DOCSLIB.ORG

Crystal Structure of a Guanine Nucleotide Exchange Factor Encoded by the Scrub Typhus Pathogen Orientia Tsutsugamushi

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Crystal Structure of a Guanine Nucleotide Exchange Factor Encoded by the Scrub Typhus Pathogen Orientia Tsutsugamushi Crystal structure of a guanine nucleotide exchange factor encoded by the scrub typhus pathogen Orientia tsutsugamushi Christopher Lima,1, Jason M. Berka,1, Alyssa Blaisea, Josie Birchera, Anthony J. Koleskea, Mark Hochstrassera,2, and Yong Xionga,2 aDepartment of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520 Edited by Peter J. Novick, University of California San Diego, La Jolla, CA, and approved October 7, 2020 (received for review September 3, 2020) Rho family GTPases regulate an array of cellular processes and are IpgB2 GEF protein activates RhoA and causes characteristic often modulated by pathogens to promote infection. Here, we membrane ruffles that are critical for Shigella invasion of the host identify a cryptic guanine nucleotide exchange factor (GEF) do- cell (7). Bacterial effector GEFs belong to either the WxxxE main in the OtDUB protein encoded by the pathogenic bacterium family (named for a conserved motif important for folding and Orientia tsutsugamushi. A proteomics-based OtDUB interaction structural integrity) or the SopE family (SopE, SopE2, and BopE). screen identified numerous potential host interactors, including These bacterial effectors share no sequence or structural homol- the Rho GTPases Rac1 and Cdc42. We discovered a domain in ogy to eukaryotic Rho GEFs, which predominantly belong to the OtDUB with Rac1/Cdc42 GEF activity (OtDUBGEF), with higher ac- Dbl homology (DH) family of GEFs that adopt a six-helix bundle tivity toward Rac1 in vitro. While this GEF bears no obvious se- with an elongated, kinked “chaise lounge” fold (8, 9). Rather, quence similarity to known GEFs, crystal structures of OtDUBGEF bacterial effector GEFs adopt a characteristic compact V-shaped alone (3.0 Å) and complexed with Rac1 (1.7 Å) reveal striking con- fold, yet activate the Rho GTPases via the same contact regions in vergent evolution, with a unique topology, on a V-shaped bacte- the GTPases that are crucial for nucleotide exchange by DH- rial GEF fold shared with other bacterial GEF domains. Structure- family GEFs (10). While substantial effort has been exerted in guided mutational analyses identified residues critical for activity detailing the molecular determinants of bacterial GEF activities and a mechanism for nucleotide displacement. Ectopic expression BIOCHEMISTRY of OtDUB activates Rac1 preferentially in cells, and expression of and specificities, no bacterial effector GEFs have been identified outside of the WxxxE or SopE-like families. the OtDUBGEF alone alters cell morphology. Cumulatively, this work reveals a bacterial GEF within the multifunctional OtDUB Recently, we identified and characterized a putative effector that co-opts host Rac1 signaling to induce changes in cytoskeletal protein, OtDUB, from the obligate intracellular bacterium that structure. causes scrub typhus, Orientia tsutsugamushi. Despite extensive characterization of the OtDUB deubiquitylase (DUB) domain Orientia tsutsugamushi | X-ray crystallography | guanine nucleotide (residues 1–259), the function of the extensive C-terminal region, exchange factor | scrub typhus | Rac1 encompassing more than 1,000 amino acids, remained elusive (11). Here, we report that OtDUB encodes a GEF domain, he Ras homologous (Rho) family of GTPases is part of the TRas superfamily of small G proteins. Rho family GTPases Significance are molecular switches that control intracellular actin dynamics and regulate a diverse array of cellular processes from cytoki- Scrub typhus is a neglected tropical disease caused by the nesis to cell migration and wound healing (1–3). These small bacterium Orientia tsutsugamushi. Although O. tsutsugamushi ∼21-kDa proteins are highly conserved in all eukaryotes, with is an emerging public health threat, its pathogenic mechanisms three founding family members that have been extensively remain markedly understudied. Bacterial pathogens subvert studied: Rac1, Cdc42, and RhoA. Each Rho family GTPase exerts host actin dynamics by encoding guanine nucleotide exchange specific effects on the actin cytoskeleton, and constitutive activa- factors (GEFs) as effector proteins, which activate cellular Rho tion of each protein leads to characteristic cellular phenotypes. GTPases. Here, we identify a GEF domain within an O. tsutsu- The signaling activity of a GTPase is controlled by its bound gamushi protein that activates the host GTPase Rac1. While the nucleotide. When GDP is bound, the GTPase is in the “inactive” overall shape of the GEF is similar to that of other bacterial state, and loading of a GTP promotes the “active” conformation effectors, the primary sequence, topology, and catalytic mecha- of the G protein. Interaction with downstream effector proteins nism are completely distinct, suggesting convergent evolution. and subsequent actin reorganization only occurs when the GTPase Our studies reveal a cryptic GEF domain encoded by O. tsutsu- is in the GTP-bound active state. The intrinsic nucleotide ex- gamushi and provide the groundwork to probe the role of cy- change (GDP to GTP) and hydrolysis (GTP to GDP) rates of Rho toskeletal modulation in this neglected pathogen. family GTPases alone are slow. Nucleotide exchange occurs on Author contributions: C.L., J.M.B., A.B., J.B., A.J.K., M.H., and Y.X. designed research; C.L., the order of 1.5 per hour (4, 5), and the intrinsic hydrolysis rate is J.M.B., A.B., and J.B. performed research; C.L., J.M.B., A.J.K., M.H., and Y.X. analyzed data; ∼0.15 per min (6). Rapid regulation of GTPases, therefore, is and C.L. and J.M.B. wrote the paper. controlled by two classes of proteins that either switch them “on” The authors declare no competing interest. or “off”: guanine nucleotide exchange factors (GEFs) promote the This article is a PNAS Direct Submission. dissociation of GDP and allow loading with GTP, and GTPase Published under the PNAS license. activating proteins (GAPs) accelerate the intrinsic GTP hydrolysis 1C.L. and J.M.B. contributed equally to this work. by the G protein. 2To whom correspondence may be addressed. Email: [email protected] or Bacterial pathogens such as certain species of Salmonella, [email protected]. Shigella and enteropathic Escherichia coli, encode and secrete This article contains supporting information online at https://www.pnas.org/lookup/suppl/ effector proteins that modulate small GTPases to benefit the doi:10.1073/pnas.2018163117/-/DCSupplemental. bacterium during infection. For instance, the Shigella flexineri www.pnas.org/cgi/doi/10.1073/pnas.2018163117 PNAS Latest Articles | 1of11 Downloaded by guest on September 26, 2021 OtDUBGEF. Using biochemical, structural, and cellular methods, bound uniquely to GST-OtDUB275–1369 and not to GST- we demonstrate that OtDUBGEF predominantly activates Rac1 OtDUB675–1369 or the GST protein control (Fig. 1C and SI Ap- in vitro and in cell culture. While the primary sequence of pendix,TableS1). Notably, several small GTPases, such as Rac1 OtDUBGEF is unrelated to WxxxE or SopE GEFs, the OtDUBGEF and Cdc42, and proteins of related functions were significantly crystal structure reveals a similar V-shaped fold despite an entirely enriched in the GST-OtDUB275–1369–bound sample compared to different topological and helical arrangement, suggesting convergent the GST-OtDUB675–1369 and GST controls (Fig. 1D). We focused evolution. We further determined the OtDUBGEF:Rac1 complex on the small GTPases as there are numerous examples of path- crystal structure and demonstrate that OtDUBGEF interacts with ogens hijacking GTPase signaling (12, 13). Rac1 at key common loci in the GTPase. The complex structure also suggested a distinct mechanism for GDP displacement, unique Identification of GTPase-Binding and GEF Activity in OtDUB. We among all GEFs characterized to date. Our work reveals that O. carried out coimmunoprecipitation (co-IP) assays using lysates of tsutsugamushi has evolved a GEF domain that expands the molec- HeLa cells ectopically expressing several OtDUB fragments to ular repertoire of bacterial effectors and suggests a critical function verify the putative interactions identified by mass spectrometry. for OtDUB in regulating Rac1 to benefit the pathogen during Flag-tagged OtDUB fragments were immunoprecipitated fol- infection. lowing incubation of whole cell lysates, and bound proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel elec- Results trophoresis (SDS-PAGE) and immunoblotted for Rac1, Cdc42, The OtDUB C-Terminal Segment Is Toxic in Yeast and Interacts with and RhoA. These co-IP experiments confirmed that full-length GTPases. The OtDUB N-terminal region contains an active DUB (FL) OtDUB and OtDUB275–1369 bound Rac1 and Cdc42 but and a high-affinity ubiquitin binding domain (UBD) within the not RhoA (Fig. 2A). Further truncations (OtDUB1–675 and first 259 residues (11). However, the remainder of the 1,369- OtDUB675–1369) abolished the interaction. To demonstrate that residue protein is devoid of any computationally predicted do- this association does not require other cellular components, we mains. To examine how the OtDUB might affect eukaryotic cells, performed direct binding assays between purified E. coli- we first generated a series of truncations (Fig. 1A) and expressed expressed recombinant OtDUB fragments and recombinant the proteins in the yeast Saccharomyces cerevisiae (Fig. 1B). Re- GST-tagged Rac1 or Cdc42. In agreement with the co-IP re- markably, expression of the full-length protein caused a complete sults with HeLa cell lysates, OtDUB275–1369-Flag
Load more

Recommended publications
  • G Protein Regulation of MAPK Networks
    Oncogene (2007) 26, 3122–3142 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc REVIEW G Protein regulation of MAPK networks ZG Goldsmith and DN Dhanasekaran Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA, USA G proteins provide signal-coupling mechanisms to hepta- the a-subunits has been used as a basis for the helical cell surface receptors and are criticallyinvolved classification of G proteins into Gs,Gi,Gq and G12 in the regulation of different mitogen-activated protein families in which the a-subunits that show more than kinase (MAPK) networks. The four classes of G proteins, 50% homology are grouped together (Simon et al., defined bythe G s,Gi,Gq and G12 families, regulate 1991). In G-protein-coupled receptor (GPCR)-mediated ERK1/2, JNK, p38MAPK, ERK5 and ERK6 modules by signaling pathways, ligand-activated receptors catalyse different mechanisms. The a- as well as bc-subunits are the exchange of the bound GDP to GTP in the a-subunit involved in the regulation of these MAPK modules in a following which the GTP-bound a-subunit disassociate context-specific manner. While the a- and bc-subunits from the receptor as well as the bg-subunit. The GTP- primarilyregulate the MAPK pathwaysvia their respec- bound a-subunit and the bg-subunit stimulate distinct tive effector-mediated signaling pathways, recent studies downstream effectors including enzymes, ion channels have unraveled several novel signaling intermediates and small GTPase, thus regulating multiple signaling including receptor tyrosine kinases and small GTPases pathways including those involved in the activation of through which these G-protein subunits positivelyas well mitogen-activated protein kinase (MAPK) modules as negativelyregulate specific MAPK modules.
    [Show full text]
  • 679514V2.Full.Pdf
    bioRxiv preprint doi: https://doi.org/10.1101/679514; this version posted August 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The large GTPase, mGBP-2, regulates Rho family GTPases to inhibit migration and invadosome formation in Triple-Negative Breast Cancer cells. Geoffrey O. Nyabuto, John P. Wilson, Samantha A. Heilman, Ryan C. Kalb, Ankita V. Abnave, and Deborah J. Vestal* Department of Biological Sciences, University of Toledo, Toledo, OH, USA 43606 *Corresponding author: Deborah J. Vestal, Department of Biological Sciences, University of Toledo, 2801 West Bancroft St., MS 1010, Toledo, OH 43606. Phone: 1-419-383-4134. FAX: 1-419-383-6228. Email: [email protected]. Running title: mGBP-2 inhibits breast cancer cell migration. Key words: Guanylate-Binding Protein, Triple-Negative Breast Cancer, migration, CDC42, Rac1. This work was supported by funding from the University of Toledo to D.J.V. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed. 1 bioRxiv preprint doi: https://doi.org/10.1101/679514; this version posted August 19, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract Breast cancer is the most common cancer in women. Despite advances in early detection and treatment, it is predicted that over 40,000 women will die of breast cancer in 2019. This number of women is still too high. To lower this number, more information about the molecular players in breast cancer are needed.
    [Show full text]
  • Role of Rho Family Gtpases in Epithelial Morphogenesis
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW Role of Rho family GTPases in epithelial morphogenesis Linda Van Aelst1,3 and Marc Symons2 1Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA; 2Center for Oncology and Cell Biology, North Shore-Long Island Jewish Research Institute and Department of Surgery, North Shore-Long Island Jewish Medical Center, Manhasset, New York 11030, USA Epithelial cell sheets line the organ and body surfaces will also discuss the participation of these GTPases in and the specialized barrier functions of these epithelia epithelial remodeling during wound-healing and epithe- regulate the exchange of substances with the outside en- lial-mesenchymal transitions. vironment and between different body compartments. As other members of the Ras superfamily, Rho Epithelia play a role in a wide range of physiological GTPases cycle between a GDP-bound (inactive) state processes such as digestion, excretion, and leukocyte and a GTP-bound (active) state. In the active state, these trafficking. In addition, during development, some epi- GTPases relay signals from growth factors, cytokines, thelia form transient primitive structures, including the and adhesion molecules to regulate a wide range of bio- neural tube and somites, which are essential for the de- logical processes, including actin cytoskeleton organiza- velopment of more complex organs. tion, transcriptional regulation, and vesicle trafficking The establishment and maintenance of epithelial cell (Van Aelst and D’Souza-Schorey 1997; Hall 1998). polarity is critical for the development and functioning The nucleotide state of Rho family proteins is con- of multicellular organisms (Nelson 2000).
    [Show full text]
  • Small Gtpases of the Ras and Rho Families Switch On/Off Signaling
    International Journal of Molecular Sciences Review Small GTPases of the Ras and Rho Families Switch on/off Signaling Pathways in Neurodegenerative Diseases Alazne Arrazola Sastre 1,2, Miriam Luque Montoro 1, Patricia Gálvez-Martín 3,4 , Hadriano M Lacerda 5, Alejandro Lucia 6,7, Francisco Llavero 1,6,* and José Luis Zugaza 1,2,8,* 1 Achucarro Basque Center for Neuroscience, Science Park of the Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/EHU), 48940 Leioa, Spain; [email protected] (A.A.S.); [email protected] (M.L.M.) 2 Department of Genetics, Physical Anthropology, and Animal Physiology, Faculty of Science and Technology, UPV/EHU, 48940 Leioa, Spain 3 Department of Pharmacy and Pharmaceutical Technology, Faculty of Pharmacy, University of Granada, 180041 Granada, Spain; [email protected] 4 R&D Human Health, Bioibérica S.A.U., 08950 Barcelona, Spain 5 Three R Labs, Science Park of the UPV/EHU, 48940 Leioa, Spain; [email protected] 6 Faculty of Sport Science, European University of Madrid, 28670 Madrid, Spain; [email protected] 7 Research Institute of the Hospital 12 de Octubre (i+12), 28041 Madrid, Spain 8 IKERBASQUE, Basque Foundation for Science, 48013 Bilbao, Spain * Correspondence: [email protected] (F.L.); [email protected] (J.L.Z.) Received: 25 July 2020; Accepted: 29 August 2020; Published: 31 August 2020 Abstract: Small guanosine triphosphatases (GTPases) of the Ras superfamily are key regulators of many key cellular events such as proliferation, differentiation, cell cycle regulation, migration, or apoptosis. To control these biological responses, GTPases activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and in some small GTPases also guanine nucleotide dissociation inhibitors (GDIs).
    [Show full text]
  • Rhogtpase Signaling in Cell Polarity and Gene Regulation
    Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 128 RhoGTPase Signaling in Cell Polarity and Gene Regulation ANN-SOFI JOHANSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 UPPSALA ISBN 91-554-6505-6 2006 urn:nbn:se:uu:diva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
    [Show full text]
  • Presentation in Dendritic Cells Small Rho Gtpases Regulate Antigen
    Small Rho GTPases Regulate Antigen Presentation in Dendritic Cells Galina V. Shurin, Irina L. Tourkova, Gurkamal S. Chatta, Gudula Schmidt, Sheng Wei, Julie Y. Djeu and Michael R. This information is current as Shurin of September 26, 2021. J Immunol 2005; 174:3394-3400; ; doi: 10.4049/jimmunol.174.6.3394 http://www.jimmunol.org/content/174/6/3394 Downloaded from References This article cites 40 articles, 18 of which you can access for free at: http://www.jimmunol.org/content/174/6/3394.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 26, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Small Rho GTPases Regulate Antigen Presentation in Dendritic Cells1 Galina V. Shurin,* Irina L. Tourkova,* Gurkamal S. Chatta,‡ Gudula Schmidt,§ Sheng Wei,¶ Julie Y. Djeu,¶ and Michael R. Shurin2*† Dendritic cells (DC) are involved in the regulation of innate and adaptive immunity.
    [Show full text]
  • Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling
    cells Article Kalirin Interacts with TRAPP and Regulates Rab11 and Endosomal Recycling Xiaolong Wang 1 , Meiqian Weng 2, Yuting Ke 1,3 , Ellen Sapp 3, Marian DiFiglia 3 and Xueyi Li 1,3,* 1 School of Pharmacy, Shanghai Jiao Tong University, Shanghai 200240, China; [email protected] (X.W.); [email protected] (Y.K.) 2 Mucosal Immunology Lab combined program in Pediatric Gastroenterology and Nutrition, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; [email protected] 3 Department of Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129, USA; [email protected] (E.S.); difi[email protected] (M.D.) * Correspondence: [email protected] or [email protected]; Tel.: +86-21-34204737 Received: 1 April 2020; Accepted: 1 May 2020; Published: 4 May 2020 Abstract: Coordinated actions of Rab and Rho are necessary for numerous essential cellular processes ranging from vesicle budding to whole cell movement. How Rab and Rho are choreographed is poorly understood. Here, we report a protein complex comprised of kalirin, a Rho guanine nucleotide exchange factor (GEF) activating Rac1, and RabGEF transport protein particle (TRAPP). Kalirin was identified in a mass spectrometry analysis of proteins precipitated by trappc4 and detected on membranous organelles containing trappc4. Acute knockdown of kalirin did not affect trappc4, but significantly reduced overall and membrane-bound levels of trappc9, which specifies TRAPP toward activating Rab11. Trappc9 deficiency led to elevated expression of kalirin in neurons. Co-localization of kalirin and Rab11 occurred at a low frequency in NRK cells under steady state and was enhanced upon expressing an inactive Rab11 mutant to prohibit the dissociation of Rab11 from the kalirin-TRAPP complex.
    [Show full text]
  • RHO Family Gtpases in the Biology of Lymphoma
    cells Review RHO Family GTPases in the Biology of Lymphoma Claudia Voena 1 and Roberto Chiarle 1,2,* 1 Department of Molecular Biotechnology and Health Sciences, University of Torino, 10126 Torino, Italy 2 Department of Pathology, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA * Correspondence: [email protected] (C.V.); [email protected] (R.C.) Received: 2 May 2019; Accepted: 20 June 2019; Published: 26 June 2019 Abstract: RHO GTPases are a class of small molecules involved in the regulation of several cellular processes that belong to the RAS GTPase superfamily. The RHO family of GTPases includes several members that are further divided into two different groups: typical and atypical. Both typical and atypical RHO GTPases are critical transducers of intracellular signaling and have been linked to human cancer. Significantly, both gain-of-function and loss-of-function mutations have been described in human tumors with contradicting roles depending on the cell context. The RAS family of GTPases that also belong to the RAS GTPase superfamily like the RHO GTPases, includes arguably the most frequently mutated genes in human cancers (K-RAS, N-RAS, and H-RAS) but has been extensively described elsewhere. This review focuses on the role of RHO family GTPases in human lymphoma initiation and progression. Keywords: RHO family GTPases; RHOA; RHOH; VAV; mutations; chromosomal translocations; lymphoma 1. Introduction RHO GTPases are highly conserved proteins in eukaryotes, and the RHO family GTPase consists of 18–22 members [1–3]. They can be classified according to their homology and structure into the following subfamilies: CDC42, RAC, RHO, RHOD, RHOU, RHOH, and RND [1,2,4,5].
    [Show full text]
  • Uncovering the Secret Life of Rho Gtpases
    INSIGHT CELL SIGNALING Uncovering the secret life of Rho GTPases New methods to directly visualize Rho GTPases reveal how a protein called RhoGDI regulates the activity of these ’molecular switches’ at the plasma membrane. JENNA A PERRY AND AMY SHAUB MADDOX regulate Rho GTPases. These voids in the field Related research article Golding AE, Visco exist partly because currently available probes I, Bieling P, Bement WM. 2019. Extraction only track active GTPases. Now, in eLife, Adriana of active RhoGTPases by RhoGDI regulates Golding and William Bement (University of Wis- spatiotemporal patterning of RhoGTPases. consin-Madison) and Ilaria Visco and Peter Biel- eLife 8:e50471. DOI: 10.7554/eLife.50471 ing (Max Planck Institute of Molecular Physiology) report how they have adapted a strategy previously used to study Rho GTPases in yeast (Bendezu´ et al., 2015) and employed it to explore how GDI interacts with Rho GTPases ells are busy places that are constantly in the frog Xenopus laevis and in vitro responding to a variety of internal and (Golding et al., 2019). C external demands. Molecules that can Golding et al. used two labeling strategies to switch back and forth between two or more image two Rho GTPases (Cdc42 and RhoA) in states have an important role in helping cells vivo and in vitro. The first strategy consisted of inserting a green fluorescent protein into the respond to these demands. An important type middle of the Rho GTPases, so that it would not of molecular switch is the Rho family of GTPases: interfere with the N- or C-termini of the proteins, these proteins cycle between an active form (in which are important for activity and localization.
    [Show full text]
  • Rho Protein Gtpases and Their Interactions with Nfκb: Crossroads of Inflammation and Matrix Biology
    Biosci. Rep. (2014) / 34 / art:e00115 / doi 10.1042/BSR20140021 Rho protein GTPases and their interactions with NFκB: crossroads of inflammation and matrix biology Louis TONG*†‡§ and Vinay TERGAONKAR1 *Singapore National Eye Center, 11 Third Hospital Avenue, Singapore 168751 †Singapore Eye Research Institute, Singapore ‡Duke-NUS Graduate Medical School, Singapore Downloaded from http://portlandpress.com/bioscirep/article-pdf/34/3/e00115/475339/bsr034e115.pdf by guest on 01 October 2021 §Yong Loo Lin School of Medicine, National University of Singapore, Singapore Institute of Molecular and Cell Biology, A*Star Institute in Singapore, Singapore Synopsis The RhoGTPases, with RhoA, Cdc42 and Rac being major members, are a group of key ubiquitous proteins present in all eukaryotic organisms that subserve such important functions as cell migration, adhesion and differentiation. The NFκB (nuclear factor κB) is a family of constitutive and inducible transcription factors that through their diverse target genes, play a major role in processes such as cytokine expression, stress regulation, cell division and transformation. Research over the past decade has uncovered new molecular links between the RhoGTPases and the NFκB pathway, with the RhoGTPases playing a positive or negative regulatory role on NFκB activation depending on the context. The RhoA–NFκB interaction has been shown to be important in cytokine-activated NFκB processes, such as those induced by TNFα (tumour necrosis factor α). On the other hand, Rac is important for activating the NFκB response downstream of integrin activation, such as after phagocytosis. Specific residues of Rac1 are important for triggering NFκB activation, and mutations do obliterate this response. Other upstream triggers of the RhoGTPase–NFκB inter- actions include the suppressive p120 catenin, with implications for skin inflammation.
    [Show full text]
  • Regulation of Cell Polarity by Exocyst-Mediated Trafficking
    Downloaded from http://cshperspectives.cshlp.org/ on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Regulation of Cell Polarity by Exocyst-Mediated Trafficking Noemi Polgar and Ben Fogelgren Department of Anatomy, Biochemistry and Physiology, John A. Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813 Correspondence: [email protected] One requirement for establishing polarity within a cell is the asymmetric trafficking of intra- cellular vesicles to the plasma membrane. This tightly regulated process creates spatial and temporal differences in both plasma membrane composition and the membrane-associated proteome. Asymmetric membrane trafficking is also a critical mechanism to regulate cell differentiation, signaling, and physiology. Many eukaryotic cell types use the eight-protein exocyst complex to orchestrate polarized vesicle trafficking to certain membrane locales. Members of the exocyst were originally discovered in yeast while screening for proteins required for the delivery of secretory vesicles to the budding daughter cell. The same eight exocyst genes are conserved in mammals, in which the specifics of exocyst-mediated traf- ficking are highly cell-type-dependent. Some exocyst members bind to certain Rab GTPases on intracellular vesicles, whereas others localize to the plasma membrane at the site of exocytosis. Assembly of the exocyst holocomplex is responsible for tethering these vesicles to the plasma membrane before their soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated exocytosis. In this review, we will focus on the role and regulation of the exocyst complex in targeted vesicular trafficking as related to the establish- ment and maintenance of cellular polarity. We will contrast exocyst function in apicobasal epithelial polarity versus front–back mesenchymal polarity, and the dynamic regulation of exocyst-mediated trafficking during cell phenotype transitions.
    [Show full text]
  • ARHGAP4 IS a SPATIALLY REGULATED RHOGAP THAT INHIBITS NIH/3T3 CELL MIGRATION and DENTATE GRANULE CELL AXON OUTGROWTH by DANIEL L
    ARHGAP4 IS A SPATIALLY REGULATED RHOGAP THAT INHIBITS NIH/3T3 CELL MIGRATION AND DENTATE GRANULE CELL AXON OUTGROWTH By DANIEL LEE VOGT Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Neuroscience CASE WESTERN RESERVE UNIVERSITY August, 2007 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the dissertation of Daniel Lee Vogt ______________________________________________________ candidate for the Ph.D. degree *. (signed) (chair of the committee)________________________________ Stefan Herlitze ________________________________________________Alfred Malouf Robert Miller ________________________________________________ ________________________________________________Thomas Egelhoff ________________________________________________Susann Brady-Kalnay ________________________________________________ (date) _______________________6-21-2007 *We also certify that written approval has been obtained for any proprietary material contained therein. ii Copyright © 2007 by Daniel Lee Vogt All rights reserved iii Table of contents Page # Title page i Table of contents iv List of figures vii Abstract 1 Chapter one: General introduction 2 Hippocampal axon pathways and development 3 Guidance cues in hippocampal axon outgrowth 6 Slit/Robo 7 Semaphorins, plexins and neuropilins 8 Ephrins and ephs 11 Other guidance cues in the hippocampus 13 GTPases: structure and function of ras superfamily members 15 Ras GTPases 17 Ran GTPases 18 Arf GTPases 18 Rab GTPases 19 Rho GTPases
    [Show full text]