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1
Schnelzer, Andreas Christof. "Untersuchungen zum kleinen G-Protein Rac1 beim Mammakarzinom." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963997610.
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2
Wang, PiLin. "Roles for Small G Protein Rac1 in Amyloid Cascade Hypothesis." 京都大学 (Kyoto University), 2009. http://hdl.handle.net/2433/124063.
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3
Hope, Hannah. "Regulation of the Rho G protein, Rac1, in the pathogenic fungus Candida albicans." Nice, 2009. http://www.theses.fr/2009NICE4093.
Повний текст джерелаАнотація:
C. Albicans est un pathogène opportuniste de l’homme, en général présent dans la flore intestinale. Un des facteurs de virulence de cet organisme est sa capacité de dimorphisme: cette levure évolue entre une forme ovoïde et une forme filamenteuse invasive. Ce changement de forme requiert une réorganisation du cytosquelette, suggérant que les facteurs clés de cette réorganisation - les protéines G de type Rho et leurs régulateurs - jouent un rôle important dans la croissance filamenteuse et la virulence de cet organisme. A la différence de l’homme, il n’y a que six de ces protéines G chez la levure. L’objectif de ce travail est d’étudier le rôle et la régulation de l’une d’entre elles, Rac1, identifiée précédemment dans le laboratoire. Nous avons tout d’abord identifié l’activateur de Rac1, Dck1, et nos résultats suggèrent que la spécificité de Rac1 est en partie déterminée par cet activateur. Dck1 fait partie de la famille d’activateurs CDM, définie par C. Elegans Ced-5, H. Sapiens Dock180 et D. Melanogaster Myoblast city. Chez les mammifères, la protéine d’échaffaudage ELMO (engulfment and cell motility) est nécessaire, en plus de Dock180, pour l’activation optimale de Rac1. Une recherche BLAST du génome de C. Albicans a permis d’identifier un analogue d’ELMO1, appelé LMO1. L’étude de la régulation de Rac1 a été poursuivie en caractérisant le rôle de Lmo1, utilisant des approches génétiques et biochimiques. Nos résultats indiquent qu’un mutant de délétion lmo1 est déficient pour la croissance filamenteuse invasive, tout comme les mutants de délétion rac1 et dck1. La sur-expression spécifique de la protéine MAP Kinase Cek1 permet de restaurer la croissance filamenteuse invasive dans chacun de ces mutants de délétion. De plus, nos résultats montrent que Rac1, Dck1 et Lmo1 sont critiques pour l’intégrité cellulaire. D’autre part, nous avons pu déterminer que Lmo1 lie Dck1 et Rac1, soit ensemble ou soit separament. . Dans l’ensemble, nos résultats permettent de proposer que Rac1, Dck1 et Lmo1 fonctionnent en complexe, en amont de la MAP kinase Cek1, lors de la croissance filamenteuse invasive de C. Albicans. Cette étude montre pour la première fois la conservation d’un complexe fonctionnel Dock180/ELMO/Rac1 de la levure à l’hommeC. Albicans is a human opportunistic pathogen that can invade host tissues, causing infections ranging from superficial epithelial mycoses to life threatening systemic infections in immuno-compromised patients. C. Albicans is a dimorphic fungus that exists either as an oval budding yeast or as a filamentous form (hyphal or pseudohyphal). The morphological switch between these two states is critical for C. Albicans pathogenicity. Rho G-proteins are well-documented regulators of the actin cytoskeleton and cell polarity in all eukaryotic cells. In C. Albicans it has been previously shown that distinct Rho G-proteins are required for filamentous growth in response to specific stimuli. Cdc42 is essential for filamentous growth in response to serum whereas Rac1 is required for filamentous growth in an agar matrix. The aim of my thesis research was to determine how Rac1 is regulated during filamentous growth in C. Albicans. First, we identified Dck1, which belongs to the Ced-5, Dock180, myoblast city (CDM) family of guanine nucleotide exchange factors, which is required for invasive filamentous growth similar to Rac1. Subsequently we showed using in vitro binding and genetic suppression studies that Dck1 can activate Rac1 during invasive filamentous growth. From these results we concluded that the requirement for distinct G-proteins for filamentous growth in reponse to different stimuli is due to the specificity of their activators. In mammals, flies and worms the ELMO (engulfment and cell motility) protein functions as a scaffold which is necessary for Rac1 signalling. Based on similarity to the mammalian ELMO1 we identified Lmo1 and showed that it is required for invasive filamentous growth. Furthermore we demonstrated that Lmo1 interacts with both Dck1 and Rac1. Overexpression of the MAP kinase Cek1 restores invasive filamentous growth in each of these three deletion mutants. In addition we identified a role for these proteins in cell integrity. Together, our results indicate that Lmo1, Rac1 and Dck1 function as a complex upstream of the Cek1 MAP kinase during invasive filamentous growth. This work shows for the first time the conservation of the Dock180/ELMO/Rac1 signalling complex between fungi and mammals
4
Schenck, Annette. "CYFIP, a protein family implicated in neuronal connectivity, links Rac1 GTPase signalling to the fragile X mental retardation protein." Université Louis Pasteur (Strasbourg) (1971-2008), 2003. http://www.theses.fr/2003STR13175.
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Le syndrome de l'X Fragile, qui constitue la forme la plus fréquente de retard mental héréditaire, est du à l'absence de FMRP, une protéine de liaison à l'ARN qui régulerait la traduction au niveau synaptique. Afin de mieux comprendre le role de FMRP, nous avons réalisé un criblage double-hybride dans la levure pour identifier certains de ses interacteurs. Deux protéines ont été isolées, CYFIP1 et CYFIP2 (Cytoplasmic FMRP Interacting Proteins 1/2). Ces deux protéines cytoplasmiques sont hautement homologues mais interagissent différement avec les deux autres protéines de la famille FXR, FXR1P et FXR2P. Le site de liaison à CYFIP recouvre le site d'homo- et d'hétérodimérisation de FMRP, suggérant que la liaison à CYFIP pourrait moduler l'activité de FMRP. D'autre part, l'interaction entre CYFIP1 et Rac1 a été démontrée précédemment. Rac1, une protéine de la famille des Rho GTPase, est un des principaux régulateurs de la réorganisation du cytosquelette d'actine et joue un role clé dans la maturation et la maintenance des épines dendritiques, structures synaptiques riches en actine anormalement développées chez les patients X-Fragile et dans les souris invalidées pour FMRP. De nombreux gènes des voies Rho/Rac étant impliqués dans des retards mentaux, Rac1, CYFIP1 et FMRP pourraient participer à une voie commune contrôlant la morphogénèse synaptique et le fonctionnement cognitif. Pour valider cette hypothèse in vivo, nous avons choisi Drosophila melanogaster comme organisme modèle. Nous avons découvert que CYFIP y est très exprimé dans le système nerveux embryonnaire, et s'accumule notamment dans les axones centraux et aux jonctions neuro-musculaires (JNM). Des mutations de CYFIP causent des défauts de croissance, de ramification et de connexion des axones et conduisent à une morphologie anormale des synapses des JNM. Ainsi, l'absence de CYFIP provoque des défauts similaires à ceux précedemment décrits chez les mutants dFMR1 et/ou dRac1. L'analyse des interactions génétiques et biochimiques entre ces trois protéines suggère que, lorsque la voie est activée, dRac1 inhibe CYFIP qui régule à son tour négativement dFMR1Fragile X Syndrome is the most frequent form of hereditary mental retardation and caused by the absence of FMRP, an RNA binding protein that seems to regulate local protein translation at synapses. To better understand the physiological function of FMRP, we conducted a yeast two-hybrid screen to determine interacting proteins. We identified CYFIP1 and CYFIP2 (Cytoplasmic FMRP Interacting Proteins 1/2), two highly homologous cytoplasmic proteins, which show a different pattern of interaction with the two FMRP-related proteins FXR1P and FXR2P. The CYFIP binding site of FMRP overlaps with its homo- and heteromerisation domain, suggesting that binding to CYFIP may modulate FMRP function. Importantly, CYFIP1 has been previously reported to interact with Rac1. Rac1, a Rho GTPase, is a key regulator of actin cytoskeleton remodelling with a well-established role in maturation and maintenance of dendritic spines, which are actin-rich synaptic structures that are abnormally developed in Fragile X patients and FMRP null mice. Since several genes of Rac/Rho signalling pathways are implicated in mental retardation, our work suggested that Rac1, CYFIP and FMRP work in a common pathway determining synapse morphogenesis and cognitive function. To address this hypothesis in vivo, we have chosen the fruitfly Drosophila melanogaster as a genetic model organism. Drosophila CYFIP, a previously undescribed gene, is highly expressed in the embryonic nervous system, where it strongly accumulates in central axons and at the neuromuscular junction (NMJ). CYFIP mutations induce defects in axon growth, branching and pathfinding and result in abnormal synapse morphology at the neuromuscular junction. Hence, loss of CYFIP involves defects that have been previously described in dFMR1 and/or dRac1 mutants. Analyses of biochemical and genetic interactions amongst these three proteins suggest that upon activation, dRac1 acts antagonistically on CYFIP, which in turn negatively regulates dFMR1
5
Mehta, Harshini. "Role of adaptor protein SLAT in Fc[gamma]R mediated phagocytosis in macrophages." Oklahoma City : [s.n.], 2009.
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6
Attias, Ortal. "The role of Rac1 in mouse podocyte cellular process formation and differentiation /." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=111588.
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The role of podocytes in glomerular permselectivity is tightly associated with their intricate morphology, featuring interdigitating foot processes from adjacent cells. The actin cytoskeleton is an integral component of podocyte foot processes and is regulated by a number of proteins expressed in podocytes. Rho-family of small GTPases are known key regulators of the actin cytoskeleton. This study, investigated the role of Rac1 in podocytes, using conditionally immortalized mouse podocytes (MPs). We studied Rho-GTPase activities and morphology/cytoskeleton of differentiating mouse podocytes stably expressing nephrin. We also studied the impact of transfection of various Rho-GTPase mutants and IQGAP1 mutants. We demonstrated that nephrin expression potentiates and sustains Rac1 activity during the differentiation process. We showed that Rac1 contributes to process formation in differentiating MPs and may have a similar role in vivo when podocytes are recovering from injuries.
7
Saito, Kelly Cristina. "Envolvimento de Rac1 na excitotoxicidade induzida por NMDA na retina de ratos." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42134/tde-10022012-132908/.
Повний текст джерелаАнотація:
A ativação excessiva dos receptores NMDA tem sido descrita no disparo da morte neuronal que ocorre em doenças, como o glaucoma. É possível que a combinação de subunidades (NR2A-D) possa ativar vias de sinalização intracelulares que resultam na morte ou sobrevivência. Nosso objetivo foi investigar o envolvimento de subunidades NR2 e Rac1, membro da família Rho GTPase, na morte de neurônios da retina. A morte induzida por glutamato in vitro foi reduzida após a inibição de Rac1 e bloqueio de NR2B, mas não das subunidades NR2C/D. Resultados semelhantes foram obtidos in vivo após injeção intravítrea NMDA, e a detecção de Rac1 ativo, principalmente, nos processos de glia de Müller foi inibida pelo bloqueio NR2B. Além disso, a produção de TNF-α após a injeção de NMDA foi reduzida pelo bloqueio de NR2B e Rac1. Assim, nossos resultados sugerem que a excitotoxicidade via receptores NR2B/NMDA ativa Rac1 em células da glia de Müller, que por sua vez controla a produção de TNF-α possível responsável pela morte de células ganglionares da retina.Overactivation of NMDA receptors has been described to trigger neuronal death that occurs in diseases such as glaucoma. It is possible that the combination of subunits (NR2A-D) activate intracellular signaling pathways that result in death or survival. Our aim was to investigate the involvement of NR2 subunits and Rac1, a member of Rho GTPase family, in retinal neuronal death. Glutamate-induced neuronal death in vitro was reduced after Rac1 inhibition and by NR2B blocking, but not NR2C/D subunits. Similar results were obtained in vivo after NMDA intravitreous injection, although active Rac1 was mainly detected in Müller glia processes, and it was inhibited by NR2B blockade. In addition, TNF-α level after NMDA injection were reduced by NR2B blocking and Rac1. Thus, our results suggest that excitotoxicity via NR2B/NMDA receptors activate Rac1 in Müller glia cells, which in turn controls the TNF-α production that triggers retinal ganglion cell death.
8
Pokorny, Morgan R. "The role of Y-box binding protein 1 in prostate cancer." Thesis, Queensland University of Technology, 2013. https://eprints.qut.edu.au/65556/1/Morgan_Pokorny_Thesis.pdf.
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This thesis examined the possible role of Y-box binding protein 1 (YBX1) in prostate cancer aggression and spread. Novel roles were uncovered for YBX1 in the regulation of several genes previously implicated in prostate cancer, as well as showing an effect for YBX1 in increasing tumour cell invasion and movement and reciprocal regulation of androgen-regulated gene networks. In addition, it was found that Y-box 1 regulated several other well-known cancer genes implicated in breast and other cancers. The work performed in this thesis has strengthened the foundations for pursuing YBX1 as a possible central target molecule in prostate cancer therapeutics.
9
Newcombe, Anthony Richard. "The biochemical role of the small G protein Rac1 in cell signalling pathways : interaction with RhoGDI and the phagocyte NADPH oxidase component, p67'p'h'o'x." Thesis, University College London (University of London), 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.342224.
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10
Arbuckle, Janeen Lynnae. "Identification and characterization of domains in non-core RAG1." Oklahoma City : [s.n.], 2007.
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11
Somesh, Baggavalli P. "Functional analysis of Dictyostelium discoideum rho-related proteins RacG and RacH." [S.l. : s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=967178355.
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12
Lam, Jonathan Lam. "Identification of mammalian cell signaling in response to plasma membrane perforation: Endocytosis of Listeria monocytogenes and The Repair Machinery." The Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1543497502225763.
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13
ZHANG, JIAOJIAO. "The study of malignant melanoma treatment on various platforms." Doctoral thesis, Università Politecnica delle Marche, 2021. http://hdl.handle.net/11566/291029.
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Il cancro rappresenta una delle principali cause di morte nel mondo, ma causa anche un enorme dispendio di risorse mediche. Sebbene negli ultimi anni siano stati sviluppati trattamenti innovativi come terapia genica, immunoterapia e trattamenti classici (cioè chemioterapia, radioterapia e rimozione chirurgica), ad oggi non esiste un trattamento efficace per curare questa patologia, anche perché i pazienti sviluppano spesso chemioresistenza, radioresistenza e alcuni resistono anche alla terapia genica e all'immunoterapia.
La pelle è l’organo più grande del corpo umano. Il carcinoma a cellule basali, il carcinoma a cellule squamose e il melanoma sono i tre tumori della pelle più comuni. Il melanoma si sviluppa originariamente dai melanociti (cellule contenenti pigmento) e la sua incidenza è aumentata notevolmente negli ultimi 30 anni con una bassa sopravvivenza a cinque anni e un basso tasso di prognosi. Gli attuali approcci terapeutici del melanoma non solo potrebbero essere inefficaci, ma anche avere gravi effetti collaterali come la vitiligine.
Nell'ambito dello studio del melanoma, l'attuale tesi ha studiato la soppressione del melanoma su diverse piattaforme. In primo luogo, il resveratrolo è stato utilizzato per identificare i potenziali biomarcatori del melanoma. In secondo luogo, attraverso studi precedenti e poi mediante studi in silico, alcuni biomarcatori sono stati ulteriormente valutati. Quindi sono stati studiati modelli di colture cellulari di melanoma in vitro. Abbiamo dimostrato che le cellule del melanoma erano inibite dall'interazione proteina-proteina. In terzo luogo, dopo analisi LC-MS/MS, il database delle proteine è stato utilizzato per analizzare e annotare le funzioni dei potenziali biomarcatori. Quarto, è stato riportato un raro caso di melanoma maligno amelanotico (AMM) che amplia la comprensione e integra i fonotipi del melanoma.
Il resveratrolo (RSV) è un tipo di fitoalessina ampiamente distribuita nella dieta mediterranea che potrebbe agire anche da soppressore del tumore. Abbiamo valutato gli effetti dell'RSV sulle cellule di melanoma (A375) e abbiamo scoperto che l'RSV potrebbe inibire la proliferazione delle cellule di melanoma modulando il ciclo cellulare e innescando l'apoptosi; in particolare, l’espressione della Cyclin D1 e PCDH9 sono stati fortemente influenzati dalla durata del trattamento dell'RSV, mentre l’espressione di Rac1 non è stata affatto alterata. Abbiamo ulteriormente esplorato il meccanismo di questi geni mirati del melanoma.
Studi in silico hanno mostrato che il PCDH9 potrebbe rappresentare il nuovo biomarcatore del melanoma. Pertanto, l'alterazione dell'espressione di PCDH9 (sovraespressione e interferenza) è stata valutata per esplorare gli effetti di PCDH9 sul melanoma. La comune metalloproteinasi della matrice (MMP) è responsabile della degradazione della matrice extracellulare. È stato dimostrato che MMP2, tra l'insieme di enzimi MMP, gioca un ruolo importante nella migrazione cellulare. I risultati della qRT-PCR hanno mostrato che PCDH9 potrebbe sopprimere le cellule di melanoma influenzando MMP2, CCND1 (Cyclin D1) e RAC1. Il melanoma e i tessuti sani sono stati analizzati analogicamente per dimostrare l'inibizione delle cellule di melanoma da parte del PCDH9. I metodi di Co-IP e LC-MS/MS sono stati utilizzati anche per indagare in profondità la correlazione tra PCDH9 e i suoi effetti di soppressione del melanoma. Abbiamo scoperto che PCDH9 e RAC1 possono predire la prognosi del melanoma maligno e abbiamo ipotizzato che PCDH9 possa modulare la progressione del melanoma attraverso MMP2 e RAC1 riducendo la generazione di ROS dipendente da RAC1 e migliorando il complesso di attività dell'ossidasi NADPH. Abbiamo anche riportato il raro caso di AMM diagnosticato per la prima volta come carcinoma cutaneo a cellule squamose. Questo caso mostra i vari fenotipi del melanoma.With the medical improvement, the life expectancy has rocketed globally. Cancer as the main Noncommunicable disease (NCDs) is the major barrier to extend longevity, causing also a huge medical resource expense. Although innovative treatments as gene therapy, immunotherapy and classical treatments (i.e. chemotherapy, radiotherapy and surgical removal), there is no effective treatment to cure cancer, even because patients usually develop chemoresistance, radioresistance and some resist to gene therapy and immunotherapy as well. Skin is the largest organ first barrier of human body. The basal cell carcinoma, squamous cell carcinoma and melanoma are three common skin cancers Melanoma is a type of cancer that originally develops from melanocytes (pigment containing cells). Melanoma has dramatically increased during last 30 years with low five-year survival and prognosis rate. The current therapeutic approaches of melanoma not only could bring treatment assistance but also have serious side effects like vitiligo. Under the setting of melanoma study, the current thesis investigated the melanoma suppression on different platforms. Firstly, resveratrol, a common bioactive compound, was used to target the potential biomarkers of melanoma. Secondly, through previous studies and then in silico research, certain biomarkers were furtherly targeted. Then in vitro melanoma-cell-culture models were investigated. We demonstrated that the melanoma cells were inhibited by protein-protein interaction. Third, after LC-MS/MS, the protein database was used to analyze and annotate the functions of the potential biomarkers. Forth, the rare case of amelanotic malignant melanoma (AMM) was reported that enlarged the understanding and supplement the phonotypes of melanoma. Resveratrol (RSV) is a kind of phytoalexin that is widely distributed in Mediterranean diet, that as a bioactive natural product, could be a tumor suppressor. We evaluated the effects of RSV on melanoma cells (A375) and found that RSV could obviously inhibit the proliferation of melanoma cells by modulating cell cycle and triggering apoptosis; Cyclin D1 and PCDH9 were strongly affected by RSV duration while RAC1 was not influenced. We furtherly explored the mechanism of these targeted genes of melanoma. In silico and reference studies exhibited the PCDH9 would be the novel biomarker of melanoma. Therefore, the alteration of PCDH9 expression (overexpression and interference) were performed to explore the effects of PCDH9 on melanoma. The common matrix metalloproteinase (MMPs) is responsible for the extracellular matrix degradation. MMP2 -among the set of enzymes (MMPs)- has been demonstrated to play important roles in cell migration. The results of qRT-PCR exhibited that PCDH9 could suppress melanoma cells by affecting MMP2, CCND1 (Cyclin D1) and RAC1. The melanoma and healthy tissues were analogically analyzed to demonstrate the inhibition of melanoma cells by PCDH9. The methods of Co-IP and LC-MS/MS were used as well to deeply investigate the correlation between PCDH9 and its suppression effects of melanoma. We found that PCDH9 and RAC1 can predict the prognosis of malignant melanoma and hypothesized that PCDH9 can modulate melanoma progression through MMP2 and RAC1 by reducing RAC1-dependent ROS generation and enhancing NADPH oxidase activity complex. The rare AMM firstly diagnosed as cutaneous squamous cell carcinoma (cSCC) was reported. According to the results of immunohistochemical examination (Ki67 (+++), Melan-A (+++), human melanoma black (HMB)45 (+), CD20 (-), cytokeratin (CK)7 (-) and CK5/6 (-) were found), the AMM was confirmed and the patient was applied surgical resection. This case showed the various phenotypes of melanoma.
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Chakrabarti, Partha Pratim. "Biochemical and biophysical analysis of the GTPase activating proteins of the small guanine nucleotide binding protein Rap1 and RheB." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=975809598.
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15
Snodgrass, Meagan Alyssa. "Analysis of the small GTP binding protein Rac2." Thesis, Montana State University, 2005. http://etd.lib.montana.edu/etd/2005/snodgrass/SnodgrassM0805.pdf.
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16
Taylor, Helena Olga Beatrice. "Sequence specificity of the yeast telomere protein RAP1." Thesis, University of Cambridge, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.625074.
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17
Bishop, Anne Louise. "Functional analysis of the Rac binding protein POSH." Thesis, University College London (University of London), 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.248466.
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18
Tsukamoto, Noriyuki. "Rap1 GTPase-activating Protein SPA-1 Negatively Regulate Cell Adhesion." Kyoto University, 1999. http://hdl.handle.net/2433/181245.
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19
Daumke, Oliver. "Structural and functional analysis of the GTPase activating protein of the small guanine nucleotide binding protein Rap1." [S.l. : s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=971950806.
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20
Nery, Flavia Cristina. "Estudos funcionais e estruturais da proteina reguladora humana Ki-1/57." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316877.
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Orientador: Jorg KobargTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: Ki-1/57 é um antígeno humano de 57kDa reconhecido pelo anticorpo Ki-1, o qual também reconhece CD30. Ki-1/57 se encontra no núcleo e no citoplasma sendo fosforilado nos resíduos de serina e de treonina após a ativação das células. Quando Ki-1/57 foi isolado da linhagem L540 de células de linfoma de Hodgkin, ela co-imunoprecipitou com atividade quinase em resíduos de Ser/Thr. Além disso, foi relatado que Ki-1/57 interage com o ácido hialurônico e conseqüentemente foi denominada de ¿proteína intracelular que se liga a hialuronato 4¿ (IHABP4). Nós usamos o sistema de duplo híbrido em levedura e encontramos que Ki-1/57 interage especificamente com a proteína com domínios cromo-helicase e de ligação ao DNA 3 (CHD3), uma proteína nuclear envolvida em remodelagem da cromatina e regulação da transcrição, e com RACK1 (¿proteína adaptadora para quinase C ativada¿). A interação entre Ki-1/57 e seus ligantes foi confirmada por outros experimentos in vitro e in vivo. Interessantemente, a interação entre Ki-1/57 e RACK1 foi abolida após fosforilação da Ki-1/57 e observamos que o estímulo de células L540 e HeLa com 4 a -forbol 12-miristato 13-acetato (PMA) resulta na saída de Ki-1/57 do núcleo. Ki-1/57 também demonstrou ser um substrato para proteína quinase C (PKC) quando ativada com PMA, e sua fosforilação foi confirmada in vitro e in vivo. Esses dados sugerem que Ki-1/57 está associada com a via de sinalização celular de RACK1/PKC e isto pode ser importante para a regulação de suas funções nucleares. Sua interação com CHD3 e com outras proteínas envolvidas na regulação transcricional, tais como: Topors, Daxx e Tip60, entre outras, sugere que Ki-1/57 pode ter uma função neste contexto funcional. RACK1 interage com p73, um parálogo de p53, inibindo sua ativação transcricional. Nós ainda encontramos que Ki-1/57 também interage com p53 não fosforilada e pode inibir sua ativação transcricional. A estrutura tridimensional da proteína Ki-1/57 é desconhecida, mas nossos estudos espectroscópicos mostraram que a proteína Ki-1/57 é predominantemente constituída por folhas b-pregueadas. Além disso, Ki-1/57(122-413) apagou o sinal do espectro de CD de RACK1 entre 229-300 nm, que é característico de proteínas ricas em triptofanos, e também diminuiu a intensidade de emissão de fluorescência de RACK1. Isso sugere que Ki-1/57 interage com os triptofanos na superfície de RACK1
Abstract: Ki-1/57, the 57-kDa human protein antigen recognized by the CD30 antibody Ki-1, is a cytoplasmic and nuclear protein, which is phosphorylated on serine and threonine residues upon cell activation. When isolated from the Hodgkin¿s lymphoma analogous cell line L540 Ki-1/57 was co-immunoprecipitated with a Thr/Ser protein kinase activity. It has been also found to interact with hyaluronic acid and has therefore been termed intracellular hyaluronan binding protein 4 (IHABP4). We used the yeast-two-hybrid system to identify proteins interacting with Ki-1/57 and found that Ki-1/57 engages in specific interactions with the Chromatin-Helicase-DNA-binding domain protein 3 (CHD3), a nuclear protein involved in chromatin remodeling and transcription regulation, and with the adaptor protein Receptor of Activated Kinase-1 (RACK1). Next, we confirmed these interactions by in vitro and in vivo experiments. Interestingly, the interaction of Ki-1/57 with RACK1 is abolished upon activation of L540 cells with 4a-phorbol 12-myristate 13-acetate (PMA), which results in the phosphorylation of Ki-1/57 and its exit from the nucleus. We demonstrated that Ki-1/57 also co-precipitates with protein kinase C (PKC) when isolated from PMA activated L540 tumor cells and is a substrate for PKC phosphorylation in vitro and in vivo. These events associate Ki-1/57 with the RACK1/PKC pathway and may be important for the regulation of its nuclear functions. Its interaction with chromatin remodeling factors such as CHD3 and other proteins involved in transcriptional regulation including Topors, Daxx and Tip 60 may suggest that Ki-1/57 has also a function in this context and that this function is subject to regulatory events involving the PKC/RACK1 signaling pathway. RACK1 also interacts with the p53 paralogue p73. In that case, the physical binding of RACK1 to p73 can inhibit its transcription activation function. We found out that Ki-1/57 interacts with unphosphorylated p53 and that this binding inhibits p53 transcription activation function. The three-dimensional structure of Ki-1/57 is still unknown but our spectroscopic studies demonstrated that Ki-1/57 consists predominantly of b-sheets. Binding of Ki-1/57(122-413) to RACK1 abolishes its positive ellipticity at 229-300 nm, which is characteristic for tryptophan-rich proteins, and decreases its emission fluorescence. This suggests that surface tryptophans of RACK1 are involved in the interaction with Ki-1/57
Doutorado
Genetica Humana e Medica
Doutor em Genetica e Biologia Molecular
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Leo, Christopher. "Differential Mechanisms of Nuclear Receptor Regulation by the Coactivator RAC3: A Dissertation." eScholarship@UMMS, 2000. https://escholarship.umassmed.edu/gsbs_diss/110.
Повний текст джерелаАнотація:
The steroid/thyroid hormone receptor superfamily is a large class of ligand-dependent transcription factors that plays a critical role in regulating the expression of genes involved in a broad range of physiological functions, including development, homeostasis, and reproduction. In the absence of cognate hormone, several receptors are able to repress transcription below the basal level via the recruitment of the nuclear receptor corepressors SMRT and NCoR. Upon hormone binding by the receptor, the corepressor complex is dissociated and a coactivator complex is subsequently recruited. This thesis details the mechanisms by which receptor-associated coactivator 3 (RAC3) interacts with nuclear receptors, particularly the vitamin D, estrogen, and retinoid receptors, and modulates their transcriptional activity. It was discovered that these receptors interact with different α-helical LXXLL motifs of RAC3 in vitro. Mutation of specific motifs differentially impairs the ability of RAC3 to enhance transcription by the receptors in vivo. In addition, the intrinsic transcriptional activation function of RAC3 was also characterized. Here, a single LXXLL motif, NR box v, was found to be essential to activation by serving as a binding surface for the general transcriptional integrator CBP/p300. Finally, the cofactor binding pocket of retinoid receptors was characterized. It was demonstrated that, to a large extent, the coactivator pocket of RARα overlaps with the corepressor pocket, with the exception of helix 12, which is required for coactivator, but not corepressor binding. Recruitment of RAC3 or SMRT also correlates directly with the ability of RARα to activate or repress transcription, respectively. Intriguingly, it was discovered that the AF-2 domain of RXRα inhibited cofactor binding to RXRα heterodimers, for deletion of this domain dramatically enhanced RAC3 and SMRT binding. In addition, it was demonstrated that the RXRα cofactor binding pocket contributed minimally to recruitment of cofactors. Conversely, the AF-2 domain of the partnering monomer and its cofactor pocket were required for these interactions. These findings suggest that the partner of RXRα is the primary docking point for cofactors at RXRα heterodimeric complexes. Taken together, this work contributes significantly to the field of nuclear receptor function in detailing the mechanisms by which the coactivator RAC3 is recruited to nuclear receptors and regulates their transcriptional activity.
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Francis, Richard Edward. "Functional and biochemical characterisation of a novel Rac binding protein." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429102.
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Fonjungo, Peter Nkong. "Human antibody responses to Plasmodium falciparum merozoite rhoptry associated protein 1 (RAP1)." Thesis, University of Edinburgh, 1998. http://hdl.handle.net/1842/12022.
Повний текст джерелаАнотація:
My main aim was to investigate human Ab responses to RAP1. For this study, I have developed nine recombinant RAP1 proteins (rRAP1) representing almost the entire sequence of mature RAP1 that have been expressed in Escherichia coli as soluble hexa-histidine or GST fusions. The antigenicity of the rRAP1 proteins was assessed by immunogenicity tests in mice and rabbits, and by P. falciparum RAP1-specific mAbs recognising a defined linear epitope. Antisera to seven of the rRAP1 proteins specifically reacted with parasites in immunofluorescence as well as parasite-derived RAP1 protein (PfRAP1) in immunoblotting. These results indicate that these rRAP1 proteins bear antigenic similarity to P. falciparum RAP1. Affinity purified Abs produced in rabbits against three rRAP1 proteins block invasion of red blood cells by merozoites, and this suggests that the proteins contain protective epitopes. Analysis of serum Abs of residents of malaria endemic regions by ELISA shows that RAP1 is antigenic during naturally transmitted malaria infection. The recombinant proteins are specifically recognised by IgG Abs, with detectable Abs directed mostly towards fragments containing N-terminal sequences of mature PfRAP1. By contrast, only few individuals had Abs to the C-terminus. Abs from malaria patients do not complete for a linear epitope recognised by an inhibitory anti-RAP1 mAb. This indicates that Abs from malaria patients bind mostly to epitopes different from that recognised by the inhibitory mAb. In a longitudinal study of individuals conducted over a period of 4 years in a region of seasonal and unstable malaria transmission, I have found that Abs to RAP1 are produced only after a documented clinical malaria.
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D'Silva, Nisha Jacinta. "Rap1, a small GTP-binding protein in the rat parotid gland : identification, investigation of function and regulation /." Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/6388.
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Shutes, Adam. "The small G-protein Rac nucleotide exchange and interactions with IQGAP." Thesis, University College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.272074.
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Rigas, Anastasia Catherine. "The role of the scaffolding protein RACK1 in the androgen receptor signalling pathway." Thesis, University of Newcastle Upon Tyne, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.413937.
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Yasuda(Yoshiki), Sayaka. "Ras and calcium signaling pathways converge at Raf1 via the Shoc2 scaffold protein." Kyoto University, 2011. http://hdl.handle.net/2433/142105.
Повний текст джерела
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Tapon, Nicolas Alexandre Marie. "Identification of new targets for the Rho and Rac GTPases." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.286591.
Повний текст джерела
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Thompson, Mary Katherine. "Functions of the eukaryote-specific ribosomal protein Asc1 /RACK1 in gene-specific translational activity." Thesis, Massachusetts Institute of Technology, 2015. http://hdl.handle.net/1721.1/101355.
Повний текст джерелаАнотація:
Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biology, 2015.Cataloged from PDF version of thesis.
Includes bibliographical references.
Although the ribosome operates as a single molecular entity, it is composed of both ribosomal RNAs and dozens of proteins. However, the individual contributions of most ribosomal components to translational regulation are unknown. In Chapter 1, I will review the current state of knowledge related to the functions of ribosomal proteins with a focus on the RACK1 protein (Asc1 in yeast), a eukaryote-specific ribosomal protein with many proposed functions in both cellular signaling and translation. In Chapter 2, I will present evidence that the Asc1 protein is required for efficient translation of a specific set of mRNAs with short open reading frames (ORFs), including those that encode ribosomal proteins and nuclear-encoded mitochondrial components. Consistent with these translation defects, ASC1 mutants are unable to grow in conditions requiring full mitochondrial function. Asc1-sensitive mRNAs are highly associated with the translational closed-loop complex, a group of proteins that promotes a loop-like conformation of the mRNA during translation by simultaneous interaction with the 5' and 3' ends of the mRNA molecule. In wild type cells, mRNAs that associate strongly with the translational closed-loop complex are much shorter than other ORFs. Thus, I hypothesize that the closed-loop is preferentially formed and/or stabilized on mRNAs with short ORFs, and that this process is enhanced by the presence of Asc1 on the small ribosomal subunit. The dependence of closed-loop formation on ORF length could also explain why short ORFs have notably higher translation efficiency than longer ORFs, a trend I observed in data collected from several eukaryotes. In Chapter 3, I will present evidence that the mammalian RACK1 protein is also required for expression of mRNAs with short ORFs and for mitochondrial function in HeLa cells, similar to my observations in yeast. These findings hint at a conserved role for the Asc1/RACK1 protein in promoting the function of the closed-loop complex and the translation of short ORFs, which encode a set of highly abundant proteins required for central metabolic functions. Chapter 4 will discuss the biochemical and cell physiological implications of these findings and suggest some avenues for future research.
by Mary Katherine Thompson.
Ph. D.
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Nanavaty, Vishal P. "Function of Telomere Protein RAP1 and Telomeric Transcript in Antigenic Variation in Trypanosoma Brucei." Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1485424039406009.
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Hennig, Sandra Elisabeth. "Insights into the ATP-dependent reductive activation of the Corrinoid/Iron-Sulfur Protein of Carboxydothermus hydrogenoformans." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2014. http://dx.doi.org/10.18452/16984.
Повний текст джерелаАнотація:
Die Verknüpfung einer exergonischen mit einer endergonischen Reaktion zur Ermöglichung der letzteren ist eine in biologischen Systemen weit verbreitete Strategie. Energetisch benachteiligte Elektronenübertragungsreaktionen im Rahmen der reduktiven Aktivierung von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen, der zu diesen verwandten Benzoyl-CoA-Reduktasen und diversen Cobalamin-abhängigen Methyltransferasen sind gekoppelt an die Hydrolyse von ATP. Der Methylgruppentransfer des reduktiven Acetyl-CoA-Weges von Carboxydothermus hydrogenoformans erfordert den Co(I)-Zustand des Corrinoid/Eisen-Schwefel Proteins (CoFeSP). Um diese superreduzierte Form nach einer oxidativen Inaktivierung zu regenerieren ist ein „Reparaturmechanismus“ erforderlich. Ein offenes Leseraster (orf7), welches möglicherweise für eine reduktive Aktivase von Corrinoid Enzymen (RACE) kodiert, wurde in dem Gencluster der am reduktiven Acetyl-CoA-Weg beteiligten Proteine entdeckt. Im Rahmen dieser Arbeit wurde dieses potenzielle RACE Protein biochemisch und strukturell charakterisiert und die ATP-abhängige reduktive Aktivierung von CoFeSP untersucht. Auf Grundlage der in dieser Arbeit gewonnenen Ergebnisse wurde ein Mechanismus für die ATP-abhängige Aktivierung entworfen. Dieser gibt Einblicke wie die durch ATP-Hydrolyse bereitgestellte Energie einen energetisch ungünstigen Elektronentransfer ermöglichen kann. Hierzu kombiniert RACo das Ausgleichen von Bindungsenergien mit Modulationen am Elektronenakzeptor. Eine vergleichbare Strategie wurde bisher in keinem anderen ATP-abhängigen Elektronenübertragungssystem wie dem von Nitrogenasen, Radikal-abhängigen β,α-Dehydratasen oder Benzoyl-CoA-Reduktasen beobachtet und könnte ein für RACE Proteine allgemein gültige Eigenschaft darstellen.The principle of coupling an exergonic to an endergonic reaction to enable the latter is a widespread strategy in biological systems. Unfavoured electron transfer reactions in the reductive activation of nitrogenases, radical-dependent β,α-dehydratases and the related benzoyl- CoA reductases, as well as different cobalamin-dependent methyltransferases are coupled to the hydrolysis of ATP. The reductive acetyl-CoA pathway of Carboxydothermus hydrogenoformans relies on the superreduced Co(I)-state of the corrinoid/iron-sulfur protein (CoFeSP) that requires a “repair mechanism” in case of incidental oxidation. An open reading frame (orf7) coding for a putative reductive activase of corrinoid enzymes (RACE) was discovered in the gene cluster of proteins involved in the reductive acetyl-CoA pathway. In this work, this putative RACE protein was biochemically and structurally characterised and the ATP-dependent reductive activation of CoFeSP was investigated. Based on the results of this study, a mechanism for the ATP-dependent reactivation of CoFeSP was deduced providing insights into how the energy provided by ATP could trigger this unfavourable electron transfer. The reductive activator of CoFeSP combines balance of binding energies and modulations of the electron acceptor to promote the uphill electron transfer to CoFeSP. A comparable strategy has not been observed in other ATP-dependent electron transfer systems like nitrogenases, radical-dependent β,α-dehydratases and benzoyl- CoA reductases and could be a universal feature of RACE proteins.
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Bryan, Steven. "Rho/Rac GTP-binding proteins and their GTPase activating proteins in humans and in Dictyostelium discoideum." Thesis, University College London (University of London), 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266164.
Повний текст джерела
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Gaullier, Guillaume. "Étude structurale de l'assemblage du complexe télomérique humain TRF2/RAP1." Thesis, Paris 11, 2015. http://www.theses.fr/2015PA114831/document.
Повний текст джерелаАнотація:
Les télomères sont les extrémités des chromosomes linéaires des eucaryotes.Ils sont constitués de répétitions en tandem d'un motif court riche enguanine, et liés par des protéines spécifiques. Chez les vertébrés cesprotéines forment un complexe appelé le shelterin et dont l'intégrité estcritique pour assurer la réplication correcte des extrémités deschromosomes, et pour les protéger contre une prise en charge illicite parles voies de réparation des cassures double-brin de l'ADN. Des dysfonctionsdes télomères engendrent une instabilité du génome qui peut conduire à lasénescence ou au cancer. Les télomères représentent une région subnucléaireoù les protéines du shelterin sont fortement enrichies, ce qui permetl'implication dans les fonctions biologiques d'interactions de basseaffinité. Parmi les protéines du shelterin, la protéine de liaison auxrépétitions télomériques TRF2 et son partenaire constitutif RAP1 sont lesfacteurs majeurs responsables de la protection des extrémités. Nous avonsétudié en détails l'assemblage du complexe TRF2/RAP1 par des approchesintégrées de biologie structurale, de biophysique et de biochimie.Nous avons montré que cet assemblage s'accompagne d'importants ajustementsde conformation des deux protéines, et implique une interaction de basseaffinité qui engage de grandes régions des deux protéines et affecte leurspropriétés d'interactionsTelomeres are the ends of eukaryotic linear chromosomes. They are made oftandem repeats of a short guanine-rich motif and bound by specific proteins.In vertebrates, these proteins form a complex called shelterin, theintegrity of which is critical to ensure proper replication of chromosomeends and to protect them against illicit targeting by DNA double-strandbreak repair pathways. Telomere dysfunctions lead to genome instability,which can ultimately cause senescence or cancer. Telomeres are a subnuclearregion in which shelterin proteins are highly enriched, enhancing lowaffinity interactions of important biological function. Among shelterinproteins, telomeric repeat-binding protein TRF2 and its constitutive partnerRAP1 are the main factors responsible for end protection. We studied indetails the assembly of TRF2/RAP1 complex by means of integrated structural,biophysical and biochemical approaches. We showed that this assemblydisplays important conformational adjustments of both proteins, and involvesa low affinity interaction engaging large regions in both proteins whichaffects their interaction properties
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ZAMBERLAN, MARGHERITA. "La piccola GTPasi Rap1 interposta tra la proteina mitocondriale Opa1 e l'inibizione dell'angiogenesi." Doctoral thesis, Università degli studi di Padova, 2022. https://hdl.handle.net/11577/3460979.
Повний текст джерелаАнотація:
OPA1 is a protein with pleiotropic functions ranging from the orchestration of mitochondrial fusion and cristae remodeling to transcriptional reprogramming 1, 2. Here we present two different mechanisms by which OPA1 exerts a transcriptional regulation activity in endothelial and breast cancer cells.
Mitochondria are dynamic organelles that are now recognized as regulators of signal transduction able to impact on cellular genetic programs 3, 4. Increasing evidence support a fundamental role for mitochondrial shape in the orchestration of cellular transcriptional programs, but how cells sense and respond to changes in mitochondrial shape is unclear 5. We recently discovered that angiogenesis is transcriptionally modulated by the key mitochondrial fusion gene OPA1 through NFκB activation1. In particular, ablation of OPA1 in vivo and in vitro leads to developmental and tumor angiogenesis inhibition 1. A deep RNA sequencing analysis identified a signature for the Ras-proximate-1 RAP1, and its cyclic AMP (cAMP)-activated nucleotide exchange factor EPAC1 upon OPA1 deletion in Human Umbilical Vein Endothelial Cells (HUVECs). Previously, several studies had reported the essential role of Rap1 in developmental angiogenesis and vessel stabilization 6, 7. After birth, Rap1 is not essential, but it participates in the maintenance of vasculature and nitric oxide (NO) homeostasis 6, 8. Albeit EPAC1 is highly abundant and cover numerous functions in endothelial cells, its role in angiogenesis remained to be clarified 9. Likewise, EPAC1 was shown to be important for endothelial cells biology 10, 11. A handful of studies retrieved EPAC1 in mitochondria, and RAP1 in mitochondria associated membranes (MAMs) by proteomics, suggesting that they might be linked to mitochondria 12, 13. Whether the EPAC1/RAP1 axis could sense changes in mitochondria driven by OPA1 deletion was unknown. Our results show that EPAC1 and RAP1 localize in proximity to mitochondria and in MAMs, that are emerging as hubs for mitochondria-derived signals. Moreover, EPAC1 accumulates on mitochondria upon pharmacological activation and following OPA1 silencing. OPA1 silencing results in an increase in Ca2+ and in localized cAMP increase in proximity of mitochondria that in turn activated EPAC1 and therefore RAP1. Notably, Ca2+ chelation by BAPTA-AM treatment suppress the EPAC1/RAP1 activation elicited by OPA1 downregulation. Furthermore, the analysis of angiogenic parameters like migration and tubulogenesis revealed that the blockage of EPAC1 and RAP1 signaling could correct the defects caused by OPA1 downregulation. Thus, our work places EPAC1/RAP1 in the retrograde signaling pathway connecting mitochondria to angiogenesis and highlights the intricate network of signals and second messengers that can execute transcriptional changes when mitochondria are perturbed.OPA1 is a protein with pleiotropic functions ranging from the orchestration of mitochondrial fusion and cristae remodeling to transcriptional reprogramming 1, 2. Here we present two different mechanisms by which OPA1 exerts a transcriptional regulation activity in endothelial and breast cancer cells. Mitochondria are dynamic organelles that are now recognized as regulators of signal transduction able to impact on cellular genetic programs 3, 4. Increasing evidence support a fundamental role for mitochondrial shape in the orchestration of cellular transcriptional programs, but how cells sense and respond to changes in mitochondrial shape is unclear 5. We recently discovered that angiogenesis is transcriptionally modulated by the key mitochondrial fusion gene OPA1 through NFκB activation1. In particular, ablation of OPA1 in vivo and in vitro leads to developmental and tumor angiogenesis inhibition 1. A deep RNA sequencing analysis identified a signature for the Ras-proximate-1 RAP1, and its cyclic AMP (cAMP)-activated nucleotide exchange factor EPAC1 upon OPA1 deletion in Human Umbilical Vein Endothelial Cells (HUVECs). Previously, several studies had reported the essential role of Rap1 in developmental angiogenesis and vessel stabilization 6, 7. After birth, Rap1 is not essential, but it participates in the maintenance of vasculature and nitric oxide (NO) homeostasis 6, 8. Albeit EPAC1 is highly abundant and cover numerous functions in endothelial cells, its role in angiogenesis remained to be clarified 9. Likewise, EPAC1 was shown to be important for endothelial cells biology 10, 11. A handful of studies retrieved EPAC1 in mitochondria, and RAP1 in mitochondria associated membranes (MAMs) by proteomics, suggesting that they might be linked to mitochondria 12, 13. Whether the EPAC1/RAP1 axis could sense changes in mitochondria driven by OPA1 deletion was unknown. Our results show that EPAC1 and RAP1 localize in proximity to mitochondria and in MAMs, that are emerging as hubs for mitochondria-derived signals. Moreover, EPAC1 accumulates on mitochondria upon pharmacological activation and following OPA1 silencing. OPA1 silencing results in an increase in Ca2+ and in localized cAMP increase in proximity of mitochondria that in turn activated EPAC1 and therefore RAP1. Notably, Ca2+ chelation by BAPTA-AM treatment suppress the EPAC1/RAP1 activation elicited by OPA1 downregulation. Furthermore, the analysis of angiogenic parameters like migration and tubulogenesis revealed that the blockage of EPAC1 and RAP1 signaling could correct the defects caused by OPA1 downregulation. Thus, our work places EPAC1/RAP1 in the retrograde signaling pathway connecting mitochondria to angiogenesis and highlights the intricate network of signals and second messengers that can execute transcriptional changes when mitochondria are perturbed.
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RINALDI, CARLO. "Functions and regulation of the MRX and Ku protein complexes at DNA ends." Doctoral thesis, Università degli Studi di Milano-Bicocca, 2023. https://hdl.handle.net/10281/402372.
Повний текст джерелаАнотація:
L'instabilità del genoma è una delle caratteristiche delle cellule tumorali e può essere causata da difetti nella riparazione del DNA. In particolare, le rotture del doppio filamento del DNA (DSBs) sono lesioni altamente citotossiche che possono formarsi accidentalmente durante la replicazione del DNA o in seguito all'esposizione ad agenti genotossici, e devono essere correttamente riparate al fine di garantire la stabilità genomica. Per far fronte a queste lesioni del DNA, le cellule eucariotiche attivano la risposta al danno del DNA (DDR) e utilizzano due meccanismi principali per la riparazione dei DSBs: l’unione terminale non omologa (NHEJ) e la ricombinazione omologa (HR). La risposta cellulare ai DSBs ha inizio con il reclutamento dei complessi Ku e MRX/MRN alle due estremità rotte di un DSB. Inoltre, il complesso MRX recluta al DSB anche Tel1/ATM, una chinasi coinvolta nel checkpoint da danno al DNA. Tel1, a sua volta, consente di promuovere e stabilizzare l'associazione del complesso MRX sia ai DSBs che ai telomeri in un ciclo a feedback positivo. Ku, MRX/MRN e Tel1/ATM sono anche necessari per mantenere la lunghezza dei telomeri, strutture nucleoproteiche specializzate situate alle estremità dei cromosomi eucariotici. Il DNA telomerico deve inoltre essere distinto dalle estremità dei DSBs intracromosomici attraverso diversi complessi proteici, i quali vengono reclutati ai telomeri al fine di prevenire l'attivazione della DDR. Nel lievito S. cerevisiae, Rif2 e Rap1 costituiscono due delle principali proteine che compongono tali complessi. Sia Rif2 che Rap1 contrastano l'attivazione di Tel1, la degradazione nucleolitica e l’unione terminale non omologa ai telomeri. Rif2 sembra esercitare tutte queste funzioni inibendo l'associazione del complesso MRX al DNA telomerico; tuttavia, restava ancora da determinare come Rap1 controllasse negativamente l'attività di MRX alle estremità del DNA. Nella prima parte del mio dottorato di ricerca, ho contribuito a dimostrare che Rif2 contrasta l'associazione del complesso MRX sia ai DSBs che ai telomeri in modo dipendente da Rap1. Rap1, a sua volta, può inibire le funzioni di MRX in modo sia dipendente sia indipendente da Rif2, e le funzioni di Rap1 alle estremità del DNA sono influenzate dalle modalità con cui questa proteina lega il DNA. In merito al NHEJ, una questione importante è rappresentata dal mantenimento delle estremità di un DSB in stretta prossimità fra loro, necessario per consentire una corretta rilegatura. Questa funzione è chiamata end-tethering e sebbene alcuni dati in E.coli abbiano suggerito un coinvolgimento del complesso Ku in questo meccanismo di controllo, restava ancora da chiarire quale fosse il suo esatto ruolo nell’end-tethering. Nella seconda parte del mio dottorato, ho quindi studiato questa problematica tramite la generazione di una variante mutante della proteina Ku70 in grado di aumentare la persistenza del complesso Ku ai DSBs. La caratterizzazione dell'allele ku70-C85Y ha consentito di dimostrare che il complesso Ku promuove l’end-tethering del DNA e la mutazione C85Y migliora tale funzione aumentando la ritenzione di Ku in stretta prossimità alle estremità di un DSB. Inoltre, la funzione svolta da Ku nel DSB end-tethering è regolata da Tel1/ATM, che antagonizza tale funzione del complesso Ku limitandone la persistenza alle estremità dei DSBs. Poiché la presenza del complesso Ku alle estremità dei DSBs impedisce l'accesso delle nucleasi di resezione, la regolazione dell'associazione di Ku alle estremità rotte del DNA mediata da Tel1 fornisce un importante livello di controllo nella scelta tra il meccanismo di NHEJ e di HR, suggerendo una nuova funzione di Tel1 nella risposta al danno al DNA. Tutti questi risultati hanno contribuito a chiarire i meccanismi molecolari che modulano la riparazione del DNA in risposta ai DSBs, con un focus specifico sulle funzioni e sulla regolazione dei complessi MRX e Ku.Genome instability is one of the hallmarks of cancer cells and it can be caused by DNA repair defects. Among several types of DNA damage, DNA double-strand breaks (DSBs) are highly cytotoxic lesions that can form accidentally during DNA replication or upon exposure to genotoxic agents. DSBs must be repaired to avoid loss of genetic information and to ensure genomic stability. Eukaryotic cells repair DSBs by activating the DNA damage response (DDR) and by using two main mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HR). The cellular response to DSBs is initiated by the recruitment of Ku (Ku70-Ku80) and MRX/N (Mre11-Rad50-Xrs2/Nbs1) complexes at the two DSB broken ends. MRX in turn recruits Tel1/ATM, a kinase involved in the DNA damage checkpoint, a surveillance mechanism that couples DSB repair and cell-cycle progression. Tel1 allows to promote and stabilize MRX association at both DSBs and telomeres in a positive feedback loop. Ku, MRX/MRN, and Tel1/ATM are also required to maintain the length of telomeres, specialized nucleoprotein complexes at the ends of eukaryotic chromosomes. Furthermore, telomeric DNA must be distinguished from intrachromosomal DSBs ends through different protein complexes, which are recruited to telomeres in order to prevent DDR activation. In S. cerevisiae, Rif2 and Rap1 are two of the main proteins that compose these complexes. Both Rif2 and Rap1 counteract Tel1 activation, nucleolytic degradation, and NHEJ at telomeres. Rif2 appears to exert all these functions by inhibiting MRX association with telomeric DNA, however how Rap1 negatively controls MRX activity at DNA ends remained to be determined. In the first part of my PhD, I contributed to show that Rif2 counteracts MRX association at both DSBs and telomeres in a Rap1-dependent manner. Rap1 in turn can inhibit MRX functions in a Rif2-dependent and -independent manner, and Rap1 functions at DNA ends are influenced by its DNA binding mode. An important issue in NHEJ is the maintenance of the DSB ends in close proximity to allow their correct re-ligation. This function is called end-tethering and some data in E.coli suggested an involvement of the Ku complex in this control mechanism. However, a Ku role in end-tethering remained to be determined. In the second part of my PhD, I investigated this issue by generating a Ku70 mutant variant that increases Ku persistence at DSBs. The characterization of the ku70-C85Y allele has allowed to show that the Ku complex promotes DSB end-tethering and the C85Y mutation enhances this bridging function by increasing Ku retention very close to the DSB ends. The function of Ku in DSB end-tethering is also regulated by Tel1/ATM, which antagonizes this Ku function by limiting Ku persistence at the DSB ends. As the presence of Ku at the DSB ends prevents the access of resection nucleases, the Tel1-mediated regulation of Ku association with the DSB ends provides an important layer of control in the choice between NHEJ and HR mechanism, suggesting a new function of Tel1 in the DNA damage response. All these findings contributed to elucidate the molecular mechanisms that modulate DNA repair and maintain genome stability in response to DSBs, with a specific focus on the functions and regulation of MRX and Ku complexes.
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Natarajan, Suchitra. "Roles of high mobility group AT-hook protein 2 (HMGA2) in human cancers." Elsevier, 2013. http://hdl.handle.net/1993/31092.
Повний текст джерелаАнотація:
High Mobility Group AT-hook protein 2 (HMGA2) is a non-histone chromatin binding protein expressed in stem cells, cancer cells but not in normal human somatic cells. The presence of HMGA2 in cancer correlates with advanced neoplastic disease and poor prognosis. HMGA2 plays important roles in Base Excision Repair (BER) and at replication forks. HMGA2 is present at mammalian metaphase telomeres and its loss induces chromosomal aberrations. However, the functional role of HMGA2 at telomeres remains elusive. We hypothesized a protective role of HMGA2 that guards telomeres and modulates DNA damage repair signaling pathways. Employing different HMGA2+ human tumor cell models, we investigated the HMGA2-mediated functions that contribute to chemoresistance in glioblastoma (GB).
This study presents a novel interaction of HMGA2 with telomeric protein TRF2 (Telomere Repeat-Binding Factor 2). This interaction retains TRF2 at telomeres, thus capping the telomeres and reducing telomere-dysfunction induced foci despite induced telomere stress. Loss of HMGA2 coincides with increased phosphorylation of TRF2, decreased TRF2 retention at telomeres and increased formation of telomeric aggregates, anaphase bridges and micronuclei. These findings provide new evidence for a unique role of HMGA2 at telomeres as a novel contributor of telomeric integrity. We show that upon DNA damage, HMGA2 causes increased and sustained phosphorylation of Ataxia Telangiectasia and Rad3-related kinase (ATR) and checkpoint kinase 1 (CHK1). Prolonged presence of pCHK1Ser296 coincides with prolonged G2/M block and increased tumor cell survival. The relationship between (ATR)-CHK1 DNA damage response pathway and HMGA2 identifies a novel mechanism by which HMGA2 can alter DNA repair function in cancer cells.
We identified HMGA2 as a novel factor contributing to temozolomide (TMZ) resistance in GB. HMGA2 knockdown sensitizes the GB cells to TMZ. We propose a specific combination of FDA-approved drugs, TMZ and Dovitinib (DOV), to increase GB cell death. We show that DOV downregulates key BER proteins, attenuates pSTAT3-coordinated Lin28A and HMGA2 expression. Our results suggest that a sequential therapeutic strategy of pretreating GB cells with DOV followed by a sequence of TMZ and DOV diminishes TMZ resistance and enhances the ability of TMZ to induce GB cell death.
Overall, we identified HMGA2 as a multifunctional survival factor in human cancer cells and showed that targeting HMGA2 is a valid strategy to combat HMGA2+ cancer cells.February 2016
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Wang, Lei [Verfasser], and Regine [Akademischer Betreuer] Kahmann. "Functional characterization of a seven-WD40 repeat protein Rak1 in Ustilago maydis / Lei Wang. Betreuer: Regine Kahmann." Marburg : Philipps-Universität Marburg, 2011. http://d-nb.info/101328867X/34.
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Ferrari, Giovanna Maria. "The interaction of the α2 chimaerin SH2 domain with target proteins." Thesis, University College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325678.
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Zheng, Xiuzhong. "Definition of Ku-interacting domains in RAG1 and RAG2 proteins in V(D)J recombination." Thesis, University of Ottawa (Canada), 2005. http://hdl.handle.net/10393/27102.
Повний текст джерелаАнотація:
V(D)J recombination is a process that generates the diversity of the immune repertoire against foreign antigens. During B and T cell development, genes encoding immunoglobulins (Ig) and T cell receptors (TCR) variable region are somatically assembled by selective V (variable), D (diversity) and J (joining) segments pre-existing in the germline. Recombination-activating genes 1 and 2 (RAG1/RAG2), the lymphocyte-specific factors, initiate V(D)J recombination by nicking at the border the heptamer sequence of RSS (recombination signal sequence) and generate four DNA double stranded breaks (DSB) in the cleavage step. After cleavage, RAG1/RAG2 complex are still bound to the DNA ends. In the joining step, DNA breaks are processed and rejoined by non-homologous end joining apparatus, which includes Ku70/Ku80, DNA-PKcs, Artemis, XRCC4 and DNA ligase IV. However, how the cleavage step is linked to the joining step is not yet known.
My results suggest that a physical interaction between RAG1/2 and Ku antigen may help coordinate the cleavage stage of V(D)J with the non homologous DNA end rejoining of the mature sequences. (Abstract shortened by UMI.)
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Diekmann, Dagmar. "Structural and functional analysis of the small GTP-binding proteins rho and rac." Thesis, Institute of Cancer Research (University Of London), 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283195.
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Asprodites, Nicole. "The Cloning and Characterization of Two ROP/RAC G-Proteins from Gossypium Hirsutum." ScholarWorks@UNO, 2005. http://scholarworks.uno.edu/td/233.
Повний текст джерелаАнотація:
Rop/Rac proteins are plant-specific monomeric guanosine triphosphate-binding proteins (G-proteins) with important functions in plant development. Until recently, only three cotton (Gossypium hirsutum) Rop/Rac G-protein genes were sequenced, representing subfamilies III and IV of the plant monomeric Gprotein family. In this project, members of subfamilies II and I were cloned, sequenced, and named GhRac2 and GhRac3, respectively. Using real-time reverse transcription PCR, expression of GhRac2 was highest during fiber elongation, decreasing significantly when cellulose biosynthesis began. Transcript abundance of GhRac3 doubled between fiber elongation and secondary wall synthesis, remaining constant until 20 days post-anthesis. Expression of GhRac2 and GhRac3 was compared between the unfertilized ovules of Gossypium hirsutum, Texas Marker 1 and two near-isogenic fiber-impaired mutants. Expression of GhRac2 and GhRac3 was significantly higher in wild type ovules than in Ligon lintless, a mutant impaired in fiber elongation, but was not different in Naked Seed, a mutant impaired in fiber initiation.
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Scavello, Margarethakay, Alexandra R. Petlick, Ramya Ramesh, Valery F. Thompson, Pouya Lotfi, and Pascale G. Charest. "Protein kinase A regulates the Ras, Rap1 and TORC2 pathways in response to the chemoattractant cAMP in Dictyostelium." COMPANY OF BIOLOGISTS LTD, 2017. http://hdl.handle.net/10150/624638.
Повний текст джерелаАнотація:
Efficient directed migration requires tight regulation of chemoattractant signal transduction pathways in both space and time, but the mechanisms involved in such regulation are not well understood. Here, we investigated the role of protein kinase A (PKA) in controlling signaling of the chemoattractant cAMP in Dictyostelium discoideum. We found that cells lacking PKA display severe chemotaxis defects, including impaired directional sensing. Although PKA is an important regulator of developmental gene expression, including the cAMP receptor cAR1, our studies using exogenously expressed cAR1 in cells lacking PKA, cells lacking adenylyl cyclase A (ACA) and cells treated with the PKA-selective pharmacological inhibitor H89, suggest that PKA controls chemoattractant signal transduction, in part, through the regulation of RasG, Rap1 and TORC2. As these pathways control the ACA-mediated production of intracellular cAMP, they lie upstream of PKA in this chemoattractant signaling network. Consequently, we propose that the PKA-mediated regulation of the upstream RasG, Rap1 and TORC2 signaling pathways is part of a negative feedback mechanism controlling chemoattractant signal transduction during Dictyostelium chemotaxis.
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Stephan, Ilona [Verfasser]. "Analyse der Aktivierung von Rac-GTPasen durch G-Protein-gekoppelte Rezeptoren in neutrophilen Granulozyten / Ilona Stephan." Ulm : Universität Ulm. Medizinische Fakultät, 2001. http://d-nb.info/1015269095/34.
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Sooman, Linda. "Prognostic Biomarkers and Target Proteins for Treatment of High-grade Gliomas." Doctoral thesis, Uppsala universitet, Institutionen för radiologi, onkologi och strålningsvetenskap, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-215079.
Повний текст джерелаАнотація:
The survival for high-grade glioma patients is poor and the treatment may cause severe side effects. A common obstacle in the treatment is chemoresistance. To improve the quality of life and prolong survival for these patients prognostic biomarkers and new approaches for chemotherapy are needed. To this end, a strategy to evade chemoresistance was evaluated by combining chemotherapeutic drugs with agents inhibiting resistance mechanisms identified by a bioinformatic analysis (paper I). The prognostic value of 13 different proteins was analyzed in this thesis (papers II-IV). Two of them, p38 mitogen-activated protein kinase (MAPK) and protein tyrosine phosphatase non-receptor type 6 (PTPN6, also known as SHP1) were analyzed for their potential as targets in combination chemotherapy (in paper III and IV, respectively). We found that: PTPN6 expression and methylation status may be important for survival of anaplastic glioma patients, p38 MAPK phosphorylation may be a potential negative prognostic biomarker for high-grade glioma patients and FGF2 expression may be a potential negative prognostic biomarker for proneural glioma patients. PTPN6 may be a useful target for combination chemotherapy with cisplatin, melphalan or bortezomib in high-grade gliomas. The following drug combinations; camptothecin combined with an EGFR or RAC1 inhibitor, imatinib combined with a Notch or RAC1 inhibitor, temozolomide combined with an EGFR or FAK inhibitor and vandetanib combined with a p38 MAPK inhibitor may be useful combination chemotherapy for high-grade gliomas.
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Freire, José Ednésio da Cruz. "Análise in silico da sequência deduzida de Mo-CBP3, uma proteína ligante à quitina de Moringa oleifera LAM." reponame:Repositório Institucional da UFC, 2013. http://www.repositorio.ufc.br/handle/riufc/18245.
Повний текст джерелаАнотація:
FREIRE, José Ednésio da Cruz. Análise in silico da sequência deduzida de Mo-CBP3, uma proteína ligante à quitina de Moringa oleifera LAM. 2013. 116 f. Dissertação (Mestrado em Bioquímica) - Universidade Federal do Ceará, Fortaleza-CE, 2013.Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-05-30T12:44:05Z No. of bitstreams: 1 2013_dis_jecfreire.pdf: 2576370 bytes, checksum: c3841faa82a44a8e3de82ce42c785dc5 (MD5)
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Moringa oleifera is a tree belonging to the Moringaceae family. This plant is native from India where it is named as drumstick tree. In Brazil M. oleifera was introduced in the 1950’s decade and it is known as moringa. Approximately 40% of the fresh weight of these seeds is formed by proteins, some of which were isolated and characterized as flocculants and antinutritional proteins. In addition, the chitin binding proteins have been identified and isolated, especially among which is the Mo-CBP3, a thermostable glycoprotein of apparent molecular mass of around 14.3 kDa, with potent inhibitory activity against phytopathogenic fungi. In order to characterize the deduced sequence of Mo-CBP3, moringa fruits were collected 65 days after anthesis and their seeds subjected to extraction of total RNA. cDNA synthesis was directed by ‘5’ RACE’-PCR. The PCR products were subcloned into appropriate vectors (pGEM-T Easy) and then introduced into Escherichia coli cloning host TOP10F'. The recombinant plasmids were purified from transformed bacterial cell and subjected to DNA sequencing. The computational analysis of the deduced protein sequence of Mo-CBP3 showed that this protein has an apparent molecular mass of 12.85 kDa and it is unstable in cytoplasmic conditions. It has derived signal sequences, one for the signal peptide with 30 amino acids, and a sequence at the C-terminus of this protein, related to the anchorage to the plasma membrane as well as the endoplasmic reticulum. Moreover, probable sites of O-glycosylation and phosphorylation were identified. One domain related to the lipid transfer functions, reserve and trypsin inhibitors and alpha-amylase was identified following Mo-CBP3, thereby contributing to the understanding of its potent action against phytopathogenic fungi.
A Moringa oleifera é uma planta pertencente à família Moringaceae. Esta planta é nativa da Índia, sendo lá conhecida como drumstick (baqueta ou bastão de tambor). No Brasil, a M. oleifera foi introduzida na década de 1950, e é conhecida como moringa. Aproximadamente 40% do peso fresco das sementes é composto por proteínas, das quais algumas foram isoladas e caracterizadas como sendo floculantes e proteínas antinutricionais. Em adição, proteínas ligantes à quitina têm sido identificadas e isoladas, destacando-se dentre estas a Mo-CBP3, uma glicoproteína termoestável de massa molecular aparente em torno de 14,3 kDa, com potente atividade inibitória contra fungos fitopatogênicos. A fim caracterizar a sequência deduzida da Mo-CBP3, frutos de moringa foram coletados após 65 dias da antese e suas sementes submetidas à extração de RNA total. A síntese de cDNA foi dirigida por meio da técnica PCR-RACE 5'. Os produtos de PCR foram subclonados em vetores apropriados (pGEM-T Easy) e, em seguida, introduzido em hospedeiro de clonagem Escherichia coli TOP10F'. Os plasmídeos recombinantes foram purificados de células bacterianas transformadas e submetidos ao sequenciamento de DNA. A análise computacional da sequência deduzida da proteína Mo-CBP3 mostrou que esta é uma proteína de massa molecular aparente em torno de 12,85 kDa e, em condições citoplasmáticas apresenta-se instável. Possui sequências sinais deduzidas, sendo uma para peptídeo sinal, com 30 aminoácidos, e uma sequência na região C-terminal relacionada à ancoragem desta proteína à membrana plasmática, bem como ao retículo endoplasmático. Ademais, prováveis sítios de O-glicosilação e de fosforilação foram identificados. Um domínio relacionado às funções de transferência de lipídeos, de reserva e de inibidores de tripsina e de alfa-amilase foi identificado na sequência de Mo-CBP3, contribuindo, desse modo, para o entendimento de sua potente ação contra fungos fitopatogênicos.
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Warren-Paquin, Maude. "Regulation of synaptic plasticity at the Drosophila larval NMJ : the role of the small GTPase Rac." Thesis, McGill University, 2008. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112319.
Повний текст джерелаАнотація:
We are interested in understanding the molecular mechanisms that govern synaptic growth and plasticity. Recent evidence from several laboratories suggests that small GTPases play an important role in the promotion of neurite outgrowth; however, their role in the control of synaptic growth and functional plasticity is not well understood. The goal of this thesis is to investigate the role of small GTPases (including Rac, Rho and Cdc42) in the regulation of synaptic growth in vivo, using the Drosophila larval neuromuscular junction (NMJ) synapses as a model system. Our results show that presynaptic overexpression of Rac, but not of Rho or Cdc42, positively regulates both synaptic structure and function. Genetic loss of Rac leads to embryonic lethality, making it impossible to assess the full loss-of-function phenotype using conventional mutants. To circumvent this, we use the MARCM (Mosaic Analysis with a Repressible Cell Marker) technique to generate single motor neuron clones devoid of all genetic copies of Rac. Our data suggest that Rac activity is crucial for normal synaptic development. In support of this conclusion, we demonstrate that genetic removal of trio, a guanine nucleotide exchange factor (GEF) that is known to activate Rac, leads to a drastic reduction in the number of synaptic boutons. In addition, genetic removal of one copy of trio is sufficient to suppress the gain-of-function phenotype of Rac. Moreover, we demonstrate that partial removal of the fragile X mental retardation gene (dfmr1), a known suppressor of Rac, enhances the gain-of-function phenotype of Rac. Taken together, our findings support a model in which Rac signaling positively regulates synaptic growth and function at the Drosophila larval NMJ.
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Khanna, Ankita, Pouya Lotfi, Anita J. Chavan, Nieves M. Montaño, Parvin Bolourani, Gerald Weeks, Zhouxin Shen, et al. "The small GTPases Ras and Rap1 bind to and control TORC2 activity." NATURE PUBLISHING GROUP, 2016. http://hdl.handle.net/10150/614747.
Повний текст джерелаАнотація:
Target of Rapamycin Complex 2 (TORC2) has conserved roles in regulating cytoskeleton dynamics and cell migration and has been linked to cancer metastasis. However, little is known about the mechanisms regulating TORC2 activity and function in any system. In Dictyostelium, TORC2 functions at the front of migrating cells downstream of the Ras protein RasC, controlling F-actin dynamics and cAMP production. Here, we report the identification of the small GTPase Rap1 as a conserved binding partner of the TORC2 component RIP3/SIN1, and that Rap1 positively regulates the RasC-mediated activation of TORC2 in Dictyostelium. Moreover, we show that active RasC binds to the catalytic domain of TOR, suggesting a mechanism of TORC2 activation that is similar to Rheb activation of TOR complex 1. Dual Ras/Rap1 regulation of TORC2 may allow for integration of Ras and Rap1 signaling pathways in directed cell migration.
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Brembu, Tore. "Genetic, molecular and functional studies of RAC GTPases and the WAVE-like regulatory protein complex in Arabidopsis thaliana." Doctoral thesis, Norwegian University of Science and Technology, Faculty of Natural Sciences and Technology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-741.
Повний текст джерелаАнотація:
Small GTP-binding proteins are molecular switches that serve as important regulators of numerous cellular processes. In animal and plant cells, the Rho family of small GTPases participate in e.g. organisation of the actin cytoskeleton, production of reactive oxygen species through the NADPH oxidase complex, regulation of gene expression. The three most extensively studied subgroups of the Rho GTPase family are Cdc42, Rho and Rac. One of the mechanisms by which animal Rac and Cdc42 GTPases regulate actin filament organisation is through activation of the ARP2/3 complex, a multimeric protein complex which induces branching and nucleation/elongation/polymerisation of actin filaments. Activation of the ARP2/3 complex by Rac and Cdc42 is mediated through the proteins WAVE and WASP, respectively.
In a search for Ras-like GTPases in Arabidopsis, we identified a family of genes with similarity to Rac GTPases. Screens of cDNA and genomic libraries resulted in the finding of 11 genes named ARACs/AtRACs. Genes encoding Rho, Cdc42 or Ras homologues were not identified. Expression analysis of AtRAC1 to AtRAC5 indicated that AtRAC1, AtRAC3, AtRAC4 and AtRAC5 are expressed in all parts of the plant, whereas AtRAC2 is preferentially expressed in root, hypocotyl and stem.
The AtRAC gene family can be divided into two main groups based on sequence similarity, gene structure and post-translational modification. AtRAC group II genes contain an additional exon, caused by the insertion of an intron which disrupts the C-terminal geranylgeranylation motif. Instead, group II AtRACs contain a putative motif for palmitoylation. Phylogenetic analyses indicated that the division of plant RACs into group I and group II occurred before the split of monocotyledonous and dicotyledonous plants. Analyses of the genes neighbouring AtRAC genes revealed that several of the plant RAC genes have been created through duplications.
The restricted/tissue-specific expression pattern of AtRAC2 led us to do a more detailed expression analysis of this gene. A 1.3 kb fragment of the upstream (regulatory) sequence of AtRAC2 directed expression of GUS or GFP to developing primary xylem in root, hypocotyl, leaves and stem. In root tips, the onset GUS staining or GFP fluorescence regulated by the AtRAC2 promoter slighty preceded the appearance of secondary cell walls. In stems, GUS staining coincided with thickening of xylem cell walls. Transgenic plants expressing constitutively active AtRAC2 displayed defects in the polar growth of leaf epidermal cells, indicating that AtRAC2 may be able to regulate the actin cytoskeleton. Surprisingly, an AtRAC2 T-DNA insertion mutant did not show any observable phenotypes. GFP fusion proteins of wild type and constitutively active AtRAC2 were both localised to the plasma membrane. The data suggest that AtRAC2 is involved in development of xylem vessels, likely through regulation of the actin cytoskeleton or NADPH oxidase.
The role of RAC GTPases in regulation of the actin cytoskeleton in plants is well documented. However, although the ARP2/3 complex had been identified in plants/Arabidopsis, the mechanisms regulating this complex were unknown. Through database searches, we identified three Arabidopsis genes, AtBRK1, AtNAP and AtPIR, which encoded proteins with similarity to subunits of a protein complex shown to regulate the activity of WAVE1 in mammalian cells. T-DNA inactivation mutants of AtNAP and AtPIR displayed morphological defects on epidermal cells undergoing polar expansion, such as trichomes and leaf pavement cells. The phenotypes were similar to those observed for ARP2/3 complex mutants, suggesting that AtNAP and AtPIR act in the same pathway as the ARP2/3 complex in plants. The actin cytoskeleton in atnap and atpir mutants was less branched than in wild type plants; instead, actin filaments aggregated in thick actin bundles.
Finally, we have recently discovered a small gene family encoding putative WAVE homologues. In mammalian cells, Rac activates WAVE1 through binding to PIR121 or Sra1 (the mammalian homologues of AtPIR). The discovery of a putative WAVE regulatory complex as well as putative WAVE homologues in Arabidopsis suggests that plant RAC GTPases regulate organisation of the actin cytoskeleton during polar growth at least partly through the ARP2/3 complex, using an evolutionarily conserved mechanism.
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Falsetti, Samuel C. "The Role of RalA and RalB in Cancer." [Tampa, Fla] : University of South Florida, 2008. http://purl.fcla.edu/usf/dc/et/SFE0002307.
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Deiß, Katharina [Verfasser], and Martin [Akademischer Betreuer] Lohse. "Die Regulation des Kinasemodulators Raf Kinase Inhibitor Protein (RKIP): Einfluss von Phosphorylierung und Dimerisierung auf die Interaktion mit Raf1 und G-Protein-gekoppelter Rezeptorkinase 2 (GRK2) / Katharina Deiß. Betreuer: Martin Lohse." Würzburg : Universitätsbibliothek der Universität Würzburg, 2012. http://d-nb.info/1028326408/34.
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