an ideal method for biologic labs, but also an approach well suited for large-scale assembly of TALENs and any other TALE-based constructions.

TNFR2 expression on CD25hiFOXP3+ T cells induced upon TCR stimulation of CD4 T cells identifies maximal cytokine-producing effectors.

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Chindu eGovindaraj

2013-08-01

Full Text Available In this study, we show that CD25hiTNFR2+ cells can be rapidly generated in vitro from circulating CD4 lymphocytes by polyclonal stimuli anti-CD3 in the presence of anti-CD28. The in vitro induced CD25hiTNFR2+ T cells express a conventional Treg phenotype FOXP3+CTLA4+CD127lo/-, but produce effector and immunoregulatory cytokines including IL-2, IL-10 and IFN-g. These induced CD25hiTNFR2+ T cells do not suppress target cell proliferation, but enhance it instead. Thus the CD25hiTNFR2+ phenotype induced rapidly following CD3/28 cross linking of CD4 T cells identifies cells with maximal proliferative and effector cytokine producing capability. The in vivo counterpart of this cell population may play an important role in immune response initiation.

Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

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Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Dinakaran, Vasudevan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2016-01-01

Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

Functional, Antigen-Specific Stem Cell Memory (TSCM CD4+ T Cells Are Induced by Human Mycobacterium tuberculosis Infection

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Cheleka A. M. Mpande

2018-03-01

Full Text Available BackgroundMaintenance of long-lasting immunity is thought to depend on stem cell memory T cells (TSCM, which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (TCM or effector (TEFF T cells. Our knowledge of TSCM derives primarily from studies of virus-specific CD8+ TSCM. We aimed to determine if infection with Mycobacterium tuberculosis (M. tb, the etiological agent of tuberculosis, generates antigen-specific CD4+ TSCM and to characterize their functional ontology.MethodsWe studied T cell responses to natural M. tb infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT converters and three cross-sectional QFT+ adult cohorts; and to bacillus Calmette–Guerin (BCG vaccination in infants. M. tb and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of M. tb-specific tetramer+ CD4+ TSCM (CD45RA+ CCR7+ CD27+ were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry.ResultsM. tb-specific TSCM were not detected in QFT-negative persons. After QFT conversion frequencies of TSCM increased to measurable levels and remained detectable thereafter, suggesting that primary M. tb infection induces TSCM cells. Gene expression (GE profiling of tetramer+ TSCM showed that these cells were distinct from bulk CD4+ naïve T cells (TN and shared features of bulk TSCM and M. tb-specific tetramer+ TCM and TEFF cells. These TSCM were predominantly CD95+ and CXCR3+, markers typical of CD8+ TSCM. Tetramer+ TSCM expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk TN and TSCM cells. M. tb-specific TSCM were also

Protecting and rescuing the effectors: roles of differentiation and survival in the control of memory T cell development

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Sema eKurtulus

2013-01-01

Full Text Available Vaccines, arguably the single most important intervention in improving human health, have exploited the phenomenon of immunological memory. The elicitation of memory T cells is often an essential part of successful long-lived protective immunity. Our understanding of T cell memory has been greatly aided by the development of TCR Tg mice and MHC tetrameric staining reagents that have allowed the precise tracking of antigen-specific T cell responses. Indeed, following acute infection or immunization, naïve T cells undergo a massive expansion culminating in the generation of a robust effector T cell population. This peak effector response is relatively short-lived and, while most effector T cells die by apoptosis, some remain and develop into memory cells. Although the molecular mechanisms underlying this cell fate decision remain incompletely defined, substantial progress has been made, particularly with regards to CD8+ T cells. For example, the effector CD8+ T cells generated during a response are heterogeneous, consisting of cells with more or less potential to develop into full-fledged memory cells. Development of CD8+ T cell memory is regulated by the transcriptional programs that control the differentiation and survival of effector T cells. While the type of antigenic stimulation and level of inflammation control effector CD8+ T cell differentiation, availability of cytokines and their ability to control expression and function of Bcl-2 family members governs their survival. These distinct differentiation and survival programs may allow for finer therapeutic intervention to control both the quality and quantity of CD8+ T cell memory. Effector to memory transition of CD4+ T cells is less well characterized than CD8+ T cells, emerging details will be discussed. This review will focus on the recent progress made in our understanding of the mechanisms underlying the development of T cell memory with an emphasis on factors controlling survival of

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

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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.