The Rho GDI Rdi1 regulates Rho GTPases by distinct mechanisms

© 2008 by The American Society for Cell Biology. Under the License and Publishing Agreement, authors grant to the general public, effective two months after publication of (i.e.,. the appearance of) the edited manuscript in an online issue of MBoC, the nonexclusive right to copy, distribute, or display the manuscript subject to the terms of the Creative Commons–Noncommercial–Share Alike 3.0 Unported license (http://creativecommons.org/licenses/by-nc-sa/3.0).The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions, including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae, contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42 effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel mechanism, which includes the glycogen synthase kinase 3β homologue Ygk3, vacuolar proteases, and the proteasome. Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.Deutsche Forschungsgemeinschaf

IL-35-producing B cells are critical regulators of immunity during autoimmune and infectious diseases

International audienceB lymphocytes have critical roles as positive and negative regulators of immunity. Their inhibitory function has been associated primarily with interleukin 10 (IL-10) because B-cell-derived IL-10 can protect against autoimmune disease and increase susceptibility to pathogens. Here we identify IL-35-producing B cells as key players in the negative regulation of immunity. Mice in which only B cells did not express IL-35 lost their ability to recover from the T-cell-mediated demyelinating autoimmune disease experimental autoimmune encephalomyelitis (EAE). In contrast, these mice displayed a markedly improved resistance to infection with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium as shown by their superior containment of the bacterial growth and their prolonged survival after primary infection, and upon secondary challenge, compared to control mice. The increased immunity found in mice lacking IL-35 production by B cells was associated with a higher activation of macrophages and inflammatory T cells, as well as an increased function of B cells as antigen-presenting cells (APCs). During Salmonella infection, IL-35- and IL-10-producing B cells corresponded to two largely distinct sets of surface-IgM(+)CD138(hi)TACI(+)CXCR4(+)CD1d(int)Tim1(int) plasma cells expressing the transcription factor Blimp1 (also known as Prdm1). During EAE, CD138(+) plasma cells were also the main source of B-cell-derived IL-35 and IL-10. Collectively, our data show the importance of IL-35-producing B cells in regulation of immunity and highlight IL-35 production by B cells as a potential therapeutic target for autoimmune and infectious diseases. This study reveals the central role of activated B cells, particularly plasma cells, and their production of cytokines in the regulation of immune responses in health and disease

Charakterisierung von Orthopocken Ankyrin-Repeat-Proteinen im modifizierten Vaccinia Virus Ankara Hintergrund

Abstract In mammalian the ankyrin motif is one of the most common motifs which functions in protein protein interactions. Ankyrin repeat proteins (ARPs) are known as adaptors between the spectrin skeleton and integral membrane proteins. They are also known to be able to bind to proteins involved in apoptosis. However, within viruses ARPs are rather rare. One exception is the family of poxviruses. Proteins, which harbour an ankyrin motif encoded by poxviruses, were identified to influence the immune response via the NF-κB pathway, to play a role in apoptosis and serve as host range factors. The aim of this thesis was the characterization of cowpox (CPXV) ARPs in a complete ARP free virus environment. The poxvirus modified vaccinia virus Ankara (MVA) is well known as the prime vaccine against smallpox and was used in Germany in the 70´s. The virus is highly attenuated since it lost about 15 % of its parental genome in the process of passaging on chicken embryo fibroblasts (CEFs). Therefore, only one published ARP is left in its genome. The attenuation comes along with the description of MVA as host restricted to baby hamster kidney (BHK) cells and CEFs. Consequently, this virus is a convenient model to study the host range function of proteins. We identified an additional still unknown ARP encoding open reading frame (ORF) in MVA. One after the other the ORFs were deleted in a bacterial artificial chromosome (BAC) of the MVA genome via en passant mutagenesis, thus allowing the generation of a complete ARP free virus. After virus reconstitution, the generated deletion viruses were characterized in terms of their growth properties and ability to spread from cell-to-cell. It could be shown that the deletion lead to a defect in cell-to-cell spread but virus replication does not seem to be affected. Using the deletion mutants as a backbone various ARP encoding ORFs from CPXV were inserted. The generated knock-in mutants were tested on a representative number of different cell lines. Thereby, three new cell lines come to be known as non-permissive for MVA. Furthermore, a host range factor for RK13 cells could be identified and characterized concerning growth properties and cell-to-cell spread ability. Additionally, a selection of knock-in mutants was examined regarding the influence in apoptosis and in the NF-κB pathway. It was published that poxvirus proteins are able to change the formation of pocks on chorion allantoic membranes (CAMs). Hence, the development of pocks on the CAM of embryonated chicken eggs was also monitored. The results of this work contribute to of the potential application of MVA for the development of new vaccines and to a better understanding of poxvirus ARPs concerning host range and virus behaviour in general.Zusammenfassung Im Säuger ist das Ankyrin-Motiv eines der häufigsten Motive, das Protein-Protein Interaktionen vermittelt. Ankyrin-Repeat-Proteine (ARPs) sind bekannt als Adaptoren zwischen dem Spektrin Zytoskelett und integralen Membranproteinen und binden ebenfalls an Proteine, die in der Apoptose involviert sind. In Viren sind ARPs eher selten. Eine Ausnahme stellt die Familie der Pockenviren dar. Für Proteine, die das Ankyrin-Motiv enthalten und von Pockenviren kodiert werden, wurde gezeigt, dass sie die Immunantwort über den NF-κB Weg beeinflussen, eine Rolle in der Apoptose spielen und als host range Faktoren fungieren. Das Ziel dieser Arbeit war die Charakterisierung von Kuhpocken ARPs in einem komplett ARP freien Virus Umfeld. Das modifizierte Vaccinia Virus Ankara (MVA), bekannt als Prime-Vaccine gegen die Pockenerkrankung, wurde in Deutschland in den späten 70igern benutzt. Das Virus ist stark geschwächt, da es ungefähr 15 % seines Ursprungsgenoms bei der Passagierung auf Hühnerembryofibroblasten (CEFs) verloren hat. Daher ist nur ein ARP in seinem Genom übriggeblieben und veröffentlicht. Die Abschwächung geht einher mit der Beschreibung von MVA als wirtseingeschränkt auf Baby- Hamster-Nieren (BHK) Zellen und CEFs. Folglich ist dieses Virus ein geeignetes Modell, um die Wirtsspezifität von Proteinen zu untersuchen. Wir identifizierten einen weiteren noch unbekannten ARP kodierenden offenen Leserahmen (ORF). Ein ORF nach dem anderen wurde in einem künstlichen bakteriellen Chromosom (BAC) des MVA Genoms mittels en passant Mutagenese deletiert, um ein komplett ARP freies Virus zu generieren. Nach der Rekonstitution der Viren wurden die generierten Deletionsmutanten bezüglich ihrer Wachstumseigenschaften und der Fähigkeit sich von Zelle zu Zelle auszubreiten untersucht. Es konnte gezeigt werden, dass die Deletion zu einem Defekt in der Ausbreitung von Zelle zu Zelle führt, wohingegen die Virusreplikation nicht beeinträchtigt zu sein scheint. In die Deletionsmutanten wurden unterschiedliche Kuhpocken ARP kodierende ORFs insertiert. Die generierten knock-in Mutanten wurden auf einer representativen Anzahl unterschiedlicher Zelllinien getestet. Dadurch konnten drei neue Zelllinien als nicht-permissiv für MVA identifiziert werden. Außerdem wurde ein host range Faktor für RK13 Zellen identifiziert und bezüglich der Wachstumsfähigkeiten und der Ausbreitung von Zelle zu Zelle charakterisiert. Zusätzlich wurde eine Auswahl von knock-in Mutanten betreffend des Einflusses auf Apoptose und des NF-κB Weges charakterisiert. Es ist publiziert worden, dass Pockenvirenproteine in der Lage sind die Ausbildung von Pocken auf der Chorion-Allantois-Membran (CAM) zu verändern. Infolgedessen wurde die Entwicklung von Pocken auf CAMs von embryonierten Hühnereiern überprüft. Die Ergebnisse dieser Arbeit tragen zur möglichen Nutzung von MVA zur Entwicklung neuer Impfstoffe und zum besseren Verständnis von Pockenvirus ARPs bezüglich Wirtsspezifität und Virusverhalten im Allgemeinen bei

Mannose phosphorylation in health and disease

Kollmann K, Pohl S, Marschner K, et al. Mannose phosphorylation in health and disease. European Journal of Cell Biology. 2010;89(1):117-123

B cells producing type i IFN modulate macrophage polarization in tuberculosis

In addition to their well-known function as antibody-producing cells, B lymphocytes can markedly influence the course of infectious or noninfectious diseases via antibody-independent mechanisms. In tuberculosis (TB), B cells accumulate in lungs, yet their functional contribution to the host response remains poorly understooFil: Bénard, Alan. Centre National de la Recherche Scientifique; FranciaFil: Sakwa, Imme. Leibniz - Institute Of Freshwater Ecology And Inland Fisheries; AlemaniaFil: Schierloh, Luis Pablo. Instituto National de Recherches Agronomiques. Centre de Recherches de Toulouse; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Colom, André. Centre National de la Recherche Scientifique; FranciaFil: Mercier, Ingrid. Instituto National de Recherches Agronomiques. Centre de Recherches de Toulouse; FranciaFil: Tailleux, Ludovic. Instituto Pasteur; FranciaFil: Jouneau, Luc. Institut National de la Recherche Agronomique; FranciaFil: Boudinot, Pierre. Institut National de la Recherche Agronomique; FranciaFil: Al Saati, Talal. Université Paul Sabatier; FranciaFil: Lang, Roland. Universitat Erlangen-Nuremberg; AlemaniaFil: Rehwinkel, Jan. University of Oxford; Reino UnidoFil: Loxton, Andre G.. Stellenbosch University; SudáfricaFil: Kaufmann, Stefan H. E.. Max Planck Institut für Infektionsbiologie; AlemaniaFil: Anton Leberre, Veronique. Institut National de la Recherche Agronomique; FranciaFil: O'Garra, Anne. National Institute for Medical Research; Reino UnidoFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Gicquel, Brigitte. Institut National de la Recherche Agronomique; FranciaFil: Fillatreau, Simon. Centre National de la Recherche Scientifique; FranciaFil: Neyrolles, Olivier. Centre National de la Recherche Scientifique; FranciaFil: Hudrisier, Denis. Centre National de la Recherche Scientifique; Franci