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Buchta, Claire Marie. "Mechanisms of TLR signaling and cooperation in B lymphocytes." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4584.
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B lymphocytes play important roles in antibody production, cytokine production, and antigen presentation to T cells. Ligation of Toll-like receptors (TLRs) on B cells stimulates cellular activation and B cell effector functions. Synergistic activation of other receptors such as CD40 or the B cell receptor (BCR) with TLR ligation further enhances B cell activation and effector functions. The tumor necrosis factor receptor associated factor (TRAF) family of proteins act as cytoplasmic signaling adaptor molecules and moderate downstream signaling from both the tumor necrosis factor receptor (TNFR) superfamily of proteins, including CD40, and the IL-1R/TLR superfamily of proteins.
To date, only TRAFs 3 and 6 have been shown to be involved in TLR signaling, with TRAF6 providing positive regulation and TRAF3 providing negative regulation of TLR signaling in B cells. Deficiency in another TRAF family member, TRAF5, has been implicated in the development of atherosclerosis, a disease developed in part due to TLR dysregulation. Here, we addressed the hypothesis that TRAF5 is a negative regulator of TLR signaling.
We found that TRAF5 negatively regulated TLR-mediated cytokine and antibody production in B lymphocytes. The enhanced cytokine production seen in TLR-stimulated TRAF5 KO B cells was not attributable to altered cellular survival or proliferation, but instead more cytokine was produced on a per-cell basis, likely due to enhanced MAPK pathways after TLR ligation. Additionally, TRAF5 deficiency did not dramatically affect cytokine production in TLR-stimulated bone marrow-derived macrophages or dendritic cells, suggesting that TRAF5 plays a greater role in TLR signaling in lymphoid versus myeloid cells. TRAF5 associated with the TLR signaling proteins MyD88 and TAB2, and negatively regulated the association of TAB2 with its binding partner TRAF6.
Furthermore, we manipulated B cell activation via ligation of various TLRs, CD40, and/or the BCR in order to activate the cells to effectively present antigen. Activated B cells pulsed with antigen served as an effective cellular vaccine and offered protection against both an infectious pathogen (Listeria monocytogenes) and a model of murine melanoma. We identified two candidate activation criteria for B cell vaccines (Bvacs): stimulation through the BCR and TLR7, and stimulation through CD40 and TLR4. Additionally, we found that high IL-6 production by the activated Bvac was essential for inducing optimal CD8+ T cell memory. These B cell activation protocols offer significant advantages over those currently being tested for clinical use. Understanding B cell activation through TLRs is a critical step in developing new therapies against cancer and infectious disease.
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Wan, Youzhong. "THE FUNCTION OF INTERLEUKIN-1 RECEPTOR ASSOCIATED KINASE 2 IN TOLL-LIKE RECEPTOR-MEDIATED SIGNALING." Case Western Reserve University School of Graduate Studies / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=case1278430683.
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Kim, Tae Whan. "The role of kinase activity of IRAK₄ in Tlr/il-1r mediated signaling." Cleveland, Ohio : Case Western Reserve University, 2008. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1228500771.
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Kim, Tae Whan. "THE ROLE OF KINASE ACTIVITY OF IRAK4 IN TLR/IL-1R-MEDIATED SIGNALING." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1228500771.
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Lai, Jen-Feng. "The essential role of macrophages and TLR signaling in the host response to Mycoplasma pneumoniae." Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2009. https://www.mhsl.uab.edu/dt/2010r/lai.pdf.
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Lampropoulou, Vasiliki [Verfasser], and Roland [Akademischer Betreuer] Lauster. "TLR/MyD88 signaling in B cells suppresses T cell-mediated CNS autoimmunity / Vasiliki Lampropoulou. Betreuer: Roland Lauster." Berlin : Universitätsbibliothek der Technischen Universität Berlin, 2012. http://d-nb.info/1021976601/34.
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Huang, Xuesong. "The Study on Signal Mechanism of Protein Kinase C zeta-involved NF-κB Activation in LPS-stimulated TLR4 Signaling Pathways." University of Toledo Health Science Campus / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=mco1193663177.
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Vaughan, Tamisha Y. "Novel Mechanisms Underlying the Inflammatory Effects of Leptin and Low Dose Endotoxin." Diss., Virginia Tech, 2010. http://hdl.handle.net/10919/28013.
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Obesity over the last several has become a major health concern in our country as well as the world. Obesity is also one of the risk factors which lead to several inflammatory complications such as diabetes, artherosclerosis, etc. Two leading factors involved in the causes of inflammatory complications include leptin and low dose endotoxin lipopolysaccharide (LPS). However, the mechanism underlying the involvement of these two mediators is not clearly understood. The purpose of this study is to understand the mechanism underlying inflammatory complications caused by leptin and low dose endotoxin most recently coined metabolic endotoxemia. Interleukin-Receptor Associated Kinase 1 (IRAK-1) is an intracellular signaling component shown to activate NFκB which leads to the induction of proinflammatory mediators. Deletion of IRAK-1 in mice has beneficial effects in alleviating inflammatory complications and human variations in IRAK-1 gene are correlated with higher risks for inflammatory diseases. Therefore, we hypothesized that IRAK-1 is critically involved for the induction of proinflammatory mediators induced by leptin and low dose LPS. IL-6 mRNA levels were measured in THP-1 (human monocytic cells) and wild type and IRAK-deficient bone marrow derived macrophages (BMDM) challenged with different combinations of leptin and LPS. Data shows that leptin alone will not induce inflammatory mediators. However, increased induction of IL-6 was observed in a synergistic manner involving both LPS and leptin in an IRAK-1 dependent manner causing a robust inflammatory response. With regard to the effect of low dose LPS, we observed that human monocytic cells treated with low concentrations of LPS showed a mild yet sustained induction of proinflammatory cytokines, which is contrast to the robust and transient induction of cytokines by a high dose LPS. To further determine the molecular mechanisms, we measured several key signaling molecules that include IRAK-1, IKKepsilon, and C/EBPdelta. Our study revealed a novel mechanism that appears to be distinct from the traditional NFï «B pathway responsible for the effect of low dose LPS.Ph. D.
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Campbell, Sara J. "Mechanisms of Moraxella catarrhalis Induced Immune Signaling in the Pulmonary Epithelium." University of Toledo Health Science Campus / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=mco1268141520.
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Faria, Camila Cristina Quinello Gomes de. "Avaliação da resposta imune após estimulação de monócitos via Toll-Like Receptor 2 (TLR-2) em recém-nascidos a termo e pré-termo." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/5/5141/tde-04022014-105154/.
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O sistema imune neonatal tem sido considerado funcionalmente imaturo e recentes estudos sugerem que a suscetibilidade do neonato às infecções pode ser devido a alterações funcionais de células apresentadoras de antígenos que podem levar a deficiências secundárias nas respostas adaptativas. A ativação das células apresentadoras de antígenos é desencadeada pela estimulação de receptores, como os Toll-like Receptors (TLRs) e alterações na ativação desses receptores podem levar a uma subsequente redução da ativação de proteínas da via de sinalização intracelular e consequente alterações dos níveis das citocinas pró- e anti-inflamatórias, contribuindo assim, para uma resposta imune ineficiente do neonato. O Toll-like receptor 2 (TLR-2) é um receptor essencial para o reconhecimento seletivo de vários antígenos bacterianos e virais, em especial, o peptideoglicano, que compreende cerca de 50% da parede celular de bactérias Grampositivas, como os estafilococos, que são agentes infecciosos que prevalecem nas Unidades de Terapia Intensiva Neonatal. O objetivo deste estudo foi avaliar a ativação e resposta de monócitos de sangue do cordão umbilical de recém-nascidos pré-termo saudáveis < 34 semanas de gestação (Grupo 1), recém-nascidos pré-termo : 34 e < 37 semanas de gestação (Grupo 2) e recém-nascidos a termo (Grupo 3) e de adultos saudáveis, como controles, após a estimulação de TLR-2 ex-vivo com Pam3CSK4. Após a estimulação dos monócitos, foram determinados os níveis de expressão dos marcadores de ativação celular, os níveis das citocinas pró- e anti-inflamatórias e a expressão de moléculas envolvidas na sinalização intracelular. A caracterização das populações leucocitárias, bem como a capacidade fagocítica de Staphylococcus aureus e geração de burst oxidativo por monócitos e neutrófilos foram analisados por Citometria de Fluxo. Os resultados demonstraram que as células dendríticas e monócitos de neonatos expressam TLR-2 em níveis semelhantes aos de adultos. A expressão adequada de TLR-2 sugere um reconhecimento antigênico eficiente que é refletido em uma ativação apropriada das moléculas da cascata de sinalização e uma potente produção de citocinas pró-inflamatórias, apesar da reduzida produção de IL-10. Fagócitos neonatais apresentaram capacidade fagocítica de S. aureus reduzida em relação aos adultos e geração do burst oxidativo semelhante entre os grupos, no entanto neonatos prétermo apresentaram produção de peróxido de hidrogênio deficiente, o que poderia contribuir com uma reduzida morte intracelular deste microrganismo. Em conclusão, o recém-nascido não apresenta uma imaturidade funcional, mas sim, um desequilíbrio em sua resposta imune inata, com uma aparente menor produção de fatores antiinflamatórios, o que pode levar a predisposição à sepseThe neonatal immune system has been considered functionally immature and recent studies suggest that susceptibility of the neonate to infections may be due to functional alterations in antigen-presenting cells that can prompt to secondary deficiencies in adaptive responses. The activation of antigen-presenting cells is triggered by stimulation of receptors such as Toll-like receptors (TLRs) and changes in the activation of these receptors may lead to a subsequent reduction in the activation of intracellular signaling pathway proteins and consequent changes in pro- and anti-inflammatory cytokine levels, thus contributing to an inefficient immune response of the neonate. Toll-like Receptor 2 (TLR-2) is an essential receptor for the selective recognition of several bacterial and viral antigens, in particular, peptidoglycan, which comprises about 50% of the Gram-positive bacteria cell wall, such as staphylococci, which are infectious agents that prevail in Neonatal Intensive Care Units. The aim of this study was to evaluate the activation and response of monocytes derived from umbilical cord blood of healthy preterm newborns <34 weeks of gestation (Group 1), preterm newborns :34 and <37 weeks of gestation (Group 2) and term newborns (Group 3) and from healthy adults, as controls, after ex-vivo TLR-2 stimulation with Pam3CSK4. After monocyte stimulation, it was determined the expression levels of cellular activation markers, pro- and anti-inflammatory cytokine levels and the expression of molecules involved in downstream intracellular signaling. The characterization of leukocyte populations, as well as the phagocytic ability of Staphylococcus aureus and generation of oxidative burst by monocytes and neutrophils were analyzed by flow cytometry. The results demonstrated that neonatal dendritic cells and monocytes express TLR- 2 at similar levels to those of adults. The proper expression of TLR-2 suggests an efficient antigen recognition which is reflected in an appropriate activation of downstream signaling molecules and potent production of pro-inflammatory cytokines, in spite of the reduced production of IL-10. Neonatal phagocytes showed reduced phagocytic capacity of S. aureus compared to adults and similar generation of oxidative burst between groups, however preterm neonates showed deficient production of hydrogen peroxide, which could contribute to a reduced intracellular killing of this microorganism. In conclusion, the newborn does not present a functional immaturity, but an imbalance in its innate immune response, with an apparent lower production of antiinflammatory factors, which can lead to a predisposition to sepsis
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Busca, Aurelia. "Antiapoptotic Proteins in Human Macrophage Survival, Differentiation, Innate Immunity and Protection from HIV-induced Apoptosis." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/23989.
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Macrophages represent long lived immune cells that are remarkably resistant to apoptosis, which allows them to perform in highly stressful environments. Apoptosis resistance is a characteristic that develops during the differentiation process from monocytes to macrophages. However, the signaling pathways that mediate the development of macrophage antiapoptotic phenotype during differentiation remain mostly unknown. Because of their decreased susceptibility to cell death, macrophages are also key viral reservoirs during HIV infection. My research aims to understand the molecular mechanisms and signaling pathways that mediate cell survival during and after monocyte to macrophage differentiation and the involvement of the main families of antiapoptotic proteins, IAPs (inhibitors of apoptosis) and Bcl2 in this process. HIV accessory protein Vpr was used as an apoptotic stimulus, due to its death inducing abilities in other cell types.
My results show that survival of macrophages is distinctively regulated during and after differentiation. I have identified a signaling pathway consisting of PI3K/Akt activation of NFκB that is important in survival of differentiating macrophages by specifically sustaining antiapoptotic Bcl-xL expression. However, once differentiated, Mcl-1, but not Bcl-xL is dependent on PI3K/Akt activation. Moreover, differentiated macrophages are resistant to the effect of HIV-Vpr, which is highly apoptotic for monocytes. In contrast, resistance to HIV-Vpr induced apoptosis of human macrophages is specifically mediated by antiapoptotic IAP proteins, with no involvement of the Bcl2 family, which maintains macrophage viability in the absence of any apoptotic stimuli.
In addition to their antiapoptotic properties, IAPs are also important regulators of macrophage function. By using chemical compounds (SMAC mimetics) that target IAPs for degradation, I have shown that IAPs positively modulate LPS-induced IL10, IL-27 and MIG (monokine induced by IFNγ) production in human macrophages, by promoting TRAF2, JNK and p38 signaling and NFκB activation. In addition, IAPs also contribute to LPS-induction of CD80/CD86 costimulatory molecules.
Overall, my results suggest that both IAPs and Bcl2 families contribute to survival of human macrophages and that IAPs are also involved in innate immune responses. Unraveling the mechanisms that control macrophage survival and function in various settings would provide therapeutic strategies aimed at eliminating cells when their survival is no longer beneficial for the host, as in the case of HIV infection or autoimmune diseases.
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Singh, J. C. I., S. M. Cruickshank, D. J. Newton, L. Wakenshaw, Anne M. Graham, J. Lan, J. P. A. Lodge, P. J. Felsburg, and S. R. Carding. "Toll-like receptor-mediated responses of primary intestinal epithelial cells during the development of colitis." The American Physiological Society, 2004. http://hdl.handle.net/10454/4048.
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noThe interleukin-2-deficient (IL-2¿/¿) mouse model of ulcerative colitis was used to test the hypothesis that colonic epithelial cells (CEC) directly respond to bacterial antigens and that alterations in Toll-like receptor (TLR)-mediated signaling may occur during the development of colitis. TLR expression and activation of TLR-mediated signaling pathways in primary CEC of healthy animals was compared with CEC in IL-2¿/¿ mice during the development of colitis. In healthy animals, CEC expressed functional TLR, and in response to the TLR4 ligand LPS, proliferated and secreted the cytokines IL-6 and monocyte chemoattractant protein-1 (MCP-1). However, the TLR-responsiveness of CEC in IL-2¿/¿ mice was different with decreased TLR4 responsiveness and augmented TLR2 responses that result in IL-6 and MCP-1 secretion. TLR signaling in CEC did not involve NF-B (p65) activation with the inhibitory p50 form of NF-B predominating in CEC in both the healthy and inflamed colon. Development of colitis was, however, associated with the activation of MAPK family members and upregulation of MyD88-independent signaling pathways characterized by increased caspase-1 activity and IL-18 production. These findings identify changes in TLR expression and signaling during the development of colitis that may contribute to changes in the host response to bacterial antigens seen in colitis.
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Komegae, Evilin Naname. "Papel dos receptores inatos TLR na formação de memória humoral e linfócitos B de longa vida: ação das proteases natterinas, toxinas majoritárias do veneno de Thalassophryne nattereri." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/42/42133/tde-01102010-120643/.
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A contribuição de células B para a memória imunológica se dá por duas distintas populações: células B de memória e células produtoras de anticorpos de longa vida (ASC). A inter-relação entre estas células bem como os mecanismos envolvidos para a manutenção destas tem sido pouco entendida. O veneno de Thalassophryne nattereri tem se mostrado capaz de induzir uma intensa resposta imune de memória. Nós avaliamos o efeito das Natterinas, que são as toxinas majoritárias e inéditas, na indução e manutenção da resposta imune de memória de células B. Este estudo, além de permitir um maior esclarecimento da resposta humoral de memória induzida pelo veneno do peixe T. nattereri, permitiu o estudo da complexa organização do compartimento de células B de memória e ASCs. Também evidenciamos a importância da atividade proteásica para a manutenção da cronicidade de resposta de células B no peritônio, no baço e na medula, como verificamos que a ativação de receptores inatos como osTLRs é decisiva para a geração e manutenção de ASCs B220pos/neg em resposta às Natterinas, dependentes das vias de sinalização MyD88 ou TRIF. Estas sinalizações controlam a magnitude, a qualidade e a longa duração da resposta humoral de memória.The contribution of B cells for the immunological memory feels for two different populations: memory B cells and long-lived antibodies secreting cells (ASC). The interrelation among these cells as well as the mechanisms involved for the maintenance of these it has been little understood. The venom of Thalassophryne nattereri possesses the ability to induce an intense memory immune response. We evaluated the effect of Natterins that are majority toxins in the venom, in the induction and maintenance of the immune memory response of cells B. The study, besides allowing a larger explanation of the humoral memory response induced by the venom of the fish, it allowed the understanding of the complex organization of the memory B cells compartment, mainly of the subtype of long-lived cells (ASC). Also, we showed the importance of the protease activity of Natterins in the maintenance of the chronic B cell responses in the three analyzed compartments. We verify that the activation of Toll like receptors is decisive for the generation and maintenance of ASCs B220pos/neg in response to Natterins, dependent on the MyD88 or TRIF signaling that control the quality and the duration of the humoral memory response.
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Liljeroos, M. (Mari). "Toll-like receptor 2 (TLR2) and TLR4 signaling in the innate response against bacterial components." Doctoral thesis, University of Oulu, 2008. http://urn.fi/urn:isbn:9789514288111.
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Abstract Toll-like receptors (TLRs) are transmembrane proteins involved in the recognition of specific microbial structures and thus the activation of signaling cascades of innate immunity. Regulation of the innate immune response is a complex biological process involving the combined synergistic and antagonistic effects of distinct signaling mediators. Although TLR signaling has been widely studied in recent years, there remain many unexplored unique features of each TLR signaling pathway. The present study evaluated the activation and regulation of TLR4 and TLR2 signaling with the aim of better understanding the molecular mechanisms that control these inflammatory signaling pathways. In the present study, the signal transduction mechanisms of TLR4 and TLR2 in response to Escherichia coli LPS and Staphylococcus aureus LTA were evaluated in mouse macrophages. The inductions, interactions, and activations of the signaling molecules and mediators in the TLR pathways were studied by using several molecular biology and protein chemistry methods. In addition, the role of TLR4 and TLR2 in the regulation of the hepatic inflammatory reaction during endotoxemia was studied. Mouse macrophages were found to induce central proinflammatory mediators in response to LPS and LTA stimulation. Specific roles for PI 3-kinase and Btk were described. These kinases were found to be activated by LPS and LTA; moreover, PI 3-kinase and Btk were found to form specific interactions with TLRs and their intracellular signaling mediators. In addition, a unique IRF2 signaling pathway for LTA-induced TLR2 was found, resulting in the activation of signal transducers and activators of transcription (Stats) and IFN-α secretion. The secreted IFN-α was shown to regulate the LTA-induced inflammatory responses, thereby combining the LTA-induced IRF proteins into NF-κB pathway. The present study provides insight into the signal transduction mechanisms of TLRs. The understanding of these molecular mechanisms that control the activation of TLR signaling cascades will in the future help to predict predisposition and outcome in infectious diseases, and to control the course of disease at an earlier stageTiivistelmä Toll:n kaltaiset reseptorit (TLR) ovat solukalvon proteiineja, jotka tunnistavat taudinaiheuttajien eli patogeenien spesifisiä rakenteita johtaen elimistön puolustusjärjestelmän, immuniteetin, aktivoitumiseen. Immuniteetin säätely on monimutkainen biologinen prosessi, joka tapahtuu kudosten, solujen ja erilaisten synnynnäiseen immuniteettiin liittyvien molekyylien vuorovaikutuksina. Tulehdusvasteen säätelyssä tasapaino positiivisten ja negatiivisten säätelysignaalien välillä on erittäin tärkeää, jotta autoimmuunisairauksien, akuuttien tai kroonisten tulehdusten sekä infektiosairauksien synty voitaisiin välttää. Tämän tutkimuksen tavoitteena oli saada lisätietoa TLR2 ja TLR4 proteiinien säätelemistä signaalireiteistä, niiden vasteista tiettyjä patogeenirakenteita vastaan ja ymmärtää paremmin synnynnäisen immuniteetin puolustusmekanismeja. Patogeenirakenteiden aiheuttamaa tulehdusvastetta tutkittiin pääosin soluviljelymallissa. Lisäksi selvitettiin immuunivasteen luonnetta fysiologisessa kokonaisuudessa ja sen korrelaatiota solutasolla nähtyihin vasteisiin käyttäen in vivo hiirimallia. Tutkimus tehtiin käyttäen useita molekyylibiologian ja proteiinikemian menetelmiä proteiini- ja mRNA-ekspressioiden sekä proteiini-interaktioiden tutkimiseen ja erilaisten aktiivisuuksien määrityksiin. Tulehdusvastetta tutkittiin etenkin sytokiinivastetta määrittämällä ja signaaliketjujen toimintaa analysoitiin estämällä spesifisesti niiden toimintaa. Tarkoituksena oli selvittää, mitkä tekijät ovat välttämättömiä kyseisten tulehdusta aiheuttavien bakteerien tunnistuksessa ja puolustusreaktiossa niitä vastaan. Tutkimuksessa havaittiin kahden kinaasin, PI 3-kinaasin ja Brutonin tyrosiinikinaasin, liittyvän oleellisesti TLR signaalireitteihin. Nämä TLR:ien stimulaation seurauksena aktivoituneet kinaasit muodostivat spesifisiä sidoksia TLR:ien ja niiden signaaliketjuihin liittyvien solunsisäisten signaalivälittäjien kanssa. Lisäksi TLR2 signaalireitillä havaittiin aktivoituvan tekijöitä, jotka johtivat interferoni-α välitteiseen tulehdusvasteen säätelyyn. TLR signaalireittien selvittäminen auttaa ymmärtämään tulehdussairauksien patofysiologiaa ja voi siten tulevaisuudessa johtaa parempien hoitomenetelmien kehittämiseen
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Lundberg, Anna Maria Cecilia. "Investigation of TLR signalling pathways in human primary cells." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.425006.
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Macleod, Charlotte Victoria. "Investigating TLR-4 signalling in response to protein ligands." Thesis, University of Cambridge, 2018. https://www.repository.cam.ac.uk/handle/1810/274540.
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Toll-like receptor (TLR)-4 is a pattern recognition receptor (PRR) that recognises the pathogen-associated molecular pattern (PAMP) lipopolysaccharide (LPS) produced by Gram-negative bacteria. LPS binds to Myeloid differentiation 2 (MD-2)/TLR-4 heterodimers, driving their dimerisation and inducing a conformational change of the intracellular TLR-4 toll/interleukin-1 receptor (TIR) domains. The adaptor protein Myeloid differentiation primary response gene 88 (MyD88)-adaptor-like (Mal)/TIR domain-containing adaptor protein (TIRAP) then binds to the TIR domains of TLR-4 and acts as a bridge for MyD88 which goes on to form the myddosome, a large protein complex of six to eight MyD88 molecules and four Interleukin-1 receptor- associated kinase (IRAK) 4 and four IRAK1/2 molecules. This triggers a signalling cascade which results in nuclear factor (NF)-κB transcription factor activation and production of pro-inflammatory effector molecules such as the cytokine Tumour Necrosis Factor (TNF)-α. Upon activation TLR-4 is also endocytosed where it interacts with a second set of adaptor proteins TIR-domain-containing adaptor- inducing interferon (IFN)-β (TRIF)-related adaptor molecule (TRAM) and TRIF to initiate the type I IFN response. How TLR-4 dimerisation results in the formation of the oligomeric myddosome is not fully understood, but it is possible that the stoichiometry of Mal/TIRAP may be important in the formation of this protein complex. The aim of my thesis was to determine the stoichiometry of Mal/TIRAP at the plasma membrane of immortalised bone marrow derived macrophages (iBMDMs) and whether this stoichiometry changes upon stimulation with different TLR-4 ligands. To investigate Mal/TIRAP stoichiometry I first developed a viral transduction experimental cell model to visualise fluorescently labelled Mal/TIRAP. Mal/TIRAP-/- iBMDMs were lentivirally transduced with a Mal/TIRAPHALO construct. The halotag was fluorescently labelled then the cells were stimulated with TLR-4 ligands, such as LPS, fixed at different time points, then imaged. Total internal reflection fluorescence (TIRF) microscopy was used to image the plasma membrane and photobleaching experiments performed to determine Mal/TIRAP stoichiometry. I developed a computer-based analysis pipeline to analyse the resulting photobleaching data. Under resting conditions, Mal/TIRAP is present at the plasma membrane in clusters of approximately ten Mal/TIRAP molecules per cluster. After five minutes of stimulation with 10 ng/ml LPS Mal/TIRAP redistributes into cluster sizes of approximately six, twelve and much larger. After ten and fifteen minutes stimulation with 10 ng/ml LPS the clusters return to the resting size of approximately ten Mal/TIRAP molecules per cluster with a few much larger clusters remaining present. This confirms the rapid time frame within which TLR-4 signalling occurs at the plasma membrane and is consistent with myddosome stoichiometry of six MyD88 molecules or proposed super myddosomes of twelve MyD88 molecules. The computer-based analysis pipeline developed can be used to analyse any protein of interest at the plasma membrane. Protein ligands have also been found to activate TLR-4; for example allergens, such as Fel d 1 and Der p 2, as well as endogenous damage associated molecular patterns (DAMPs), such as extracellular matrix (ECM) proteins, for example fragments of fibronectin and tenascin-C. The mechanism by which these proteins interact with TLR-4 and induce signalling is unclear. Proteins from the ECM (fragments FNIII1c, FNIII13-14, FNIII9-E and FNIII9-E-14 from fibronectin and the fibrinogen-like globe (FBG) domain of tenascin-C) were tested using a transient transfection assay in HEK293 cells and shown to activate TLR-4. In conclusion, I have developed new tools and methodology to investigate how TLR-4 signals in response to LPS and DAMPs in living cells. Whether DAMP- activated TLR-4 forms similar signalling complexes to those induced by LPS will form part of a future study.
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Kreem, Shrook Abdulla. "Immunomodulatory effects of progesterone on TLR3 and TLR4 mediated signalling in macrophages and dendritic cells." Thesis, University of Strathclyde, 2013. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=18982.
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Sexual dimorphism in the immune response is at least in part related to the effects of sex hormones. The current studies investigate the ability of progesterone to manipulate signalling events in macrophages and dendritic cells following TLR3 or TLR4 ligation. Progesterone can mediate its action by binding both progesterone and glucocorticoid receptors. To differentiate these effects, norgestrel, a synthetic progesterone specific receptor agonist and dexamethasone a glucocorticoid receptor agonist were utilised. Progesterone and norgestrel, but not dexmethasone increased poly-riboinosinic-ribocytidylic acid (polyI:C), a TLR3 agonist, induced interferon regulatory factor 3 (IRF3) phosphorylation in BMDCs, suggesting that it may be mediated by progesterone receptor. Subsequent experiments, showed inhibitory effects of both progesterone and norgestrel on numerous elements downstream in the TLR3 and TLR4 signalling pathways, specifically at the transcriptional level represented by inhib ition of polyI:C-induced p65 and IRF3 nuclear translocation. Progesterone also inhibited LPS-induced IRF3 nuclear translocation. Progesterone and norgestrel inhibited LPS- and polyI:C-induced ISRE DNA-binding activity and may contribute to modulation of specific target gene promoters bearingISRE consensus elements including IL-12, IL-27 and IFNβ. The effects of progesterone were evident at the level of transcription. Thus, polyI:C-induced IL-12p35, EBI3 and IFNβ mRNAexpression were inhibited by progesterone and norgestrel and LPS-induced IFNβ mRNA expression was inhibited by progesterone. Progesterone inhibited LPS- and polyI:C-induced IL-6, but not norgestrel. However, IL-12p70 production was reduced by both progesterone and norgestrel following LPS and polyI:C stimulation. These results indicate that progesterone selectively functions as an immunomodulatory agent through both progesterone and glucocorticoid receptors. Progesterone potentially has a greater ability to affect TLR3 rather than TLR4 signalling. These results could have therapeutic implications for control of the innate immune response, inflammation and infectious diseases.
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Mariathasan, Sanjeev. "TCR-mediated signaling in thymocyte selection." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2001. http://www.collectionscanada.ca/obj/s4/f2/dsk3/ftp05/NQ63683.pdf.
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Rivera, Reyes Brenda Mariola. "Regulation of the TCR signaling pathway." Connect to text online, 2006. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1132588714.
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Mallaun, Michel. "Proximal TCR signaling in self tolerance /." [S.l.] : [s.n.], 2008. http://edoc.unibas.ch/diss/DissB_8729.
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Groves, Tim C. "Pre-TCR and TCR-Ãß signaling during T cell development." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ27657.pdf.
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Carlsson, Emil Karl Viktor. "Biochemical, molecular and cellular studies on negative regulators of TLR-signalling." Thesis, Imperial College London, 2015. http://hdl.handle.net/10044/1/38528.
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Toll-like receptors (TLRs) recognise pathogenic microorganisms through conserved pathogen associated molecular patterns, which activates the innate immune response. TLR signalling is mediated by cytoplasmic adaptor proteins via Toll/interleukin-1 receptor (TIR) domains. Sterile α- and armadillo-motif-containing protein (SARM) is the fifth TLR adaptor protein identified in humans and has been described as a negative regulator of the innate immune response. Several pathogenic bacteria are also known to express proteins with TIR- domains, which are believed to be involved in disruption of TLR signalling. This raises the question of whether SARM functions in a similar manner, as phylogenetic studies have shown that SARM is closely related to bacterial proteins. In this project, functional characterisation of SARM and a bacterial TIR domain protein from Bacillus anthracis (BaTdp) have been performed using both recombinantly expressed and purified proteins, as well as cellular assays. The TIR domains of both SARM and BaTdp were found to form heterotypic TIR-TIR interactions with multiple human TLR adaptors, including Myeloid differentiation factor 88 (MyD88). SARM and MyD88 both localised to mitochondria when overexpressed in mammalian cells, and SARM overexpression was associated with a reduction of TLR2-, TLR4- and MyD88- induced cytokine activation. A single amino acid residue in the SARM BB-loop motif, G601, was also identified as being critical for SARM's anti-inflammatory effect. A short peptide derived from this motif was able to target MyD88 in vitro and slightly reduce TLR4-mediated cytokine activation. Overexpression of BaTdp in mammalian cells had no significant effect on TLR-mediated cytokine activation. Instead, the protein targeted microtubular networks in the cell and BaTdp expression was associated with a significant increase in cellular autophagy activity. The findings further enhance our understanding of the underlying mechanisms by which SARM suppress the innate immune response, and also describe previously unknown functions of BaTdp.
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Pechenick, Jowers Tali. "Investigation of murine cytomegalovirus modulation of TLR/IL-1β signalling pathways." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6478.
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Cytomegaloviruses (CMV), the prototypical β-herpesviruses, have co-evolved with their hosts and thus acquired multiple strategies for modulation of the immune response. Viral engagement of pattern recognition receptors (PRR), such as toll-like receptors (TLRs) and cytosolic nucleic acids sensors, initiates the host immune response through activation of elaborate signalling programs. The ensuing inflammatory response is further sustained and amplified through cytokines, such as IL-1β, activating signalling pathways greatly overlapping those utilized by TLRs. The central hypothesis of this thesis is that a viral counter-measure by murine CMV (MCMV) involves specific targeting of TLR- and IL-1β-induced signalling along the MyD88 to NF-κB pathway. To test this hypothesis MCMV inhibition of IL-1β signalling was initially investigated in a fibroblast cell line. It was demonstrated that in MCMV infected cells IL-1β-induced IκBα degradation is largely inhibited. Comparison of productive and non-productive infection showed this modulation requires de-novo viral gene expression beyond the immediate early region. Further investigations utilising a ORF M45 deletion mutant identified viral gene M45 as necessary for mediating the observed modulation of IL-1β- induced IκBα degradation. To further test the hypothesis, studies were extended to include TLR stimulation in the context of bone marrow-derived macrophages (BMDM) infection. It was found that TLR7/9-induced NF-κB activation is inhibited in MCMV infected BMDM. Overall, data presented in this study demonstrate a previously unrecognised MCMV inhibition of IL-1β- and TLR7/9-induced NF-κB activation, and indicate a role for viral gene M45 in mediating this effect.
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Borger, J. G. "Visualising early signaling events during TCR activation." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/758822/.
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T cell activation, differentiation and expansion are induced by signals generated upon engagement of TCR by agonist peptide:MHC. At the site of T cell interaction with an antigen presenting cell, TCR, accessory proteins such as CD28 and LFA-1, and the Src family kinases (SFK) Lck and Fyn, accumulate and form the immunological synapse. Lck is the key signaling molecule, initiating TCR signals by phosphorylating the ITAM in the CD3/ subunits resulting in the recruitment and activation of Zap-70 and further propagation of the signaling cascade. Fyn is a positive mediator of T cell signaling, sharing specific substrates in common with Lck, and also acts as a negative regulator by phosphorylating the adaptor protein, PAG. Phosphorylated PAG binds Csk and its recently identified binding partner, the protein phosphatase PEP, recruiting them to the plasma membrane where they can inhibit the SFK. The differential localisation of the kinases, with Fyn compartmentalised to lipid rafts in mature T cells and the majority of Lck being non-lipid raft associated could explain the unique functions attributed to each kinase. In order to examine the interactions between SFK and their negative regulators in T cell activation, CD8+ T cells from mice expressing the class I MHC-restricted TCR, F5, were investigated. In naïve T cells Lck, Fyn, Csk and PEP were shown to co-localise to the site of activation-induced tyrosine phosphorylation following early TCR crosslinking. In contrast, in memory T cells Csk, PEP and Fyn not only co-localised with the site of TCR activation but also distributed at the distal end of the cell, suggesting differential redistribution of key negative regulators away from the site of TCR engagement in these cells. In the absence of Fyn there was no change in Csk localisation despite the loss of PAG phosphorylation, suggesting another adaptor protein can recruit Csk to the plasma membrane. Caveolin-1, a cholesterol-rich membrane protein was investigated as a possible candidate for recruiting Csk and was shown to be present in CD8+ T cells. Moreover, caveolin-1 was shown to be phosphorylated by Lck, Fyn and Abl kinases upon TCR engagement and to redistribute and polarise to the cSMAC, co-localising with Csk. In summary we have shown that there are alterations in the distribution of the negative regulators Csk, PEP and Fyn, once T cells have encountered antigen, and that this could be associated to the more efficient responses of memory cells upon re-exposure to antigen. Furthermore, its appears there is redundancy of both signaling molecules and adaptor proteins, which may contribute to the fine tuning of the TCR signaling pathway, such that signals derived from the TCR can still modulate an appropriate immune response even in the absence of key regulators.
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Vispute, Ketki. "CD180/RP105 Toll-like Receptor (TLR) mediated signalling in Chronic Lymphocytic Leukaemia." Thesis, University of Westminster, 2013. https://westminsterresearch.westminster.ac.uk/item/8z1y8/cd180-rp105-toll-like-receptor-tlr-mediated-signalling-in-chronic-lymphocytic-leukaemia.
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The role of the microenvironment in the development and progression of chronic lymphocytic leukaemia (CLL) is currently of major interest. Pathogen- and damage-associated molecular patterns (PAMPs and DAMPs, respectively) represent exogenous and endogenous microenvironmental factors acting via a range of receptors, including Toll-like receptors (TLR). CD180/RP105 is a membrane-associated orphan receptor that belongs to the TLR family, is expressed by professional antigen-presenting cells, and drives normal B-cell activation and proliferation. We have previously shown that approximately 60% of CLL samples expressed surface CD180 but only half responded to ligation with anti-CD180 monoclonal antibody (mAb) resulting in activation, cycling, and reduced basal apoptosis and were termed responders (R). In contrast, CD180+CLL samples that failed to respond to anti-CD180 mAb, despite expressing a high density of CD180 receptors, were termed non-responders (NR). We further demonstrated that in R-CLL cells, CD180 ligation significantly induced phosphorylation of ZAP70/Syk, ERK, p38MAPK, and AKT. In contrast, CD180-mediated signalling in NR CLL cells did not progress downstream from ZAP70/Syk phosphorylation indicating a block in activation of downstream protein kinases, and possible anergy. To further clarify the CD180-mediated signalling pathways in CLL, here we studied signal transduction downstream from ZAP70/Syk by delineating CLL samples into R and NR through their proximal ability to activate AKT. We have studied major signalling protein kinases associated with the BCR signalling pathway: PI3K, Btk, ERK, p38MAPK and AKT. Segregation of CLL samples responding to CD180 ligation by signalling via pAKT, rather than by CD86 upregulation, revealed that CD180 ligation on CLL cells can activate two alternative signalling pathways: pro-survival that operates via PI3K-Btk-AKT protein kinases, or mostly pro-apoptotic, that operates via p38MAPK but not through Btk. This may have implications for CLL therapy where Btk inhibitors are being used. Here we demonstrate that albeit ligation of sIgM alone also activates pro-survival PI3K-Btk-AKT pathway pre-engagement of CD180 redirected BCR-mediated signalling towards the potentially pro-apoptotic p38MAPK pathway that opens new horizons for immunotherapy. Since the tissue microenvironment plays a crucial role in generation and survival of the CLL clones, studies pertaining to CD180 expression in the lymphoid tissues were undertaken. Our pilot data suggests that in normal tonsils CD180 is expressed by the mantle zone (MZ) B cells and not the germinal centre (GC) B cells. However in CLL lymph nodes complete obliteration of the normal tissue architecture and a weak expression of CD180 has been detected, whilst expression of CD180 on bone marrow CLL cells was heterogeneous. Since CLL cells migrate to and from the solid tissues into the peripheral circulation in any CLL clone, there is always an intra-clonal kinetic heterogeneity through a suggested continuum between the 'proliferative' or CXCR4dimCD5bright, and 'resting' CXCR4 brightCD5dim fractions. Here we report that the 'resting' compartment was enriched for CD180+ cells compared to the 'proliferating' subset. In contrast, sIgM+ cells were more frequent in the proliferating fraction. Since the “resting” subset of CLL cells is also considered as the one “returning” to the solid tissues supported by the increased expression of CXCR4, our data might suggest possible attraction of the CD180+ cells towards the putative ligand in the lymphoid tissues. It is becoming apparent that intraclonal diversity plays an important role in the clinical outcome of patients with CLL. Subsets of the CLL clone that respond more robustly to external stimuli may well gain a growth and survival advantage and possibly promote clonal evolution. Identification of these CLL subpopulations was therefore of prime importance, as these cells may be preferred targets for future therapeutics. We have established that CD180 expression on CLL cells helps identifying different subsets and delineating their physiological status. Our findings on modulation of signalling pathways through CD180 and sIgM and the temporal effects of their ligation is consistent with multiple ligands in the, in vivo, microenvironment playing an important role in the survival of CLL cells. Since TLR can shuttle between inhibition and promotion of leukemic growth they may play a key role in immune evasion impacting on clinically relevant tumour-host microenvironment interactions. The identification of distinct CD180-mediated signalling pathways that promote tumour cell proliferation and survival will allow specific targeting of key players in the pathways with immunotherapy and chemotherapy.
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Barry, A. C. "Regulation of TCR signalling by SOCS." Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479241.
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Wong, Tsz-yeung, and 王子揚. "IBDV-mediated antiviral responses by TLR3 signaling pathways." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2009. http://hub.hku.hk/bib/B41897201.
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Wong, Tsz-yeung. "IBDV-mediated antiviral responses by TLR3 signaling pathways." Click to view the E-thesis via HKUTO, 2009. http://sunzi.lib.hku.hk/hkuto/record/B41897201.
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Bruger, Annika Målin. "TCR signalling in response to affinity stimulation." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:5f9c1001-6c43-472f-a495-c9573b54a84a.
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T cells are an essential part of the adaptive immune system and protect the body from intracellular infections. The specificity with which αßTCR-bearing T cells recognize cognate antigen presented on MHC molecules is paramount to maintaining the balance between mounting effector functions against pathogens and establishing peripheral tolerance to self. The mechanism by which T cells translate qualitative differences in TCR:pMHC binding to sensitive proximal signalling events which ultimately result in specific Tcell effector responses to infected cells but not to self is mostly unknown. To address how T cell signalling responds to qualitative differences in TCR triggering by pMHC, I established a system of stimulating T cells bearing the 1G4 TCR specifically in vitro with a panel of four NY-ESO-1156-165 peptide variant MHC tetramers. Single amino acid substitutions to the NY-ESO-1156-165 peptide conferred a maximum 35-fold difference in the monomeric affinity for the 1G4 TCR. The system allows the highly controlled investigation of very rapid TCR proximal signalling events simultaneously and quantitatively using flow cytometry. Stimulations with pMHC tetramers showed rapid sensitive sequential signalling responses which were able to confer ligand discrimination. Very early signalling events such as CD3ζ phosphorylation showed analogue responses to the different affinity pMHC tetramers. Later signalling events including phospho-ERK showed a distinct on/off switch-like response. The amplitude of the very early analogue signalling responses determined the extent of later digital ERK signals. This indicates that a certain analogue signalling threshold must be passed to result in T cell activation. The thymocyte protein Themis has been shown proximal TCR signalling to modulate thymocyte selection thresholds. Its deletion results in profound defects in positive thymocyte selection. Themis locates to the LAT signalosome of the TCR signalling cascade via Grb2, yet its molecular function is unknown. Employing the system I established, I demonstrate that Themis-k/d cells show increased levels of CD3z-chain phosphorylation, phospho-ERK signalling and signal-induced apoptosis which was independent of the ERK signal. This shows that Themis is a global attenuator of proximal TCR signalling. We are currently investigating possible associations of Themis to proteins phosphastases such as SHP-1 which could attenuate TCR proximal protein tyrosine signalling events.
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Downes, Joan Ellen. "Role of TLR Signalling in the Induction of Changes in Haematopoiesis by Viral and Bacterial Agents." Thesis, University of Liverpool, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507513.
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Divanovic, Senad. "Negative Regulation of TLR4/MD-2 Signaling by RP105/MD-1." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1124119013.
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Cheng, Gordon W. "Functions of CD45 in TCR signaling in CD4§+CD8§+ double-positive thymocytes." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq29256.pdf.
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Okabe, Namiko. "Suppressor of TCR signaling-2 (STS-2) suppresses arthritis development in mice." Kyoto University, 2018. http://hdl.handle.net/2433/232089.
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Sepulveda, Garrido Fernando. "Critical role for Asparagine Endopeptidase in endocytic TLR9 signaling in dentritic cells." Paris 5, 2009. http://www.theses.fr/2009PA05T051.
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Les récepteurs de reconnaissance du pathogène (PRR) protègent l'organisme des infections provoquant une réponse antimicrobiennes variée qui permet la destruction du pathogène par l'activation coordonnée du système immunitaire inné et acquis. Entre les différents types de PRR, la famille des récepteurs de type Toll (TLRs) sont les mieux caractérisés. Le TLR9 est situé dans les endosomes et reconnaît l'ADN bactérien ou viral. L'interaction entre le TLR9 et ses ligands, induit des changements conformationnels qui permettent l'association de la molécule adaptatrice MyD88, conduisant à la sécrétion de cytokines inflammatoires et à l'activation de l'immunité adaptative. Récemment, il a été montré que dans des macrophages, après la reconnaissance de son ligand, TLR9 est clivé dans son domaine extracellulaire pour générer un fragment C-terminale, lequel s'associe avec MyD88 pour activer sa cascade de signalisation. Ce processus est effectué par des protéases endosomales. Toutefois, l'identité de ces protéases responsables de ce traitement est encore controversée. L'objectif de ce travail est de déterminer le rôle de l'Asparragine Endopeptidase (AEP) dans la signalisation du TLR9 chez les cellules dendritiques (CD) après l'activation parl'ADN. Nos résultats montrent que l'AEP a un rôle essentiel dans la réponse induite par le TLR9 puisque : a) le TLR9 est un substrat pour l'AEP in vitro, b) les cellules dendritiques dérivées de moelle osseuse déficientes d'AEP présentent un retard dans la génération de l'extrême C-Terminal de TLR9, c) l'absence d'AEP conduit à une diminution de la sécrétion de cytokines inflammatoires in vitro et in vivo et d) en absence d'AEP il ce produit un défaut dans l'activation des cellules T CD4+. Il est important de souligner, que le défaut de signalisation par TLR9 en absence d'AEP n'est pas dû à des modifications dans l'activité d'autres protéases endosomales mais probablement à un effet direct de l'AEP sur le clivage du TLR9. En résumé, ces données montrent que l'AEP joue un rôle crucial dans la signalisation du TLR9 chez les cellules dendritiques et suggèrent AEP comme une possible cible pharmacologique pour réguler la fonction du TLR9Pathogen Recognition Receptors (PRRs) protect the host from infection by triggering a variety of antimicrobial responses that allow pathogen destruction through the coordinated activation of the innate and adaptive immune system. Among the different types of PRRs, the family of the Toll-like Receptors (TLRs) is the best characterized. TLR9 is located in the endosomes and recognizes bacterial or viral DNA. The association between TLR9 and its ligands induces a conformational change that allows the recruitment of the adaptor molecule MyD88, resulting in the secretion of inflammatory cytokines and activation of adaptive immunity. Recently, it has been shown in macrophages that TLR9 is cleaved in its luminal part after DNA recognition generating a fully active C-terminal fragment, which interacts with MyD88 to trigger the signaling cascade. This critical processing step is mediated by endosomal proteases. However, the identity of the proteases responsible for this cleavage is still controversial. The objective of this work was to determine the role played by Asparagine EndoPeptidase (AEP) in TLR9 processing and signaling in dendritic cells (DCs) upon CpG DNA treatment. Our results demonstrated that AEP plays a crucial role in TLR9 response in DCs because a) TLR9 is substrate for AEP in vitro; b) bone marrow-derived DCs deficient for AEP present a delay in the generation of the C-terminal fragment of TLR9; c) AEP deficiency decreases cytokines secretion by DCs upon CpG DNA activation either in vitro and in vivo and d) lack of AEP impairs T cells activation after CpG DNA treatment. Importantly, impaired TLR9 signaling in absence of AEP is not due to alterations in the activity of other endosomal proteases but probably due to a lack of direct processing of TLR9 by AEP. In summary, these results h ighlight AEP as a key regulator of TLR9 signaling probably by directly processing full-length TLR9 upon CpG activation in DCs
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Lucas, Elizabeth A. "TLR4 Stimulation Induces SLAMF9-Mediated Regulation of Cytokine Production and Ras Signaling." Miami University / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=miami1590144828229019.
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Katsura, Yoshichika. "mDia1/3-dependent actin polymerization spatiotemporally controls LAT phosphorylation by Zap70 at the immune synapse." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263571.
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Huang, Elizabeth Chi-Fang. "Organisation of, and ligand-independent signalling by, the TCR, with a special emphasis on the pre-TCR." Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:0c01e3d4-2002-487c-a0b6-09ed20cb223b.
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Understanding how immune signalling is initiated and regulated spatiotemporally is likely to be helped by investigations of receptor stoichiometry. The pre-TCR expressed by thymocytes shares similarities in structure and signalling components with the mature TCR but, in contrast to the TCR, it has no known ligands. This thesis has therefore analysed the stoichiometry of the pre-TCR using mutagenesis- and imaging-based approaches, and explored how ligand-independent signalling might be initiated by both the TCR and pre-TCR. The mutational analysis, which required considerable optimisation because the pre-TCR is very weakly expressed in transfected cells, suggested that only one contiguous surface on pre-Tα needs to be buried in the pre-TCR complex in order for it to reach the cell surface. This comprised the surface buried at the interface with the constant region of TCRβ and therefore was incompatible with previous suggestions that the pre-TCR assembles into a 'head-to-tail' dimer. Unexpectedly, super-resolution imaging experiments combined with cluster analysis, and the method of single-molecule cross-colour coincidence detection were suggestive that, rather than dimers, the pre-TCR forms higher-order oligomers in transfected cells and possibly also in thymocytes. Similar analyses showed that the organisation of the TCR was heterogeneous, depending on the level of expression of the receptor. Overall, technical limitations that emerged in the course of the study highlighted some of the difficulties in studying native receptor stoichiometry on resting cells generally. The final part of the thesis investigated ligand-independent signalling by the TCR, using a novel assay based on the binding of a superagonistic antibody to a non-signalling form of the receptor CD28. Using CRISPR/Cas-mediated gene editing, it was shown that ligand-independent signalling by the mature TCR and pre-TCR was dependent on Lck and Zap70, indicating that it is reliant on the triggering of conventional signalling pathways. Ways in which the pre-TCR might initiate signalling, that could be tolerant of complexes exhibiting a range of stabilities, are also discussed.
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Poltorak, Mateusz Pawel [Verfasser], and Luca [Akademischer Betreuer] Simeoni. "Analysis of the TCR-mediated signaling dynamics / Mateusz Pawel Poltorak. Betreuer: Luca Simeoni." Magdeburg : Universitätsbibliothek, 2014. http://d-nb.info/1057913898/34.
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Schneider, Olivia Dawn. "An Analysis of the Effects of Pertussis Toxin on T Cell Signaling." University of Cincinnati / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1258667926.
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Salles, Audrey. "Influence de l'organisation latérale de la membrane sur l'activation lymphocytaire T." Thesis, Aix-Marseille 2, 2010. http://www.theses.fr/2010AIX22137.
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Les rafts lipidiques sont des nanodomaines membranaires enrichis en cholestérol et en sphingolipides impliqués dans la régulation de la signalisation médiée par le TCR. Néanmoins l'existence et la fonction de ces domaines sont sujettes à controverse compte tenu des difficultés expérimentales pour les étudier in vivo. En utilisant des traitements non invasifs ciblant spécifiquement la voie de biosynthèse de ces lipides, nous avons étudié l'influence de l'organisation latérale de la membrane sur l'activation lymphocytaire T. Par des approches biophysiques, nous avons démontré au sein de lymphocytes T primaires CD4+, que les molécules TCR, CD4 et Lck sont constitutivement partitionnées dans les rafts lipidiques dont est exclue CD45. De plus, cette préorganisation moléculaire modifie les paramètres d'adhésion entre le TCR. Pour étudier le rôle de ces structures au sein de cellules individuelles, nous avons développé une nouvelle méthodologie permettant de détecter et d'analyser à haut débit et de manière automatique la réponse calcique des cellules T. Nous avons confirmé l'influence des rafts membranaires dans la signalisation TCR par les rafts lipidiques joue un rôle majeur dans l'initiation de la reconnaissance antigénique des cellules TLipid rafts are membrane nanodomains enriched in chrolesterol and sphingolipids, which ahave previously been implanted in TCR signaling mechanisms. This contention, however, has beacome highly controversial due to experimental difficulties to study these membrane organizations in vivo. Using non invasive treatments that target specific lipid biosynthesis, we have studied the influence of lateral membrane organization in T lymphocyte activation. By using biophysical approaches, we have demonstrated that in murine CD4+Tcelles, TCR, CD4 and Lck are constitutively and dynamically trapped in lipid rafts, whereas CD45 is excluded. Moreover, this pre-organization impacts binding of TCR to the MHC II-peptide complex and controls the initiation of early TCR signaling. To investigate the role of these structures within individual live cells, we have developed a new high throughput methodology to monitor the calcium mobilization in T cells. We have confirmed the influence of membrane rafts in TCR signaling. Our results have thus demonstrated that pre-organization of TCR signaling protagonists by lipid rafts play a major role in the initiation of T cell antigen recognition
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Esparza, Greg Angel. "The molecular requirements for activation of specific toll-like receptor 4 signaling pathways." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2866.
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Endotoxins (E) are a unique and abundant family of glycolipids located in the outer leaflet of the outer membrane of Gram-negative bacteria. Host immune responses to endotoxin depend on ordered endotoxin-host protein interactions, resulting in delivery of an endotoxin monomer to MD-2 which acts as a potent agonist of Toll-Like Receptor (TLR) 4. Activated TLR4 is unique among TLRs in its ability to mobilize two distinct intracellular signaling pathways: the MyD88- and TRIF-dependent pathways. The regulated action of both pathways is likely important for optimal host immune responses to Gram-negative bacterial infection, but how this is achieved is not well understood Recent studies have indicated an essential role for host CD14 in TRIF-dependent signaling by activated TLR4 but the extent to which these observations reflect a general role of CD14 in endotoxin-triggered TRIF signaling or one more narrowly restricted to the specific endotoxins and/or cell types used is uncertain. We have addressed this question by identifying a novel CD14-independent mechanism for efficient delivery of E monomer to MD-2 and TLR4 activation, that is mediated by endotoxin.albumin complexes. We have used these complexes to demonstrate CD14-independent activation of MD-2⋅TLR4 by a wider range of endotoxin species than previously thought possible and activation of both MyD88- and TRIF-dependent pathways. Taken together, the findings in this thesis indicate that the molecular structure and physical presentation of endotoxin as well as CD14-independent properties of the host cell help determine the extent to which CD14 is required for TRIF-dependent signaling by activated TLR4.
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Meijer, Lisa. "Signalling and activation of TLR4 by Gram-negative bacteria in epithelial cells /." Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-560-3/.
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Westcott, Sarah Louise. "Structural and functional studies of SER/THR kinase signalling in mycobacterium tuberculosis." Thesis, University College London (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.411741.
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Paquette, Sara Montminy. "Evasion of LPS-TLR4 Signaling as a Virulence Determinate for Yersinia pestis." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/458.
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Yersinia pestis, the gram-negative causative agent of plague, is a master of immune evasion. The bacterium possesses a type three secretion system which translocates Yop effector proteins into host immune cells to inhibit a number of immune and cell signaling cascades. Interestingly, this apparatus is not expressed at low temperatures such as those found within the flea vector and is therefore neither in place nor functional when the bacteria are first transmitted into a mammalian host. However, the bacterium is still able to avoid activating the immune system, even very early during infection.
When grown at 37°C (human body temperature) Y. pestis produces a tetra-acyl lipid A molecule, which is antagonistic to the human Toll like receptor 4/MD2, the major lipopolysaccharide recognition receptor. Although tetra-acyl lipid A binds this receptor complex, it does not induce signaling, and in fact inhibits the receptors interaction with other stimulatory forms of lipid A. The work undertaken in this thesis seeks to determine if the production of tetra-acyl lipid A by Y. pestis is a key virulence determinant and was a critical factor in the evolution of Y. pestis from its ancestral parent Yersinia pseudotuberculosis.
By examining the enzymes involved in the lipid A biosynthesis pathway, it has been determined that Y. pestis lacks LpxL, a key enzyme that adds a secondary acyl chain on to the tetra acyl lipid A molecule. In the absence of this enzyme, Y. pestis cannot produce a TLR4 stimulating form of lipid A, whereas Y. pseudotuberculosis does contain the gene for LpxL and produces a stimulatory hexa acyl lipid A. To determine if the absence of LpxL in Y. pestis is important for virulence, LpxL from E. coli and Y. pseudotuberculosis were introduced into Y. pestis. In both cases the addition of LpxL led to bacterium which produced a hexa-acylated lipid A molecule and TLR4/MD2 stimulatory LPS. To verify the LpxL phenotype, lpxL was deleted from Y. pseudotuberculosis, resulting in bacteria which produce tetra-acylated lipid A and nonstimulatory LPS. Mice challenged with LpxL expressing Y. pestis were found to be completely resistant to infection. This profound attenuation in virulence is TLR4 dependent, as mice deficient for this receptor rapidly succumb to disease. These altered strains of the bacterium also act as vaccines, as mice infected with Y. pestis expressing LpxL then challenged with wild type Y. pestis do not become ill. These data demonstrate that the production of tetra-acyl lipid A is a critical virulence determinant for Y. pestis, and that the loss of LpxL formed a major step in the evolution of Y. pestis from Y. pseudotuberculosis.
These bacterial strains were also used as tools to determine the contributions of different innate immune receptors and adaptor molecules to the host response during Y. pestis infection. The use of LpxL expressing Y. pestis allowed identification of the innate immune pathways critical for protection during Y. pestis infection. This model also established that CD14 recognition of rough LPS is critical for protection from Y. pestisexpressing LpxL, and activation of the IL-1 receptor and the induction of IL-1β plays a major role in this infection as well.
The lipid A acylation profile of gram negative bacteria can have a direct and profound effect on the pathogenesis of the organism. This work illustrates a previously unknown and critical aspect of Y. pestis pathogenesis, which can be extended to other gram-negative pathogens. The greater detail of the contributions which different host adaptor and receptor molecules make to the overall innate immune signaling pathway will allow a better insight into how gram negative infections progress and how they are counteracted by the immune system. Alterations of the lipid A profile of Y. pestis have important implications for the production of vaccines to Y. pestis and other gram negative pathogens. Taken together, this work describes a novel mechanism for immune evasion by gram negative bacteria with consequences for understanding the immune response and the creation of more effective vaccines, both of which will decrease the danger posed by this virulent pathogen.
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Ziegler, Kira [Verfasser]. "Inflammatory processes and TLR4 signaling influenced by nitrated proteins and related substances / Kira Ziegler." Mainz : Universitätsbibliothek der Johannes Gutenberg-Universität Mainz, 2021. http://d-nb.info/123664090X/34.
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Gallagher, Michael P. "Differential TCR signaling dynamics tune graded gene expression in early-activating CD8+ T cells." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1115.
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The strength of peptide:MHC interactions with the T cell receptor (TCR) is correlated with the time to first cell division, the relative scale of the effector cell response, and the graded expression of activation-induced proteins. The TCR proximal tyrosine kinase ITK simultaneously influences many biochemically separate signaling cascades. T cells lacking ITK exhibit selective impairments in effector T cell responses after activation, but under the strongest signaling conditions ITK activity is dispensable. To gain insight into whether TCR signal strength and ITK activity tune observed graded gene expression through unequal activation of disparate signaling pathways, I examined NFAT, NF-κB and MAP kinase pathways during early activation of individual naïve OT-I CD8+ T cells using peptide-loaded antigen presenting cells. Utilizing both measurement of transcription factor translocation in single T cell nuclei and conventional phospho-flow cytometry, I observed digital activation of Erk-MAPK and NFAT1 at all peptide doses and avidities. However, NF-κB activation showed a graded response to variation in TCR signal strength and was more sensitive to treatment with an ITK inhibitor. Inhibitor-treated cells showed poor induction of AP-1 factors Fos and Fosb, NF-κB response gene transcripts, and survival factor Il2 transcripts. ATAC-seq analysis revealed genomic regions most sensitive to ITK inhibition are enriched for NF-κB and AP-1 motifs. Together, these data indicate a key role for ITK in orchestrating optimal activation of separate TCR downstream pathways, specifically aiding NF-κB activation. More broadly, I describe a mechanism by which variation in TCR signal strength can produce patterns of graded gene expression in activated T cells.
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Conley, James M. "TCR Signal Strength Controls Dynamic NFAT Activation Threshold and Graded IRF4 Expression in CD8+ T Cells." eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1019.
Full textAbstract:
TCR signal strength is critical for CD8+ T cell clonal expansion after antigen stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. Here, using multiple methods to vary TCR signal strength and measure changes in transcriptional activation in single CD8+ T cells, we connect antigen potency to the kinetics of NFAT activation and Irf4 mRNA expression. T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. The activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the signal transduction pathway accounting for the reduced clonal expansion of low affinity CD8+ T cells following infection. We also describe another context for ITK activity, autoreactive T cell migration. Here, we connect TCR signaling strength to modulation of selectin binding and autoreactive T cell-mediated pathology in an adoptive transfer model system of autoimmune disease. Understanding the signaling mechanisms linking changes in TCR signaling to CD8 T cell function is important in furthering the understanding of vaccine development and T cell adoptive immunotherapy.
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Ball, Corbie. "The Role of Transforming Growth Factor Beta Signaling in Inflammation-Dependent Colon Cancer." Diss., The University of Arizona, 2015. http://hdl.handle.net/10150/593463.
Full textAbstract:
Chronic inflammatory conditions such as Crohn's disease (CD) and Ulcerative colitis (UC) are risk factors for colon cancer. TGFβ has been shown to be dysregulated in colon cancer. Bacteria-induced inflammation is necessary for the induction of colon cancer in TGFβ mouse models. However, the mechanism by which TGFβ regulates the inflammatory response in these models is not well elucidated. It was our thought that we needed to be able to distinguish what was TGFβ dependent and what was inflammation dependent. To do this we created 2 colonies of Smad3 mice. One colony was housed with normal colonic bacteria (Smad3-uninfected animals) and the other colony (Smad3-infected animals) had chronic H. hepaticus infection. As previously seen the Smad3⁻/⁻- infected animals developed colitis and carcinoma (~40%). In the absence of H. hepaticus infection SMAD3 was found to negatively regulate TLR4 expression. This was then exacerbated with the addition of H. hepaticus resulting extreme up-regulation of TLR4 and the downstream effectors IRAK4 and NF-κB in Smad3⁻/⁻-infected colonic tissues. Examination of adaptive immune regulation in this model demonstrated that SMAD3 was necessary for FOXP3 expression in H. hepaticus-infected splenocytes. Loss of SMAD3 resulted in up-regulation of IL17 and reduced iTreg populations. These data demonstrate the important role SMAD3 has in maintaining tolerance to microbial populations through both the innate and adaptive immune systems.
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Wallis, Alicia M. "TRAF3 as a regulator of T lymphocyte activation." Diss., University of Iowa, 2017. https://ir.uiowa.edu/etd/5873.
Full textAbstract:
T cells are an essential component of the adaptive immune system, which evolved to facilitate development of long-term, effective protection against infectious diseases. Upon activation, T cells play an important role in clearing infections, and especially, in preventing establishment of subsequent infections with the same pathogen. Because this is such a powerful response, it must be tightly regulated. Our lab has long been interested in how signaling molecules regulate the function of T and B lymphocytes. Our prior studies stimulated an interest in the signaling adapter molecule, Tumor necrosis factor receptor (TNFR)-associated factor 3 (TRAF3). Our group previously produced a T cell-conditional (CD4-Cre) TRAF3-/- mouse, which demonstrated that TRAF3 unexpectedly plays an important positive role in T cell functions, including providing help for B cell responses, protection from infectious pathogens, cytokine production and proliferation. After TCR engagement, TRAF3 associates with the T Cell Receptor (TCR)/CD28 complex. These data identified a new role for TRAF3 in T cell activation. There are three signals that are required for full T cell activation. The three types of receptors that deliver these signals are the TCR, co-stimulatory receptors and cytokine receptors. This dissertation explores the regulatory role of TRAF3 in the 3 signals required for T cellsactivation. In signal 1, TRAF3 enhances TCR signaling by regulating the localization of the TCR inhibitors, PTPase non-receptor type 22 (PTPN22) and the c-Src kinase (Csk). Our lab previously reported that recruitment of TRAF3 to the TCR complex requires co-stimulation of CD28, the primary receptor for signal 2. In this dissertation, we show that TRAF3 associates with the Linker of Activated T cells (LAT) complex, demonstrating preference for distinct LAT-associated proteins. For delivery of signal 3, T cells require stimulation of a cytokine receptor, such as IFNαR, for differentiation of a T cell to an effector cell. Upon IFN stimulation, TRAF3 inhibits IFNαR-induced early molecular events, which results in the regulation of both canonical and non-canonical IFNαR signaling pathways. The results presented in this dissertation highlight the dynamic roles of TRAF3 as a regulator of T cell activation, by regulating multiple T cell signaling pathways.
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Pracheil, Tammy. "Regulation of the Target of Rapamycin Signaling Pathway in Saccharomyces cerevisiae." ScholarWorks@UNO, 2013. http://scholarworks.uno.edu/td/1662.
Full textAbstract:
An integrative, biochemical, genetic, and molecular biology approach utilizing gene manipulation, gene knock outs, plasmid based protein expression, and in vivo protein localization of fluorescence tagged proteins was employed to determine the function of an essential protein, Lst8, in TORC1 and TORC2 signaling and a previously uncharacterized complex, the Far3-7-8-9-10-11 complex (Far complex) in the budding yeast, Saccharomyces cerevisiae. Mutations in SAC7 and FAR11 suppressed lethality of both lst8 and tor2-21 mutations but not TORC1 inactivation, suggesting that the essential function of Lst8 is linked only to TORC2.
Far11, a component of a six-member complex, was found to interact with Tpd3 and Pph21, conserved components of Protein Phosphatase 2A (PP2A) via co-immunoprecipitation. Mutations in FAR11 and RTS1, which encodes a PP2A regulatory B subunit, restore phosphorylation to the TORC2 substrate Slm1 in a tor2-21 mutant. These data suggest that TORC2 signaling is antagonized by Far11-dependent PP2A activity.
To characterize the assembly of the Far complex in vivo, intracellular localization of the Far complex was examined by fluorescence microscopy. It was found that the Far complex localizes to the endoplasmic reticulum (ER). The data show that Far9 and Far10 are tail-anchored proteins that localize to the ER first and recruit a Far8-Far7-Far3 pre-complex. Far11 is found at the ER only when all other Far proteins are assembled at the ER. Surprisingly, ER localization is required for the Far Complex’s role TORC2 signaling because deletion of the tail-anchor domain of Far9 results in partial bypass of the tor2-21 mutant growth defect at 37 ˚C.