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Manabu Negishi - One of the best experts on this subject based on the ideXlab platform.
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Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation
Molecular biology of the cell, 2012Co-Authors: Izumi Oinuma, Kana Kawada, Kiyoka Tsukagoshi, Manabu NegishiAbstract:The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.
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Different Requirement for Rnd GTPases of R-Ras GAP Activity of Plexin-C1 and Plexin-D1
The Journal of biological chemistry, 2009Co-Authors: Kanami Uesugi, Hironori Katoh, Izumi Oinuma, Manabu NegishiAbstract:Plexins, comprising Plexin-A, -B, -C, and -D subfamilies, are receptors for semaphorins governing cell adhesion, migration, and axon guidance. Among plexin subfamilies, Plexin-A1 and Plexin-B1 have been shown to function as an R-Ras GAP, inducing repulsive responses, and the expression of R-Ras GAP activity requires the binding of Rnd1, a member of Rnd subfamily of Rho GTPases. However, signaling pathways of Plexin-D1 and Plexin-C1 still remain obscure. Here, we found that Plexin-D1 displayed R-Ras GAP activity and inhibited migration of COS-7 cells, and these actions required Rnd2, another Rnd subfamily GTPase. Rnd2 bound to Plexin-D1 in cortical neurons, and Sema3E/Plexin-D1-induced inhibition of axon outgrowth of cortical neurons required Rnd2 and down-regulation of R-Ras activity. On the other hand, Plexin-C1 displayed R-Ras GAP activity and inhibited cell migration of COS-7 cells without Rnd proteins. Therefore, R-Ras GAP activity is a common function of plexin subfamilies but the regulation of R-Ras GAP activity of plexins by Rnd proteins is different among plexin subfamilies.
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Small GTPase Rnd1 is involved in neuronal activity-dependent dendritic development in hippocampal neurons.
Neuroscience letters, 2006Co-Authors: Yukio Ishikawa, Hironori Katoh, Manabu NegishiAbstract:Rho family small GTPases are key regulators for neuronal morphogenesis including dendritogenesis. We recently have shown that Rnd1, a member of the Rho family, is highly expressed in brain during the synaptogenic stage and is involved in dendritic spine formation. However, the mechanism by which Rnd1 regulates dendritic development including spine morphogenesis remains unknown. Here we report that Rnd1, a member of the Rho family, plays a critical role in neuronal activity-dependent dendritic development in hippocampal neurons. Overexpression of Rnd1 promoted dendritic growth and branching in cultured hippocampal neurons. On the other hand, suppression of endogenous Rnd1 expression by RNA interference significantly inhibited neuronal activity-dependent dendritic development and this inhibitory effect was canceled by inhibition of RhoA effector ROCK. In addition, knockdown of Rnd1 also abolished dendritic development promoted by treatment with brain-derived neurotrophic factor in hippocampal neurons. Our findings demonstrate that Rnd1 is involved in signaling pathways of neuronal activity-dependent dendritic development.
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Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity.
The Journal of biological chemistry, 2006Co-Authors: Hiroko Tanaka, Hironori Katoh, Manabu NegishiAbstract:The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.
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In vivo function of Rnd2 in the development of neocortical pyramidal neurons.
Neuroscience research, 2005Co-Authors: Kazuhiro Nakamura, Hironori Katoh, Yoko Yamashita, Nobuaki Tamamaki, Takeshi Kaneko, Manabu NegishiAbstract:The present study examined the in vivo role of Rnd2, a Rho family small GTPase, in brain development. Rnd2 was expressed by radially migrating cells, which primarily develop to pyramidal neurons, during their stay in the subventricular zone of embryonic cerebral cortex and hippocampus. Exogenous expression of wild-type and a constitutively active Rnd2, but not a negative mutant of Rnd2, in radially migrating cells by in utero electroporation disturbed their morphology and migration to upper layers. These results indicate that Rnd2 functions in vivo as a regulator of the migration and morphological changes associated with the development of pyramidal neurons.
Hironori Katoh - One of the best experts on this subject based on the ideXlab platform.
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Different Requirement for Rnd GTPases of R-Ras GAP Activity of Plexin-C1 and Plexin-D1
The Journal of biological chemistry, 2009Co-Authors: Kanami Uesugi, Hironori Katoh, Izumi Oinuma, Manabu NegishiAbstract:Plexins, comprising Plexin-A, -B, -C, and -D subfamilies, are receptors for semaphorins governing cell adhesion, migration, and axon guidance. Among plexin subfamilies, Plexin-A1 and Plexin-B1 have been shown to function as an R-Ras GAP, inducing repulsive responses, and the expression of R-Ras GAP activity requires the binding of Rnd1, a member of Rnd subfamily of Rho GTPases. However, signaling pathways of Plexin-D1 and Plexin-C1 still remain obscure. Here, we found that Plexin-D1 displayed R-Ras GAP activity and inhibited migration of COS-7 cells, and these actions required Rnd2, another Rnd subfamily GTPase. Rnd2 bound to Plexin-D1 in cortical neurons, and Sema3E/Plexin-D1-induced inhibition of axon outgrowth of cortical neurons required Rnd2 and down-regulation of R-Ras activity. On the other hand, Plexin-C1 displayed R-Ras GAP activity and inhibited cell migration of COS-7 cells without Rnd proteins. Therefore, R-Ras GAP activity is a common function of plexin subfamilies but the regulation of R-Ras GAP activity of plexins by Rnd proteins is different among plexin subfamilies.
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Small GTPase Rnd1 is involved in neuronal activity-dependent dendritic development in hippocampal neurons.
Neuroscience letters, 2006Co-Authors: Yukio Ishikawa, Hironori Katoh, Manabu NegishiAbstract:Rho family small GTPases are key regulators for neuronal morphogenesis including dendritogenesis. We recently have shown that Rnd1, a member of the Rho family, is highly expressed in brain during the synaptogenic stage and is involved in dendritic spine formation. However, the mechanism by which Rnd1 regulates dendritic development including spine morphogenesis remains unknown. Here we report that Rnd1, a member of the Rho family, plays a critical role in neuronal activity-dependent dendritic development in hippocampal neurons. Overexpression of Rnd1 promoted dendritic growth and branching in cultured hippocampal neurons. On the other hand, suppression of endogenous Rnd1 expression by RNA interference significantly inhibited neuronal activity-dependent dendritic development and this inhibitory effect was canceled by inhibition of RhoA effector ROCK. In addition, knockdown of Rnd1 also abolished dendritic development promoted by treatment with brain-derived neurotrophic factor in hippocampal neurons. Our findings demonstrate that Rnd1 is involved in signaling pathways of neuronal activity-dependent dendritic development.
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Pragmin, a novel effector of Rnd2 GTPase, stimulates RhoA activity.
The Journal of biological chemistry, 2006Co-Authors: Hiroko Tanaka, Hironori Katoh, Manabu NegishiAbstract:The Rho family of small GTPases has been implicated in the reorganization of actin cytoskeleton and subsequent morphological changes in various cells. Rnd2 is a member of the Rnd subfamily, comprising Rnd1, Rnd2, and Rnd3. In contrast to Rnd1 and Rnd3, displaying an antagonistic action for RhoA signaling, signaling pathways of Rnd2 are not well known. Here we have performed a yeast two-hybrid screen using Rnd2 as bait and identified a novel Rnd2 effector protein, predominantly expressed in neurons, including cortical and hippocampal neurons. We named it Pragmin (pragma of Rnd2). In in vivo and in vitro binding assays, Pragmin specifically binds to Rnd2 among the Rho family GTPases in a GTP-dependent manner. Rnd2-bound Pragmin significantly stimulates RhoA activity and induces cell contraction through RhoA and the Rho-kinase pathway in HeLa cells. In PC12 cells, expressing Pragmin inhibits nerve growth factor-induced neurite outgrowth in response to Rnd2, and knock-down of Pragmin by Pragmin-specific small interfering RNA enhances neurite elongation. Therefore, Rnd2 regulates neurite outgrowth by functioning as the RhoA activator through Pragmin, in contrast to Rnd1 and Rnd3 inhibiting RhoA signaling.
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In vivo function of Rnd2 in the development of neocortical pyramidal neurons.
Neuroscience research, 2005Co-Authors: Kazuhiro Nakamura, Hironori Katoh, Yoko Yamashita, Nobuaki Tamamaki, Takeshi Kaneko, Manabu NegishiAbstract:The present study examined the in vivo role of Rnd2, a Rho family small GTPase, in brain development. Rnd2 was expressed by radially migrating cells, which primarily develop to pyramidal neurons, during their stay in the subventricular zone of embryonic cerebral cortex and hippocampus. Exogenous expression of wild-type and a constitutively active Rnd2, but not a negative mutant of Rnd2, in radially migrating cells by in utero electroporation disturbed their morphology and migration to upper layers. These results indicate that Rnd2 functions in vivo as a regulator of the migration and morphological changes associated with the development of pyramidal neurons.
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Rho Family GTPases and Dendrite Plasticity
The Neuroscientist, 2005Co-Authors: Manabu Negishi, Hironori KatohAbstract:Dendrite structures exert a profound influence on neuronal information processing. The Rho family GTPases have been implicated in the regulation of dendritic development. Among them, Rho, Rac, and Cdc42 have been characterized extensively, and Rac and Cdc42 promote dendrite growth and branching, whereas Rho acts as a negative regulator for dendrite growth. Recently, other members of Rho family GTPases, including Rnd1 and Rnd2, have also been shown to be involved in the regulation of dendrite development. Rnd1 promotes spine maturation, and Rnd2 stimulates dendrite branching through its specific effector, Rapostlin. Thus, a variety of Rho family GTPases play important roles in dendritic development.
Laetitia Mouly - One of the best experts on this subject based on the ideXlab platform.
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The Rnd1 Small GTPase: Main Functions and Emerging Role in Oncogenesis.
International journal of molecular sciences, 2019Co-Authors: Laetitia Mouly, Julia Gilhodes, Anthony Lemarié, Elizabeth Cohen-jonathan Moyal, Christine Toulas, Gilles Favre, Olivier Sordet, Sylvie MonferranAbstract:The Rho GTPase family can be classified into classic and atypical members. Classic members cycle between an inactive Guanosine DiPhosphate -bound state and an active Guanosine TriPhosphate-bound state. Atypical Rho GTPases, such as Rnd1, are predominantly in an active GTP-bound conformation. The role of classic members in oncogenesis has been the subject of numerous studies, while that of atypical members has been less explored. Besides the roles of Rnd1 in healthy tissues, recent data suggest that Rnd1 is involved in oncogenesis and response to cancer therapeutics. Here, we present the current knowledge on Rnd1 expression, subcellular localization, and functions in healthy tissues. Then, we review data showing that Rnd1 expression is dysregulated in tumors, the molecular mechanisms involved in this deregulation, and the role of Rnd1 in oncogenesis. For several aggressive tumors, Rnd1 presents the features of a tumor suppressor gene. In these tumors, low expression of Rnd1 is associated with a bad prognosis for the patients. Finally, we highlight that Rnd1 expression is induced by anticancer agents and modulates their response. Of note, Rnd1 mRNA levels in tumors could be used as a predictive marker of both patient prognosis and response to anticancer agents.
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Rnd1 regulates migration of human glioblastoma stem-like cells according to their anatomical localization and defines a prognostic signature in glioblastoma
Oncotarget, 2018Co-Authors: Sabrina Boyrie, Julia Gilhodes, Anthony Lemarié, Caroline Delmas, Vincent Lubrano, Perrine Dahan, Laure Malric, José Luis, Marie Tosolini, Laetitia MoulyAbstract:Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ-). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, Rnd1 in PVZ GSCs compared to CT GSCs. Overexpression of Rnd1, dramatically inhibited PVZ GSC migration and conversely, downregulation of Rnd1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of Rnd1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of Rnd1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of Rnd1, we identified an Rnd1 low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the Rnd1 low signature as key targets genes to impede tumor relapse after treatment.
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Rnd1 regulates migration of human glioblastoma stem-like cells according to their anatomical localization and defines a prognostic signature in glioblastoma
Oncotarget, 2018Co-Authors: Sabrina Boyrie, Julia Gilhodes, Anthony Lemarié, Caroline Delmas, Vincent Lubrano, Perrine Dahan, Laure Malric, José Luis, Marie Tosolini, Laetitia MoulyAbstract:// Sabrina Boyrie 1, 2 , Caroline Delmas 1, 2 , Anthony Lemarie 1, 3 , Vincent Lubrano 4, 5 , Perrine Dahan 1 , Laure Malric 1, 3 , Jose Luis 6 , Julia Gilhodes 2 , Marie Tosolini 1 , Laetitia Mouly 1, 3 , Maxime Lehmann 7 , Christine Toulas 1, 2, * , Elizabeth Cohen-Jonathan Moyal 1, 2, 3, * and Sylvie Monferran 1, 3, * 1 INSERM UMR1037, Cancer Research Center of Toulouse, Oncopole, Toulouse, France 2 Institut Claudius Regaud, IUCT-O, Toulouse, France 3 Universite Toulouse III, Toulouse, France 4 INSERM UMR825, Universite Toulouse III, Toulouse, France 5 Service de Neurochirurgie, Centre Hospitalier de Purpan, Universite Toulouse III, Toulouse, France 6 Centre de Recherche en Oncologie biologique et Oncopharmacologie (CRO2), INSERM UMR 911, Aix-Marseille Universite, Marseille, France 7 UMR 7213 CNRS, Laboratoire de Biophotonique et Pharmacologie, Tumoral Signaling and Therapeutic Targets, Universite de Strasbourg, Faculte de Pharmacie, 67401 Illkirch, France * These authors contributed equally to this work Correspondence to: Sylvie Monferran, email: sylvie.monferran@inserm.fr Christine Toulas, email: toulas.christine@iuct-oncopole.fr Elizabeth Cohen-Jonathan Moyal, email: moyal.elizabeth@iuct-oncopole.fr Keywords: glioblastoma stem-like cells; periventricular zone; migration; prognostic signature; Rnd1 Received: February 20, 2018 Accepted: July 31, 2018 Published: September 18, 2018 ABSTRACT Despite post-operative radio-chemotherapy, glioblastoma systematically locally recurs. Tumors contacting the periventricular zone (PVZ) show earlier and more distant relapses than tumors not contacting the PVZ. Since glioblastoma stem-like cells (GSCs) have been proposed to play a major role in glioblastoma recurrence, we decided to test whether GSC migration properties could be different according to their anatomical location (PVZ+/PVZ–). For that purpose, we established paired cultures of GSCs from the cortical area (CT) and the PVZ of glioblastoma patient tumors. We demonstrated that PVZ GSCs possess higher migration and invasion capacities than CT GSCs. We highlighted specific transcriptomic profiles in PVZ versus CT populations and identified a down-regulation of the RhoGTPase, Rnd1 in PVZ GSCs compared to CT GSCs. Overexpression of Rnd1, dramatically inhibited PVZ GSC migration and conversely, downregulation of Rnd1 increased CT GSC migration. Additionally, transcriptomic analyses also revealed a down-regulation of Rnd1 in glioblastoma compared to normal brain. Using the glioblastoma TCGA database, low levels of Rnd1 were also shown to correlate with a decreased overall survival of patients. Finally, based on signaling pathways activated in patients with low levels of Rnd1 , we identified an Rnd1 low signature of six genes (MET, LAMC1, ITGA5, COL5A1, COL3A1, COL1A2) that is an independent prognostic factor in glioblastoma. These findings contribute to explain the shorter time to progression of patients with PVZ involvement and, point out genes that establish the Rnd1 low signature as key targets genes to impede tumor relapse after treatment.
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PARP-1-dependent Rnd1 transcription induced by topoisomerase I cleavage complexes confers cellular resistance to camptothecin
Cell death & disease, 2018Co-Authors: Laetitia Mouly, Gilles Favre, Olivier Sordet, Kenza Mamouni, Rémi Gence, Agnese Cristini, Julia Cherier, Adrien Castinel, Morgane Legrand, Sylvie MonferranAbstract:RHO GTPases regulate essential functions such as the organization of the actin cytoskeleton. The classic members cycle between an active GTP-bound and an inactive GDP-bound conformation whereas atypical members are predominantly GTP-bound. Besides their well-established role, the classic RHO GTPases RHOB and RAC1, are rapidly induced and/or activated by genotoxic stress and contribute to the DNA damage response. Here we used camptothecin, a selective topoisomerase I (TOP1) inhibitor that stabilizes TOP1 cleavage complexes (TOP1cc), to search for other potential early DNA damage-inducible RHO GTPase genes. We identified that an atypical RHO GTPase, Rnd1, is rapidly induced by camptothecin. Rnd1 induction is closely associated with the presence of TOP1cc induced by camptothecin or by DNA lesions that elevate TOP1cc levels such as UV and hydrogen peroxide. We further demonstrated that camptothecin increases Rnd1 gene transcription and mRNA stability. Camptothecin also increases poly(ADP-ribose) polymerase 1 (PARP-1) activity, whose inhibition reduces Rnd1 transcription. In addition, overexpression of Rnd1 increases PARP-1, suggesting a cross-talk between PARP-1 and Rnd1. Finally, Rnd1 protects cells against camptothecin-induced apoptosis, and hence favors cellular resistance to camptothecin. Together, these findings highlight Rnd1 as an atypical RHO GTPase early induced by TOP1cc, and show that the TOP1cc-PARP-1-Rnd1 pathway protects cells against apoptosis induced by camptothecin.
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role de la gtpase rho Rnd1 dans la reponse aux dommages a l adn
2018Co-Authors: Laetitia MoulyAbstract:La famille des GTPases Rho, comprenant 20 membres, controle la dynamique du cytosquelette d'actine et differents processus cellulaires comme la migration. En plus de leurs roles bien etablis, certaines GTPases Rho, notamment RhoB et Rac1, ont emerge en tant que genes de reponse precoce aux dommages a l'ADN. En effet, RhoB est induite en reponse a divers stress genotoxiques, y compris la camptothecine (CPT), les UV et le cisplatine, et protege principalement les cellules de l'apoptose. Le role des autres GTPases Rho en reponse precoce aux genotoxiques reste largement meconnu. Dans ce projet, nous avons utilise la camptothecine, un inhibiteur de la topoisomerase I (TOP1), qui stabilise selectivement les complexes de clivage TOP1-ADN (TOP1cc) sur la chromatine, afin de cribler les GTPases Rho induites de facon precoce par les dommages a l'ADN. En plus de RhoB, nous avons identifie Rnd1 comme un gene rapidement induit par la CPT. L'induction de Rnd1 est reversible et etroitement correlee a la presence de TOP1cc induit par la CPT. En accord avec ces observations, les rayons UV et le peroxyde d'hydrogene, qui stabilisent indirectement les TOP1cc, induisent egalement Rnd1. La CPT augmente la transcription de Rnd1 independamment de l'activite de son promoteur minimal. De plus, la CPT augmente l'activite de la poly ADP-ribose polymerase (PARP1), dont l'inhibition previent la transcription de Rnd1. La surexpression de Rnd1 augmente egalement l'expression de PARP1, suggerant une regulation positive entre PARP1 et Rnd1 en reponse aux TOP1cc. Ainsi, nous proposons qu'en reponse a la CPT, les TOP1cc activent PARP1, qui a son tour favorise la transcription de Rnd1, initiant ainsi une boucle de retrocontrole positive. Enfin, nous avons montre que Rnd1 protege les cellules contre l'apoptose induite par la CPT et entraine leur resistance a la CPT. L'ensemble de ces resultats ont permis d'identifier Rnd1 comme nouvelle GTPase Rho impliquee dans la reponse au stress et proposent un nouveau mecanisme de regulation de la transcription des genes en reponse aux TOP1cc via l'activation de PARP1. Ces resultats suggerent par ailleurs qu'inhiber la signalisation de Rnd1 pourrait sensibiliser les cellules tumorales aux derives cliniques de la CPT.
Matthias Buck - One of the best experts on this subject based on the ideXlab platform.
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Molecular Dynamics Simulations Reveal Isoform Specific Contact Dynamics between the Plexin Rho GTPase Binding Domain (RBD) and Small Rho GTPases Rac1 and Rnd1
The journal of physical chemistry. B, 2017Co-Authors: Liqun Zhang, Matthias BuckAbstract:The Plexin family of transmembrane receptors are unique in that their intracellular region interacts directly with small GTPases of the Rho family. The Rho GTPase binding domain of plexin (RBD)—which is responsible for these interactions—can bind with Rac1 as well as Rnd1 GTPases. GTPase complexes have been crystallized with the RBDs of plexinA1, -A2, and -B1. The protein association is thought to elicit different functional responses in a GTPase and plexin isoform specific manner, but the origin of this is unknown. In this project, we investigated complexes between several RBD and Rac1/Rnd1 GTPases using multimicrosecond length all atom molecular dynamics simulations, also with reference to the free forms of the RBDs and GTPases. In accord with the crystallographic data, the RBDs experience more structural changes than Rho-GTPases upon complex formation. Changes in protein dynamics and networks of correlated motions are revealed by analyzing dihedral angle fluctuations in the proteins. The extent of thes...
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Allosteric Communication in Rnd1 and Rac1 Association with the Plexin-B1 RhoGTPase Binding Domain Revealed by Hydrogen Exchange Mass Spectroscopy and by Solution NMR
Biophysical Journal, 2015Co-Authors: Shufen Cao, Matthias BuckAbstract:Rho family GTPases play important roles in the regulation of the cell's actin cytoskeleton and mediate the repulsive and attractive effects of guidance molecules. Their interaction with single transmembrane receptors Plexins are of particular importance, since Plexin receptors are important for axon guidance, angiogenesis and also cancer. Our previous study mapped the interactions of small Rho GTPases, Rnd1 and active Rac1, with the plexin-B1 through a common region, the Rho GTPase Binding Domain (RBD)[1]. And NMR relaxation experiments on the RBD: Rac1 complex revealed that the Rac1 is less dynamic in the Plexin bound state[2]. To further address the mechanism under the specificity and different functions of different Rho GTPase involving Plexin-B1, we present here the dynamical behavior of Rac1 and Rnd1, and the changes upon their association with plexin-B1 RBD. Solution NMR as well as mass spectrometry experiments are conducted to monitor the amide hydrogen exchange process. Both Rnd1 and Rac1 become rigid in general, especially in the switch I and switch II regions. In addition, both GTPases show complimentary dynamics changes distal from the binding sites, indicating an allosteric signaling mechanism. This also suggests a more direct role of Rho GTPase upon binding to Plexin-B1, rather than simply being sequestrated by Plexin-B1. More importantly, the dynamical study revealed the binding with Plexin-B1 RBD induces different changes in the dynamics and in different regions of the two GTPases. These are in accord with previous thermodynamic measurements and implies a difference in the mechanism of action of the two Rho GTPases-Rac1 and Rnd1, upon interaction with Plexin-B1[3]. These studies together suggest the origin of the specificity of the GTPase- protein interaction and signaling in the plexin-B1 system.
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Backbone assignment and secondary structure of Rnd1, an unusual Rho family small GTPase
Biomolecular NMR assignments, 2012Co-Authors: Shufen Cao, Xi’an Mao, Deli Liu, Matthias BuckAbstract:Rho GTPases have attracted considerable interest as signaling molecules due to their variety of functional roles in cells. Rnd1 is a relatively recently discovered Rho GTPase with no enzymatic activity against its bound GTP nucleotide, setting it apart from other family members. Research has revealed a critical role for Rnd1 not only in neurite outgrowth, dendrite development, axon guidance, but also in gastric cancer and in endothelial cells during inflammation. Structural information is crucial for understanding the mechanism that forms the basis for protein–protein interactions and functions, but until recently there were no reports of NMR studies directly on the Rnd1 protein. In this paper we report assignments for the majority of Rnd1 NMR resonances based on 2D and 3D NMR spectra. Rnd1 assignment was a challenging task, however, despite optimization strategies that have facilitated NMR studies of the protein (Cao and Buck in Small GTPase 2:295–304, 2012). Besides common triple-resonance experiments, 3D HNCA, 3D HN(CO)CA, 3D HNCO which are usually employed for sequence assignment, 3D NOESY experiments and specific labeling of 13 kinds of amino acids were also utilized to gain as many 1H(N), 13C, and 15N resonances assignments as possible. For 170 cross peaks observed out of 183 possible mainchain N–H correlations in the 1H–15N TROSY spectrum, backbone assignment was finally completed for 127 resonances. The secondary structure was then defined by chemical shifts and TALOS+ based on the assignments. The overall structure in solution compares well with that of Rnd1 in a crystal, except for two short segments, residues 77–83 and residues 127–131. Given that some features are shared among Rho GTPases, Rnd1 assignments are also compared with two other family members, Cdc42 and Rac1. The overall level of Rnd1 assignment is lower than for Cdc42 and Rac1, consistent with its lower stability and possibly increased internal dynamics. However, while the Rnd1 switch II region remained un-assigned, the switch I region could be more fully assigned compared to Cdc42 and Rac1. The NMR assignment and structure analysis reported here provides a robust basis for future study of the binding between Rnd1 and other proteins, as well as for further studies of the molecular function of this unusual GTPase.
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Optimization and stabilization of Rho small GTPase proteins for solution NMR studies: The case of Rnd1
Small GTPases, 2011Co-Authors: Matthias BuckAbstract:Rho GTPases of the Ras superfamily have important roles in regulating the organization of the actin filament system, morphogenesis and migration of cells. Structural details for these proteins are still emerging, and information on their dynamics in solution is much needed to understand the mechanisms underlying their signaling functions. This report reviews conditions for solution NMR studies of Rho GTPases and describes our optimization and stabilization of Rnd1 for such experiments. Rnd1 belongs to the Rnd protein subfamily branch of Rho small GTPases and functions in neurite outgrowth, dendrite development and in axon guidance. However, as we report here, solution NMR studies of this protein are challenging. Multiple methods have been employed to enhance the stability of Rnd1, including by cleavage of an N-terminal His expression tag and by addition of non-hydrolysable GMPPNP (β: γ-imidoguanosine 5'-triphosphate) nucleotide. Further stabilization of Rnd1 against aggregation was achieved through a structure informed point mutation while maintaining its conformation and binding affinity for a partner protein. The NMR spectrum of the optimized protein reveals significant improvement in NMR signal dispersion and intensity. This work paves the way for structural and protein-protein/protein-ligand interaction studies of Rnd1 by solution NMR and also provides a guide for optimization and stabilization of other Rho GTPases.
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Structural Basis of Rnd1 Binding to Plexin Rho GTPase Binding Domains (RBDs)
The Journal of biological chemistry, 2011Co-Authors: Hui Wang, Prasanta K. Hota, Yufeng Tong, Limin Shen, Lyudmila Nedyalkova, Susmita Borthakur, Soonjeung Kim, Wolfram Tempel, Matthias BuckAbstract:Plexin receptors regulate cell adhesion, migration, and guidance. The Rho GTPase binding domain (RBD) of plexin-A1 and -B1 can bind GTPases, including Rnd1. By contrast, plexin-C1 and -D1 reportedly bind Rnd2 but associate with Rnd1 only weakly. The structural basis of this differential Rnd1 GTPase binding to plexin RBDs remains unclear. Here, we solved the structure of the plexin-A2 RBD in complex with Rnd1 and the structures of the plexin-C1 and plexin-D1 RBDs alone, also compared with the previously determined plexin-B1 RBD.Rnd1 complex structure. The plexin-A2 RBD·Rnd1 complex is a heterodimer, whereas plexin-B1 and -A2 RBDs homodimerize at high concentration in solution, consistent with a proposed model for plexin activation. Plexin-C1 and -D1 RBDs are monomeric, consistent with major residue changes in the homodimerization loop. In plexin-A2 and -B1, the RBD β3-β4 loop adjusts its conformation to allow Rnd1 binding, whereas minimal structural changes occur in Rnd1. The plexin-C1 and -D1 RBDs lack several key non-polar residues at the corresponding GTPase binding surface and do not significantly interact with Rnd1. Isothermal titration calorimetry measurements on plexin-C1 and -D1 mutants reveal that the introduction of non-polar residues in this loop generates affinity for Rnd1. Structure and sequence comparisons suggest a similar mode of Rnd1 binding to the RBDs, whereas mutagenesis suggests that the interface with the highly homologous Rnd2 GTPase is different in detail. Our results confirm, from a structural perspective, that Rnd1 does not play a role in the activation of plexin-C1 and -D1. Plexin functions appear to be regulated by subfamily-specific mechanisms, some of which involve different Rho family GTPases.
Naoto Ueno - One of the best experts on this subject based on the ideXlab platform.
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xenopus Rnd1 and rnd3 gtp binding proteins are expressed under the control of segmentation clock and required for somite formation
Developmental Dynamics, 2009Co-Authors: Tadahiro Goda, Chiyo Takagi, Naoto UenoAbstract:The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor-8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP-binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant-negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation-related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis. Developmental Dynamics 238:2867–2876, 2009. © 2009 Wiley-Liss, Inc.
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Xenopus Rnd1 and Rnd3 GTP‐binding proteins are expressed under the control of segmentation clock and required for somite formation
Developmental dynamics : an official publication of the American Association of Anatomists, 2009Co-Authors: Tadahiro Goda, Chiyo Takagi, Naoto UenoAbstract:The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor-8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP-binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant-negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation-related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis. Developmental Dynamics 238:2867–2876, 2009. © 2009 Wiley-Liss, Inc.
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TGF-β signaling-mediated morphogenesis: modulation of cell adhesion via cadherin endocytosis
Genes & development, 2007Co-Authors: Souichi Ogata, Naoto Ueno, Christof Niehrs, Junji Morokuma, Tadayoshi Hayata, Gabriel Kolle, Ken W.y. ChoAbstract:The molecular mechanisms governing the cell behaviors underlying morphogenesis remain a major focus of research in both developmental biology and cancer biology. TGF-beta ligands control cell fate specification via Smad-mediated signaling. However, their ability to guide cellular morphogenesis in a variety of biological contexts is poorly understood. We report on the discovery of a novel TGF-beta signaling-mediated cellular morphogenesis occurring during vertebrate gastrulation. Activin/nodal members of the TGF-beta superfamily induce the expression of two genes regulating cell adhesion during gastrulation: Fibronectin Leucine-rich Repeat Transmembrane 3 (FLRT3), a type I transmembrane protein containing extracellular leucine-rich repeats, and the small GTPase Rnd1. FLRT3 and Rnd1 interact physically and modulate cell adhesion during embryogenesis by controlling cell surface levels of cadherin through a dynamin-dependent endocytosis pathway. Our model suggests that cell adhesion can be dynamically regulated by sequestering cadherin through internalization, and subsequent redeploying internalized cadherin to the cell surface as needed. As numerous studies have linked aberrant expression of small GTPases, adhesion molecules such as cadherins, and TGF-beta signaling to oncogenesis and metastasis, it is tempting to speculate that this FLRT3/Rnd1/cadherin pathway might also control cell behavior and morphogenesis in adult tissue homeostasis.
