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1
Blackburn, Elizabeth Anne. "Biophysical studies of protein-ligand interactions and the discovery of FKBP12 inhibitors". Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/6504.
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The principal aim of this study was to discover, through virtual screening, new nonimmunosuppressive inhibitors for the human immunophilin FKBP12, a target of the immunosuppressant drugs rapamycin and FK506. The enzyme acts as peptidyl-prolyl isomerase catalysing protein folding in the cell. Structurally similar isomerase domains are important for molecular recognition in multi-domain chaperone proteins. FKBP inhibitors have been shown to have protective effects against nerve damage and are therefore interesting targets for the treatment of neurodegenerative diseases. Virtual screening has been used to discover novel inhibitors for protein drug targets. Recent advances in computational power and the availability of large virtual libraries, such as the EDULISS database at Edinburgh University, have enhanced the appeal of this approach. X-ray structures of known protein-ligand complexes were examined to obtain an understanding of the key non-covalent interactions in the FKBP12 binding pocket. Virtual screening hits were selected using macromolecular docking and programs that employed a ligand-based approach. The bulk of the virtual screening in this study used Edinburgh University’s in-house program LIDAEUS. In the course of this study nearly three hundred compounds were screened in the laboratory using biophysical and biochemical binding assays. Thirty four compounds were found to have an affinity for FKBP12 of less than one hundred micromolar. To test virtual hits, it was necessary to select the most appropriate medium-throughput biophysical assay. The aim was to employ methods with sufficient sensitivity to detect compounds with affinity in the order of one hundred micromolar, coupled with the capacity to screen hundreds of compounds in a week. This study used a wide variety of biophysical techniques, these including: electrospray ionisation mass spectrometry, surface plasmon resonance and isothermal titration calorimetry. There was a particular emphasis on the quality of data from electrospray ionisation mass spectrometry. A correlation was found between the cone voltages that gave 50 % dissociation of the complex with the enthalpic contribution to the free energy of binding. From the careful examination of the differences in charge-state distributions between a pure protein and a protein-ligand mixture, it was possible to determine if a protein-ligand complex had been present in solution prior to dissociation during the electrospray process. This observation provides the basis for an assay that could be of general utility in detecting very weak inhibitors.
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Zibrova, Darya. "Adenovirus-mediated gene transfer of FK506-binding proteins FKBP12.6 and FKBP12 in failing and non-failing rabbit ventricular myocytes". Doctoral thesis, [S.l.] : [s.n.], 2004. http://deposit.ddb.de/cgi-bin/dokserv?idn=972602275.
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Main, Ewan Ralph Gibson. "Studies on the immunosuppressant binding protein FKBP12 and the nuclear/steroid receptors vitamin D3 and oestrogen". Thesis, University of Cambridge, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.621749.
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Chaurasia, S. "IN SILICO STUDY OF PROTEIN PROTEIN INTERACTION STABILIZATION AND MECHANICAL FORCE APPLICATION ON BIOMOLECULES". Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229253.
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Targeting protein-protein interactions is a challenging task in drug discovery process. Despite the challenges, several studies have provided evidences for the development of small molecules modulating protein-protein interactions. In Part I, it is demonstrated that how a small molecule can induce the formation of an otherwise unstable protein-protein complex. A study of the stabilization of a FKBP12-FRB complex by a small molecule rapamycin is presented. The stability of the complex is analyzed and its interactions are characterized at the atomic level by performing free energy calculations and computational alanine scanning. It is shown that rapamycin stabilizes the complex by acting as a bridge between the two proteins; and the complex is stable only in the presence of rapamycin. The reported results and the good performance of standard molecular modeling techniques in describing the model system can be interesting not only in the design and development of improved molecules acting as FKBP12–FRB protein interaction stabilizers, but also in the somehow neglected study of protein-protein interactions stabilizers in general.
In Part II, studies regarding computational modeling of the application of mechanical force to biomolecules is presented. This part is further divided into two chapters since the investigations have been performed on two biological systems. In the first chapter of Part II (chapter 6), it is described that how the osmolyte molecules affect the mechanical unfolding of a peptide. The mechanical unfolding of peptide has been performed by using Steered Molecular Dynamics. In this study, the effect of four different osmolytes on the free energy difference between the folded and the denatured state have been calculated. The observed trend mirrors the expected behavior of the studied osmolytes and unfolding pathways analysis allows an insight into the mechanism of action of osmolytes.
After the successful application of Steered molecular dynamics technique on the β-hairpin peptide, the same is applied on tubulin heterodimers for the in-depth study of the lateral and longitudinal interactions which are responsible for the stability and dynamics of the microtubules. In the other chapter of Part II (chapter 7), these interactions are studied with the help of mechanical dissociation of the tubulin heterodimers. These studies have allowed the identification of the critical interactions responsible for the binding of tubulin heterodimers laterally as well as longitudinally. The observations obtained could be important for the design of compounds that target these interactions and acts as microtubule inhibitors or stabilizers.
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Kim, Ju Young. "M1 muscarinic acetylcholine receptor regulation of endogenous transient receptor potential-canonical, subtype 6 (TRPC6) channels". Connect to resource, 2005. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1117570788.
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Thesis (Ph. D.)--Ohio State University, 2005.Title from first page of PDF file. Document formatted into pages; contains xviii, 178 p.; also includes graphics. Includes bibliographical references (p. 163-178). Available online via OhioLINK's ETD Center
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De, Cicco Maristella Verfasser], Sonja A. [Akademischer Betreuer] [Gutachter] Dames y Aymelt [Gutachter] [Itzen. "NMR characterization of the membrane-localized interaction network between the kinase TOR, the GTPase Rheb and the FKBP12-like protein FKBP38. / Maristella De Cicco ; Gutachter: Aymelt Itzen, Sonja A. Dames ; Betreuer: Sonja A. Dames". München : Universitätsbibliothek der TU München, 2017. http://d-nb.info/1147566178/34.
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Davies, Todd Howard. "Regulation of Glucocorticoid Receptor Function by TPR-domain Proteins". University of Toledo Health Science Campus / OhioLINK, 2004. http://rave.ohiolink.edu/etdc/view?acc_num=mco1098292002.
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Olivieri, Lilian. "Recherche et caractérisation par dynamique moléculaire d'états intermédiaires pour la complexation entre la protéine FKBP12 et des ligands de haute affinité". Thesis, La Réunion, 2012. http://www.theses.fr/2012LARE0011/document.
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FKBP12 est une protéine ubiquitaire, principalement cytosolique, qui est au carrefour de plusieurs voies signalétiques. Son abondance naturelle dans les tissus nerveux peut être reliée à son implication dans les maladies neurodégénératives telles que les maladies d'Alzheimer et de Parkinson ainsi que dans les neuropathies périphériques et diabétiques ou dans des blessures des cordons spinaux. De nombreuses études ont montré que des molécules exogènes (ligands) venant se fixer sur cette protéine permettent la régénération d'un grand nombre de connexions neuronales endommagées. Une difficulté provient cependant du fait que, pour un ligand donné, il n'existe aucune relation claire entre sa structure et sa capacité de liaison à FKBP12. Notre étude vise ainsi à rationaliser la relation entre la structure d'un ligand et son affinité pour cette protéine. Deux complexes modèles, formés entre FKBP12 et chacun des deux ligands 8 et 308, ont été utilisés. Ces deux ligands de haute affinité ont des structures différentes. Notre travail s'est appuyé sur des simulations de dynamique moléculaire pour caractériser l'état intermédiaire qui est formé transitoirement lors du processus de complexation entre la protéine et son ligand. Dans cet état particulier, l'identification des interactions naissantes entre les partenaires a permis (i) de comprendre l'implication des différentes parties du ligand dans le mécanisme de reconnaissance avec FKBP12 et (ii) de rationaliser les affinités de certains ligands apparentésFKBP12 is an ubiquitous, mostly cytosolic, protein found at the crossroads of several signaling pathways. Its natural abundance in the nervous tissues can be related to its implication in neurodegenerative diseases like Alzheimer's and Parkinson's as well as in peripheral neuropathies and diabetes or in injuries of the spinal cords. Several studies have demonstrated that exogenous molecules (ligands) that can bind to FKBP12 allow the regeneration of many damaged neuron connections. However, there is no clear relationship between the structure of a ligand and its ability to bind to FKBP12. Our study aims at rationalizing the relationship between the structure of a ligand and its affinity to FKBP12. Two model complexes, formed between FKBP12 and each of the two high-affinity ligands 8 and 308, were studied. These two ligands are structurally different. We used molecular dynamics simulations to characterize the intermediate state that is transiently formed during the binding process between the protein and its ligand. In this state, the analysis of the nascent interactions allowed (i) to unravel the role played by the various ligand moieties in the recognition process with FKBP12 and (ii) to rationalize the affinities of related ligands
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Belnou, Mathilde. "Études biophysiques des propriétés et des interactions entre trois protéines impliquées dans la maladie d’Alzheimer : récepteur des oestrogènes α, Calmoduline et FKBP52". Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066262.
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Nous nous sommes intéressés à plusieurs protéines impliquées dans la maladie d’Alzheimer, notamment la protéine FKBP52, la calmoduline et le ROα. Nous nous sommes attachés à apporter quelques éléments de réponse quant à la formation d'un éventuel hétérocomplexe ROα/Ca4CaM/FKBP52. Dans une première partie, nous avons voulu étudier quelques bases moléculaires de l'interaction entre FKBP52 et la Ca4CaM, afin de mieux comprendre la pertinence biologique de cette affinité. Après avoir produit différents domaines de la protéine FKBP52 et la Ca4CaM, différentes techniques d’interaction protéine/protéine ont été utilisées. L'approche protéique de ce travail a été confortée par une approche peptidique. Elles ont permis de cibler le troisième domaine comme lieu de l’interaction. Pour la première fois, il a été totalement attribué par RMN et les sites concernés par l’interaction ont pu être discriminés. Par ailleurs, il a été montré que le premier domaine de FKBP52 pouvait interagir intermoléculairement avec ROα, par un motif en coude β de type II. Le ROα est un facteur de transcription dont l'activité dépend d'un certain nombre de coactivateurs parmi lesquels Ca4CaM. Le peptide issu de la séquence de recrutement de la calmoduline au sein du ROα (séquence 298-310) a fait l’objet au sein du groupe de nombreuses publications. Il a été montré que ce peptide possédait un caractère amyloïde. Bien qu’il n’existe aucun lien apparent entre cette caractéristique et une quelconque pathologie associée, la cinétique de formation des fibres issues de ce peptide dans différentes conditions de pH et de concentrations a été étudiéeWe are interested in several proteins involved in the Alzheimer disease, in particular the FKBP52, calmodulin and ERα. We have provided some answers concerning the formation of a possible ROα/Ca4CaM/FKBP52 heterocomplex. In a first part, we wanted to study the molecular basis of the interaction between FKBP52 and Ca4CaM, to better understand the biological relevance of this affinity. After producing different domains of the FKBP52 protein and Ca4CaM, various techniques such as ITC, SPR, fluorescence or NMR were used. The protein approach of this work was supported by a peptide based study. These approaches have made it possible to target the third domain as the place of interaction. For the first time, the TPR domain was assigned by NMR spectroscopy and the sequences involved in the interaction could be discriminated. Furthermore, it was shown that the first domain of FKBP52 could interact intermolecularly with the ROα, by a type II β-turn motif. ROα is a transcription factor whose activity depends on a number of coactivators including Ca4CaM. The peptide resulting from the recruitment sequence of calmodulin within ROα (sequence 298-310) has been the subject of numerous publications within the group. It has been shown that this peptide has an amyloid character. Although there is no apparent link between this feature and any associated pathology, the kinetics of fiber formation from this peptide under different pH and concentration conditions has been studied
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Stechschulte, Lance A. "The Co-chaperones FKBP51 and PP5 Control Nuclear Receptor Phosphorylation and Adipogenesis". University of Toledo Health Science Campus / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=mco1370871316.
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Mathioudakis, Nikolaos. "Etudes fonctionnelles sur le composant de la voie des piRNA TDRD1". Phd thesis, Université de Grenoble, 2012. http://tel.archives-ouvertes.fr/tel-00907417.
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Les ARN interagissants avec Piwi (ARNpi) sont des petits ARN non-codants qui sont exprimes dans la ligne grrminale des animaux. Ils interagissent avec les proteines de la branche Piwi de la famille des Argonautes en formant des complexes des ribonucleproteines impliques dans le maintien de l'intégrité du génome. La region N-terminale des quelques proteines Piwi contiennent symetriquement des arginines diméthylées. Il est considere que ce status symmetrique de la dimethylation est responsable du recrutement des proteines possédant des domaines Tudor (TDRDs). Ces domaines peuvent avoir un role comme platforme pour medier les interactions entre les proteines de la voie de l'ARNpi. Nous avons mesure indivindiuellemnt l'affinite de liaison des quatres domaines etendus Tudor (TD) de la proteine murine TDRD1 pour les trois differents peptides de la protein murine Mili qui contiennent de la methyl-arginine. Les resultats montrent une preference des TD2 et TD3 pour les peptides consecutives Mili alors que TD4 et TD1 ont une affinite plus bas et plus faible respectivement pour tous les peptides. Ces observations ont ete confirmees par des experiences pull-down en utilisant des proteines Piwi endogenes et des proteines-interagissent avec Piwi. L'affinite de TD1 pour les peptides qui contiennent de la methyl-arginine peut etre restoree par une seul mutation ponctuelle dans la cage aromatique pour revenir a la sequence consensus. La structure de cristal de la proteine TD3 lie au peptide methyle Mili montre une orientation inattendue de la peptide de liaison et de la chaine latérale de l'arginine methyle dans la cage aromatique. Finalement, le model SAXS des quatres domains tandem Tudor de TDRD1 revele une forme de la proteine flexible et elongee. Globalement, les resultats montrent que la proteine TDRD1 peut accommoder des differents peptides des differentes proteines et ainsi de fonctionner comme une protéine d'échafaudage dans la voie de l'ARNpi. La proteine FKBP6 (FK506 Binding Protein) a ete recemment identifiee comme un nouvel facteur interagissent dans la voie de l'ARNpi. FKBP6 est constituee d'une domaine d'isomerase FK et une domaine de tetratricopeptide (TPR). Une perte de la Fkbp6 conduit a la de -repression des transposons et a la sterilite masculine des souris. Le domaine TPR est implique dans l'interaction avec la proteine chaperone Hsp90 et le domaine FK est une isomerase inactive qui a ete evolue a une module structurale. En effectuant des exepriences biochimiques preliminaires nous avons identifie la region N-terminal du domaine MYND de TDRD1 comme le partenaire d'interaction du domaine FK de la FKBP6. Nous proposons que la proteine TDRD1 est une plateforme moleculaire qui reconnait des marques de methylation de MILI et elle recrute FKB6 pour promouvoir la formation d'un complexe indispensable pour la fonction de la voie de l'ARNpi.
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Stein, Walter von. "Charakterisierung von PTEN, FKBP59 und CG4420, Interaktionspartnern des PDZ-Domänen-Proteins Bazooka aus Drosophila melanogaster". [S.l.] : [s.n.], 2006. http://deposit.ddb.de/cgi-bin/dokserv?idn=981590128.
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McClelland, K. "Characterisation of a Novel Protein FKBPL/DIR1; Implications for Pathways Controlling Cell Growth and Survival". Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.501322.
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Bennett, Rachel. "Modulation of the anti-angiogenic protein FKBPL : implications for a host of diseases, including cancer". Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.680888.
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FKBPL is a secreted anti-angiogenic protein, with a therapeutic peptide, ALM201, based on the active domain of FKBPL shortly entering phase I clinical trials. The aim of this thesis was to characterise the effects of FKBPL modulation on physiological and pathological angiogenesis, to identify FKBPL-associated pathways and to determine how FKBPL transcription, translation and secretion is mediated. To study the effects of FKBPL modulation on angiogenesis and tumour growth, a novel amphipathic peptide, RALA, was used to deliver FKBPL siRNA and FKBPL cDNA in vitro and in vivo, to ZR-75-1 xenografts; increased FKBPL was I associated with delayed tumour growth, prolonged survival and decreased microvessel density (MVD), whilst decreased I expression resulted in increased MVD and stemness. The physiological impact of endogenous FKBPL was established by development of a Fkbpl+/- mouse; Fkbpl-/- mice I were embryonically lethal prior to E8.5, suggesting a critical role for FKBPL in embryonic development. However, whilst Fkbpl+/- embryos showed some vascular irregularities, the mice developed normally. In murine angiogenesis models including the aortic ring, sponge, and tumour growth assays, Fkbpl+/- mice exhibited significantly increased sprouting, enhanced vessel recruitment and faster tumour growth, respectively, compared to their wild -type littermates, supporting the anti-angiogenic function of FKBPL. Furthermore, Fkbpl+/ mice were more prone to obesity and were less able to regulate glucose levels; interestingly, ALM201 was able to normalise this phenotype. SIRT1, a key gene involved in ; obesity and diabetes was positively regulated by FKBPL, both in vitro and in vivo, going some way to explaining these effects. Furthermore, manipulation of the SIRT pathway also potentiated the anti-tumour activity of FKBPL. The regulation of FKBPL by pro-angiogenic stimuli, and its secretory pathway was,also investigated. FKBPL was secreted via the Golgi body to a greater extent in human microvascular endothelial cells compared to tumour cells, in keeping with its anti-angiogenic role. Protein and mRNA expression was unaffected by hypoxia and other angiogenic cytokines, VEGF, bFGF or IL8; whilst hypoxia inhibited its secretion in a normal endothelial cell line, but not in a cancer cell line. In conclusion, this indicates that FKBPL is a potent secreted anti-angiogenic protein, and is essential for normal 1 physiological development. As well as being an anti-angiogenic drug with potential for use in cancer treatment, ALM201 also has the potential to reduce weight gain and to normalise blood glucose in patients deficient in FKBPL, opening up further opportunities for future study.
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Miyadahira, Eduardo Hideki. "Efeito da remoção cirúrgica das lesões de endometriose profunda na expressão dos microRNAs-21, -451 e -29c e da proteína FKBP52". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/5/5139/tde-22082016-154558/.
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INTRODUÇÃO: O tratamento cirúrgico da endometriose profunda tem se mostrado benéfico para os resultados de Reprodução Assistida. O motivo que leva aos melhores resultados ainda é desconhecido, mas remete à etiologia multifatorial dessa doença. Observa-se, então, a oportunidade de investigar mecanismos moleculares que possam justificar este fato. Nesse contexto, os microRNAs podem desempenhar papel fundamental na medida em que alteram a expressão de diferentes genes por meio da inibição póstranscricional. OBJETIVO: Analisar o efeito da remoção cirúrgica das lesões de endometriose profunda na expressão dos microRNAs -21, -451 e -29c, além da expressão da proteína FKBP52. MÉTODOS: Trata-se de estudo clínico, prospectivo, longitudinal e comparativo no qual foram incluídas 26 pacientes que foram divididas em dois grupos, segundo o resultado da ultrassonografia transvaginal com preparo intestinal. As pacientes que não apresentaram alterações sugestivas de endometriose compuseram o grupo controle (n = 11) e foram submetidas à coleta de amostra de endométrio. As pacientes do grupo de estudo (n = 15) foram submetidas à amostragem endometrial antes e após o tratamento cirúrgico da endometriose, além de ter sido realizada a coleta de amostra da lesão profunda durante o ato operatório. Foi realizada a extração total de RNA dessas amostras e, posteriormente, PCR em tempo real para análise de expressão dos microRNAs -21, -451 e -29c, além da proteína FKBP52. RESULTADOS: A comparação da expressão relativa dos microRNAs -21 e -451 entre as amostras, de forma geral, não mostrou diferença estatisticamente significante. A expressão relativa do microRNA-29c foi maior no endométrio de pacientes com endometriose em relação ao grupo controle e ainda maior nas lesões de endometriose profunda. Após a cirurgia, a expressão do microRNA-29c no endométrio, foi equivalente à do grupo controle. A expressão da FKBP52 foi menor no endométrio das mulheres com endometriose e nas lesões da doença em comparação ao grupo controle. Após o tratamento cirúrgico houve aumento da expressão de FKBP52 nas amostras de endométrio nas pacientes com endometriose que passou a ser semelhante à do grupo controle. CONCLUSÕES: Os resultados sugerem que a presença das lesões de endometriose pode aumentar a expressão do microRNA-29c no endométrio e, por conseguinte, diminuir a expressão de um dos seus RNA mensageiros alvos que codifica a proteína FKBP52. Após o tratamento cirúrgico, com remoção das lesões de endometriose, a expressão do microRNA-29c e da FKBP52 se assemelhou à do grupo controleINTRODUCTION: Surgical treatment of deeply infiltrating endometriosis appears to yield benefits to the affected women and also to the assisted reproduction treatments. Although the mechanisms involved on the improvement of the outcomes are still unknown; yet, they are similar to the multifactorial origin of the endometriosis. Considering that the microRNAs can modify different genes expression, it was investigated their role concerning the molecular pathways leading to endometriosis and the clinical betterment of the assisted reproductive procedures after the surgical treatment. OBJECTIVE: The aim of this study was to evaluate the effect of surgical treatment in women with endometriosis focusing the microRNAs -21, -451 and -29c expression, as well as the protein FKBP52 expression. METHODS: This is a clinical, prospective, longitudinal and comparative study which included 26 patients that were divided into two groups according to the findings of the transvaginal ultrasound with bowel preparation. Eleven women without evidence of DIE composed the control group and were submitted to eutopic endometrium sampling. Fifiteen women presented with evidence for DIE detected by pelvic ultrasound were also submitted to eutopic endometrium sampling before and after surgical treatment. Surgical procedures revealed the presence of relevant DIE. Total RNA extraction of all samples was performed and followed by real time PCR to evaluate microRNAs -21, -451, -29c and protein FKBP52 expression. RESULTS: MicroRNAs -21 and -451 expression analysis did not show statistically significant difference among samples. Nonetheless, expression of microRNA-29c was elevated in eutopic endometrium of women suffering from DIE in comparison to the control group. After surgery, the microRNA- 29c expression in eutopic endometrium became equivalent to the control group. The expression for FKBP52 was lower in women having DIE than those without endometriosis. The surgical treatment increased the expression of the protein FKBP52 in eutopic endometrial samples, turning it similar to the control group. CONCLUSIONS: The results of this study suggest that the presence of DIE might increase the expression of microRNA-29c in eutopic endometrium and consequently decrease the expression of one of its target messenger RNA that codifies the protein FKBP52. After surgical removal of endometriosis lesions, the microRNA-29c and the FKBP52 expression returned to a level similar to the control group
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McAlpine, Kerry Elizabeth. "Characterisation of the novel hsp90 interacting protein FKBPL and its potential role in steroid receptor complexes". Thesis, Queen's University Belfast, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484996.
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The FKBPL protein was recently identified as an hsp90 interacting protein and shares significant homology to the immunophilins FKBP52 and cyclophilin 40. The FKBPs are involved in many cellular processes, one of the most documented being their role in hsp90-steroid 'receptor complexes. It was therefore thought logical to investigate a potential role for FKBPL in these complexes. The main aims were to evaluate potential interactions between FKBPL the glucocorticoid receptor (GR) and dynamitin. The functional role played by FKBPL would be addressed in overexpression studies. Since a single nucleotide polymorphic variant and a 12 bp insertion mutant of FKBPL had been identified, the ability of these variants to interact and function within steroid receptor complexes was also evaluated. Finally. a small study was carried out to determine if a correlation existed between the frequency of the FKBPL SNP and the incidence of cancer. Co-localisation studies revealed that FKBPL co-Iocalisea with the glucocorticoid receptor, dynamitin and tubulin. Interactions of FKBPL and its variants with dynamitin was verified using the biomolecular fluorescence complementation assay. furthermore, the cytoplasmic localisation of the interaction with hsp90 was determined in this assay. This assay also confirmed interactions of the FKBPL variants with hsp90. Interactions between endogenous FKBPL and polymorphic FKBPL with GR/dynamitin were confirmed by co-immunoprecipitation. These data suggested a role for FKBPL in steroid hormone receptor complexes. Co-localisation of GR and FKBPL was examined after treatment with the GR ligand dexamethasone. Both proteins translocated from the cytoplasm to the nucleus of DU145 cells within 10 min, indicative of retrograde transport. FKBPL overexpression was found to decrease GR protein levels and transactivity in L132 cells, and increase GR protein levels in DU145 cells. FKBPL overexpression also caused a dexamethasone . dose dependent increase in GR transactivity in DU145 cells. These data suggest FKBPL is involved in steroid rec~ptor complexes and modulates GR protein levels and signalling. Finally, we demonstrated that although the homozygous SNP variant was more prevalent in DNA from endometrial cancer and HNPCC patients, compared to controls, it \Vas not significant.
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Song, Zhi-Ning. "Development of novel affinity-guided catalysts for specific labeling of endogenous proteins in living systems". Kyoto University, 2017. http://hdl.handle.net/2433/228238.
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Neumann, Jacob Trevor. "THE ROLE OF ATP AND FK-506 BINDING PROTEIN IN THE COUPLED GATING OF SKELETAL RYANODINE RECEPTORS". OpenSIUC, 2011. https://opensiuc.lib.siu.edu/dissertations/348.
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During skeletal muscle stimulation, there is a summation of local events of Ca2+ release from the sarcoplasmic reticulum, known as Ca2+ sparks. Ca2+ sparks originate from groups of skeletal ryanodine receptors (RyR1) that activate and close in synchrony. This synchrony allows for the rapid and massive release of Ca2+ from the sarcoplasmic reticulum to initiate contraction and, more important, would provide a mechanism to terminate Ca2+ release under conditions where independent RyR1 are normally active. RyR1 mutations can result in abnormal intracellular Ca2+ signaling that is associated with numerous skeletal muscle disorders including malignant hyperthermia and central core disease. Therefore, investigating the mechanisms that control RyR1 function can help identify how these mutations cause deleterious Ca2+ handling. Currently, most published research on RyR1s gating utilizes single RyR1 reconstituted into planar lipid bilayers to test isolated RyR1. However, in vivo, arrays of RyR1 function in synchrony. Attempts to reconstitute RyR1s into planar lipid bilayers result in experiments that contain multiple channels, which under specific conditions may gate in synchrony, also known as coupled gating. Coupled RyR1 gating was first reported by A. Marks' laboratory and attributed to FK-506 binding protein 12 (FKBP12) associating with neighboring RyR1s the stabilization of RyR1-RyR1 interactions that promote coupled gating. Previous studies suggested that ATP is required for coupled RyR1 gating; however, the mechanism by which ATP promotes the coordinated activity of RyR1s has not been elucidated and is the focus of this thesis. Therefore, my hypothesis is that the agonist action of ATP and FKBP12 bound to RyR1 are required for coupled RyR1 gating. In addition, new pharmacological tools are required to better understand coupled RyR gating. Thus, an additional goal is to identify pharmacological agents that modulate RyR1s in an innovative manner, i.e., help to uncover novel aspects of RyR1 gating and conduction. This investigation suggests that the adenosine based nucleotides, ATP, ADP and AMP, are agonists of RyR1s and promote coupled RyR1 gating in planar lipid bilayers. However, ADP and AMP were unable to maintain coupled RyR1 gating with physiological levels of Mg2+. This suggests that coupled gating would be impaired when the levels ATP decrease, as in muscle fatigue. When ATP was compared to other nucleotides (GTP, ITP, and TTP), the results suggest that the nucleotide agonist action on RyR1s is dependent on the phosphate groups and amino group on the nucleobase. As ATP is the most efficient nucleotide for coupled gating, I also investigated the indirect action of ATP to act as a kinase substrate or alter the cytoskeletal network. The addition of kinases, phosphatases and cytoskeletal modulators did not produce a significant disruption of coupled RyR1 gating. I also tested the role of addition of exogenous FKBP12 to RyR1s that gated independently or had partial coupling, but coupled gating was never improved. Also, the addition of high doses of rapamycin to remove FKBP12 from coupled RyR1 failed to functionally uncouple the channels. Finally, I attempted to find pharmacological agents that could aid in the understanding of coupled RyR1. Some agents were found to modulate RyR1s; however, I did not find a probe that would affect kinetics/conductance of RyR1s and was suitable for comparing coupled gating in bilayers with Ca2+ sparks in cells. Overall, coupled RyR gating is dependent on the physiological modulators ATP and Mg2+. This thesis represents a step forward in identifying the requirements for coupled RyR1 gating and understanding how RyR1s function in cells. Until an understanding of how these receptors communicate in cells is obtained, how different mutations alter the Ca2+ leak will continue to be quite difficult to study.
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Donley, Christopher Blair. "The role of the oestrogen receptor interacting proteins, FKBPL and RBCK1, in breast cancer signalling". Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.601364.
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The aim of this thesis was to further characterise the role of FKBPL and RBCKl in the ERa: signalling pathway. Both RBCKl and FKBPL were found to interact with Hsp90 and ER and this interaction was enhanced by E2. Both RBCKl and FKBPL appear [0 be regulated by the oestrogen. However, both FKBPL and RBCKl bind along with ER« to the promoter of pS2, suggesting a potential role for FKBPL and RBCKl in ERa: trans-activation. RBCKl was shown [0 be the ubiquitin ligase for FKBPL. RBCKl was shown to facilitate the attachment of linear chains of ubiquitin to FKBPL. SiRNA knockdown of RBCKl resulted in increased levels of FKBPL in the T47D cell line, but inversely down-regulation of RBCKl in the MCF-7 cell line resulted in a reduction in FKBPL protein levels, suggesting that there is differential regulation of FKBPL by RBCKl depending on the cell line. FKBPL also appears to have a potential role in regulating the self-ubiquitination of RBCK1. RBCKl was also shown to regulate p21 transcription in a mechanism that was independent of p53. RBCKl siRNA knockdown resulted in increased p21 levels but overexpression of RBCKl in the MCF-7 cell line also increased p21 protein levels, suggesting that RBCKl regulates p21 levels through some post-translation modification, possibly through Akt. High levels of FKBPL and RBCKl were independently shown to correlate with increased patient survival in microarray data sets, and a combination of both high RBCKl and FKBPL was the most favourable phenotype. Finally the levels of RBCKl were shown to reduce the efficacy of tamoxifen in the MCF-7 and T47D cell lines, high RBCKl correlated with decreased tamoxifen efficacy. This effect was mirrored in a • micro-array data set, showing high RBCKl predicted worse response for tamoxifen therapy. Therefore RBCKl and FKBPL have potential as novel biomarkers for endocrine therapy.
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Haupt, Katja [Verfasser], C. [Akademischer Betreuer] Lücke, J. [Akademischer Betreuer] Balbach y B. [Akademischer Betreuer] Ludwig. "NMR-spektroskopische Untersuchungen der Protein-Ligand-Wechselwirkungen von FKBP38 und DnaK / Katja Haupt. Betreuer: C. Lücke ; J. Balbach ; B. Ludwig". Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2011. http://d-nb.info/1025230515/34.
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Martinelli, Silvia [Verfasser] y Mathias [Akademischer Betreuer] Schmidt. "Effect of stress on protein homeostasis mediated by FKBP51 as a possible mechanism underlying stress-related disorders / Silvia Martinelli ; Betreuer: Mathias Schmidt". München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1223369757/34.
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Nelson, Laura. "Evaluating the prognostic and predictive potential of FKBPL and associated proteins as biomarkers in breast and ovarian cancer". Thesis, Queen's University Belfast, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.677957.
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Despite adjuvant systemic therapies improving breast cancer survival rates, some patients are over- or under-treated. This is particularly problematic for the management of early stage ER+/LN- breast cancers. Identifying patients at a higher risk of relapse for whom additional adjuvant chemotherapy is necessary, remains a significant challenge. The primary focus of this thesis was to evaluate the prognostic potential of FKBPL within breast TMA cohorts, and as part of a meta-analysis. FKBPL was shown to be an independent prognostic marker, which is able to predict breast cancer specific survival in ER positive, node positive patients. I As many prognostic tests now focus on the inclusion of multiple markers, the prognostic ability of two FKBPL-associated proteins, phospho-ERα and p21, were also assessed, however both failed to correlate with survival. In an attempt to evaluate the prognostic potential of additional FKBPL-associated proteins, the relationship between FKBPL and an FKBPL-interacting protein, USP19, was characterised in breast cancer cell lines. After an interaction between the two proteins was confirmed, subsequent analysis identified a role for USP19 in the deubiquitination and stabilisation of FKBPL. USP19 was shown to positively regulate FKBPL protein expression in breast cancer cell lines, functionally suppressing breast cancer cell proliferation . . ALM201, an anti-angiogenic peptide based on FKBPLs active anti-angiogenic domain, is soon to enter Phase 1111 clinical trials in ovarian cancer patients. Therefore, the current study also aimed to evaluate FKBPL's prognostic potential within an ovarian cancer TMA cohort, with the potential to utilize the FKBPL biomarker as a companion diagnostic. Unfortunately, FKBPL was not prognostic in the ovarian cancer setting. In conclusion, FKBPL has the ability to further stratify ER+/LN- breast cancer patients, identifying patients who may benefit from additional adjuvant chemotherapy. Validating the prognostic potential of USP19, within the breast cancer setting might prove beneficial to further sensitise the marker.
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Edvardsson, Anna. "Peptidyl-prolyl cis-trans Isomerases in the Chloroplast Thylakoid Lumen". Doctoral thesis, Linköping : Univ, 2007. http://www.bibl.liu.se/liupubl/disp/disp2007/med983s.pdf.
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BIZZARRI, MARCO. "Advanced in silico techniques in Rational Drug Design. Application to immunophilin ligands". Doctoral thesis, 2013. http://hdl.handle.net/2158/794848.
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This thesis presents the application of advanced molecular dynamics techniques
as "tool" in design of small molecules that bind to protein targets. In
the first part, our initial goal was to understand the factors governing the
molecular recognition of FKBP12 binding domain, through the study of the
conformational space of known ligands with disparate affinity costants. In
the second part of this thesis, results of the former part, were applied to in
silico design of new high-affinity ligands. Subsequent synthesis and characterization
confirm that all novel ligands have affinity in the nano molar
range. Moreover, among these ligands, the compound Elte378 is, for our
knowledge, the most potent synthetic inhibitor of FKBP12 to date.
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LI, YI-NI y 李宜妮. "Modulate polyglutamine protein aggregation by Trigger factor and FKBP12". Thesis, 2013. http://ndltd.ncl.edu.tw/handle/68461287922832798868.
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碩士國立中央大學
化學學系
101
Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease caused by the mutational expansion of CAG triplet repeat in mutant Huntingtin (mHTT) protein. These proteins form aggregates in the affected neurons of patient brains that correlate with disease progression and toxicity. Recent studies reported chaperone can prevent protein misfolding and serve as powerful inhibitor mutant HTT-induced neurotoxicity. According to the recent studies, FKBP12 chaperone had PPIase activity and be decreased the protein level in HD mouse. Another chaperone, Trigger factor (TF), the TFPPIase and FKBP12 are structural homology, can decrease protein aggregates. Here, we investigated whether the presence of FKBP12/TF chaperone could effectively change amounts and properties of polyQ aggregates by biophysical/biochemical technique. We have successfully established the GST-polyQ system and apply in vitro aggregation assay to unravel the effect of chaperone in aggregation process. Turbidity assay and filter assay revealed TF can significantly suppress HTT43Q aggregates, while FKBP12 increase HTT43Q aggregates. Moreover, we examined the morphology of HTT25Q and HTT43Q in the presence/absence of TF/FKBP12 under Transmission Electron Microscopy (TEM). Massive fibrils can be observed in HTT43Q only. To our surprise, TF significantly changed the morphology of HTT43Q and formed short protofibril structures. Meanwhile, FKBP12 formed amorphous aggregate structures. We further used Thioflavin T (ThT) fluorescence to detect amyloidogenic fibrils. Results showed that TF can FKBP12 can both suppress amyloid fiber at day 1. The inhibition effect can still be seen in FKBP12 but not in TF at day 7, indicating TF can only retard the amyloidogenic process while FKBP12 can shift the process to form non-amyloid aggregates. From the FT-Raman spectroscopy,β-sheet composition both decreased in TF and FKBP12 compared with HTT43Q only. Here, we propose TF and FKBP12 can modulate mHTT protein aggregation in different pathway. While TF can only retard the amyloidogenic process, FKBP12 can shift the process to form non-amyloid aggregates. This may shed light on the future therapeutical treatments of Huntington’s disease.
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Jen, Shan-Ni y 任珊妮. "Harnessing chemically and biochemically inducible strategies to study how FKBP12 protein influences mutant Huntingtin". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/d8kmjm.
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碩士國立臺灣科技大學
化學工程系
106
The polyglutamine-expanded huntingtin, also known as mutant Huntingtin, is the causative agent to blame for Huntington’s Disease. Previously we have revealed that FKBP12 can alter the amyloid property of mHTT IBs (composed of huntingtin-exon1-109Q-eYFP), thereby being neuroprotective. Since FKBP12 possess no substrate-binding domain and pocket like regular enzyme or other FKBPs, how it impacts protein folding and conformation remain obscure. In this study, two strategies were exploited to enhance the interaction between FKBP12 and mHTT to address it function toward mHTT species. To monitor FKBP12 gets into proximity of mHTT species, rapamycin-mediated chemically inducible tethering (CIT) was devised. With time-lapse confocal microscopy, CIT provokes rapid co-localization of FKBP12 and mHTT IBs in cells. Strikingly, both the amyloid-specific dye NIAD-4 staining pattern and fluorescence life-time imaging (FLIM) suggest that the amyloid property and compactness of mHTT IBs would significantly decrease as FKBP12 being tethered. In addition to CIT, we also made use of the nanobody recognizing GFP moiety to confer nanobody-directed tethering (NDT) to 109Q-eYFP mHTT species. Flow cytometry analysis (FACs) suggests that FKBP12 tethering significantly reduce intracellular reactive oxygen species (ROS). In addition, FACs also suggested that FKBP12 tethering may promote mHTT IBs formation since the cells harboring with mHTT IBs increased and the cells harboring with oligomers declined. Of note, the morphology of FKBP12-tethered mHTT IBs were scrutinized by direct stochastic optical reconstruction microscopy (dSTORM) also confirmed this tendency. Take evidence altogether, FKBP12 may 1) impacts amyloid property of IBs 2) forces oligomer to become the higher order structure to provoke the fusion of inclusion bodies. The biological and biophysical consequence are also discussed.
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Zibrova, Darya [Verfasser]. "Adenovirus-mediated gene transfer of FK506-binding proteins FKBP12.6 and FKBP12 in failing and non-failing rabbit ventricular myocytes / vorgelegt von Darya Zibrova". 2004. http://d-nb.info/972602275/34.
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Gudavicius, Geoffrey. "Identification of FKBP25 as a pre-ribosome associated prolyl isomerase". Thesis, 2016. http://hdl.handle.net/1828/7684.
Texto completoResumen
The FK506-binding proteins (FKBPs) are a class of peptidyl-prolyl isomerase enzyme (PPIs) that catalyze the cis-trans inter-conversion of peptidyl-prolyl bonds in proteins. This non-covalent post-translational modification is a reversible mechanism to modulate protein structure and function. PPIs have been implicated in a wide variety of processes from protein folding to signal transduction. Despite these enzymes being ubiquitous, the substrates and functions of most PPIs have yet to be described.
FKBP25 is a nuclear FKBP that has been shown to associate with transcription factors and chromatin modifying enzymes, however its functions and substrates remain largely unresolved. FKBP25 is the human ortholog of S. cerevisiae Fpr4, which has been shown to regulate the chromatin landscape by two distinct mechanisms: 1. Acting as a histone chaperone at ribosomal DNA, and 2. Isomerizing histone prolines. Based on these observations, I hypothesized FKBP25 regulates chromatin and/or ribosome biogenesis through isomerization of histone prolines and a discrete collection of substrate proteins.
While small molecule inhibitors exist for FKBPs, applying them to dissect the specific function(s) of any given FKBP is confounded by the fact that multiple FKBPs are found in each organism, and several are inhibited by these molecules. In Chapter 2, I biochemically and structurally characterize a set of FKBP25 loss-of-function mutants, yielding a toolset capable of distinguishing between catalytic and non-catalytic functions. These reagents provide the tools necessary to analyze potential substrates of FKBP25 identified in my research going forward. In Chapter 3, I present the first unbiased proteomic screen of FKBP25 associated proteins and show that it interacts with a large number of ribosomal proteins, ribosomal processing factors and a smaller subset of chromatin proteins. I focus on the interaction between FKBP25 and nucleolin, a multi-functional nucleolar protein, and show that FKBP25 interacts with nucleolin and the pre-60s ribosomal subunit in an RNA dependent fashion. In Chapter 4, I gain insight into the role of FKBP25 in ribosome biology, and demonstratex that FKBP25 regulates RNA binding activity of nucleolin, however this does not appear to involve cis-trans prolyl isomerization.
Collectively, my work establishes FKBP25 as the first human FKBP to be implicated in the maturation of the pre-60S ribosomal subunit in the nucleus. My data supports a model whereby FKBP25 associates with the assembling large ribosomal subunit, where it is likely to chaperone protein-RNA interactions.Graduate
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Malone, Jenna Moira. "Analysis of signal pathway protein-protein interactions during biotic and abiotic stress". 2009. http://hdl.handle.net/2440/60985.
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The overall objective of the work described in this thesis was to characterise the three genes Hv14.3.3c, HvMAPKK1 and HvFKBP41, in terms of a role in defence and stress response signalling. These genes had previously been found to be differentially expressed in compatible versus incompatible interactions of barley with the fungus Rhynchosporium secalis, suggesting a possible role in the plant defence response, while current literature suggests these genes may also play a role in signal transduction, possibly under a broad range of stresses, including abiotic as well as biotic. Two main approaches were undertaken to characterise gene function: expression analysis and the identification of protein-protein interactions. To facilitate expression analysis, full length cDNA fragments of each gene were first obtained using bioinformatics, RACE and genomic walking techniques. Expression was then investigated using quantitative real-time RT-PCR. The results of the expression analysis confirmed that the candidate genes were in fact differentially expressed during infection, suggesting a role in the defence response of barley against R. secalis. Analysing their expression in the context of other stresses and treatments, namely frost, drought and ABA, indicated their role may not be limited only to biotic stress, but include abiotic stress as well. To investigate the possibility that these genes are involved in signalling during the defence response, protein-protein interaction techniques such as yeast two-hybrid and affinity pulldowns were used to identify interacting proteins in an attempt to place the genes within a known signalling network and build and extend on these networks. Y2H screening was used successfully to identify two putative interactors of Hv14.3.3c; an EPSP (5-enolpyruvylshikimate-3-phosphate) synthase and a putative wound-induced protein, and two interactors of HvFKBP41; a Rab-type GTPase and the same wound-induced protein. From what is known about the function of these genes in the literature, they fit well with a role in stress response signalling and the potential to be involved in signalling networks with the candidate gene products and also with each other. Through the trial of many different affinity pulldown techniques, a method for identifying interacting proteins from plant extracts was successfully established, however, issues with protein identification meant that interacting proteins were not identified using this technique. Steps were then made towards confirming the interactions identified using the Y2H system. Full length cDNA sequences of the identified interactors were obtained and expression analysis performed, in the aim of investigating co-expression patterns between the genes encoding the interacting proteins and the three candidate genes, to support a potential interaction. To confirm the Hv14.3.3c-HvEPSP interaction, co-immunoprecipitation and BRET were then used, however confirmation was unsuccessful due to issues with non-specific binding in co-immunoprecipitation and technical issues trying to establish the BRET analysis system in barley. In summary, the results of this study place the candidate genes Hv14.3.3c, HvMAPKK1 and HvFKBP41 as players in signal transduction during the plant defence/stress response. With the identification of previously uncharacterised protein interactions, some progress has also been made towards placing these genes within known signalling networks and identifying potential downstream genes that could possibly play a more specific role in defence response signalling and therefore be potential targets for the generation of resistant or stress tolerant plants.Thesis (Ph.D.) -- University of Adelaide, School of Agriculture, Food and Wine, 2009
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Cree, Tabitha. "Investigating the role of FK506 binding protein 25 in cell proliferation and differentiation". Thesis, 2021. https://vuir.vu.edu.au/42901/.
Texto completoResumen
Peptidyl prolyl isomerases (PPIase) are a class of enzymes that are required to catalyse the conversion of proline residues from cis to trans conformation. There are several classes of PPIase molecules, including parvulins, cyclophilins, and FK506 binding proteins (FKBPs). Among these PPIase molecules each class contains a conserved PPIase domain that facilitates protein to protein interactions. These PPIase molecules have diverse functions in cellular function and disease progression. FKBPs are a group of immunophilin molecules that are known to interact with immunosuppressant molecules FK506 and rapamycin to stop the immune response and inhibit mTOR, respectively. The structure and function of FKBPs is diverse, these proteins act to facilitate protein to protein interactions, act as co-chaperones, translocate throughout the cell in response to stress events, and bind to DNA. Importantly, FKBPs have been implicated in the pathogenesis of cancer, largely through their roles in co-chaperoning hormone receptors in hormone responsive cancers i.e. breast and prostate cancers. Of particular interest, FKBP25, a 25kDa protein that consists of two functional domains, an N terminal basic helix–loop–helix and C terminal PPIase domain. FKBP25 is known to be involved in protein folding, cytoskeletal dynamics, DNA damage repair, double stranded RNA binding, interacting with the pre-ribosome, and cellular stress responses. Despite the variety of roles that FKBP25 is known to play, there is limited research regarding FKBP25 role in disease and cell differentiation.
To address this, initial studies investigated the role of FKBP25 in breast cancer progression and epithelial to mesenchymal transition (EMT). Here it was found that FKBP25 protein expression is reduced in both mesenchymal breast cancer cell types, including BT-549, Hs578t, MDA-MB-231. To further understand the potential role of FKBP25 in breast cancer pathogenesis, a variety of mutations that contribute to malignant transformation were examined. Here it was found that the oncogenic mutations, that are associated with growth pathways in fact increased FKBP25 expression. However, in an epidermal growth factor mediated model EMT in MDA- MB-468 breast cancer cells, it was identified that FKBP25 protein expression was reduced. This implies that the loss of FKBP25 protein expression may be required for de-differentiation and progression of cancer cells. As such, it was hypothesised that FKBP25 protein expression was correlated with the level of cellular differentiation. To examine this hypothesis, next a model of mesenchymal to epithelial transition (MET) was analysed.
The C2C12 model of myogenesis to study the role of FKBP25 in an MET-like example of cell differentiation. Previous studies have identified that FKBP25 is the most highly expressed FKBP in skeletal muscle and is expressed in the top 10% of the skeletal muscle proteome. Here it was identified that in proliferative myoblasts there is a higher level of FKBP25 protein expression compared to that of post mitotic myotubes. This was further demonstrated in a model of C2C12 quiescence where it was demonstrated that upon removal from the cell cycle, myoblasts accumulate greater levels of FKBP25 protein expression, which is then reduced upon re-entry to the cell cycle. Interestingly, this trend was not observed in human primary myoblasts, however, was identified in human rhabdomyosarcoma cells which may be due to the presence of p53 and MyoD mutations. Furthermore, in vivo models of muscle plasticity were examined to assess the impact of FKBP25 on skeletal muscle regeneration considering FKBP25 is the most highly expressed FKBP in mature skeletal muscle. Here it was discovered that FKBP25 protein expression is increased in models of regeneration including, chronic mechanical loading, murine muscular dystrophy (mdx), and denervation. It is hypothesised that this was observed due to extensive cytoskeletal remodelling to repair structural damage caused by hypertrophy and atrophy of fibres.
Next, we examined the impact of FKBP25 knockdown (25KD) on cell biology and function of MDA-MB-468 and C2C12 cells. 25KD cells were developed using doxycycline inducible SMARTvector (Dharmacon, CO, USA) short hairpin RNA technology. After confirming adequate 25KD, it was observed that in both cell lines 25KD resulted in an increase in proliferation compared to respective non-targeting (NT) cells. Furthermore, in MDA-MB-468 cells, it was observed that there were no changes to invasion outgrowth or migration in vitro. However, it was demonstrated that 25KD resulted in decreased anchorage dependent growth, which could be explained by alterations to cytoskeletal stability. Conversely, in C2C12 myoblasts it was found that 25KD resulted in a significant increase in wound healing migration. Upon investigation of myogenic regulatory factor expression in differentiated 25KD myotubes it was revealed that there were no changes in protein expression. Furthermore, upon measurement of fibre diameter and fusion index it was found that there were no discernible changes to myotube formation.
Finally, the influence of 25KD on tubulin regulation and dynamics was assessed. Initially, the presence of microtubule (MT) post-translational modifications was assessed, including detyrosination and acetylation which are associated with MT stability. Both C2C12 and MDA-MB-468 25KD cells showed no changes to stabilising modifications. Similarly, upon examination of MT stabilising protein stathmin, both C2C12 and MDA-MB-468 25KD showed no change to stathmin expression. After this, the impact of 25KD on tubulin polymerisation under control and paclitaxel treated (induction of maximal polymerisation) conditions was explored. However, here no differences in MT polymer content was found in either 25KD in either C2C12 or MDA- MB-468 cells.
In conclusion, this thesis has examined the potential role of FKBP25 in cell differentiation and de-differentiation in EMT and MET-like models. It was found that FKBP25 is required for some cell processed including proliferation, anchorage dependent growth, and migration. It was hypothesised that this was a result of cytoskeletal reorganisation and altered MT dynamics, however, this was unable to be demonstrated. Further studies should further examine the impact of 25KD on MT dynamics using methods less prone to error. Nonetheless, FKBP25 was demonstrated to have a role in cell proliferation and differentiation. Maintenance of FKBP25 protein in both cancers and skeletal muscle could help to preserve epithelial-like phenotype and maintain structural integrity, respectively.
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Dilworth, David. "Functional characterization of the nuclear prolyl isomerase FKBP25 : A multifunctional suppressor of genomic instability". Thesis, 2017. https://dspace.library.uvic.ca//handle/1828/8473.
Texto completoResumen
The amino acid proline is unique – within a polypeptide chain, proline adopts either a cis or trans peptide bond conformation while all other amino acids are sterically bound primarily in the trans configuration. In proteins, the isomeric state of a single proline can have dramatic consequences on structure and function. Consequently, cis-trans interconversion confers both barrier and opportunity – on one hand, isomerization is a rate limiting step in de novo protein folding and on the other can be utilized as a post-translational regulatory switch. Peptidyl-prolyl isomerases (PPIs) are a ubiquitous superfamily that catalyzes the interconversion between conformers. Although pervasive, the functions and substrates of most PPIs are unknown. The two largest subfamilies, FKBPs and cyclophilins, are the intracellular receptors of clinically relevant immunosuppressant drugs that also show promise in the treatment of neurodegenerative disorders and cancer. Therefore, narrowing the knowledge gap has significant potential to benefit human health.
FKBP25 is a high-affinity binder of the PPI inhibitor rapamycin and is one of few nuclear-localized isomerases. While it has been shown to bind DNA and associate with chromatin, its function has remained largely uncharacterized. I hypothesized that FKBP25 targets prolines in nuclear proteins to regulate chromatin-templated processes. To explore this, I performed high-throughput transcriptomic and proteomic studies followed by detailed molecular characterizations of FKBP25’s function. Here, I discover that FKBP25 is a multifunctional protein required for the maintenance of genomic stability. In Chapter 2, I characterize the unique N-terminal Basic Tilted Helical Bundle (BTHB) domain of FKBP25 as a novel dsRNA binding module that recruits FKBP25’s prolyl isomerase activity to pre-ribosomal particles in the nucleolus. In Chapter 3, I show for the first time that FKBP25 associates with the mitotic spindle apparatus and acts to stabilize the microtubule cytoskeleton. In this chapter, I also present evidence that this function influences the stress response, cell cycle, and chromosomal stability. Additionally, I characterize the regulation of FKBP25’s localization and nucleic acid binding activity throughout the cell cycle. Finally, in Chapter 4, I uncover a role for FKBP25 in the repair of DNA double-stranded breaks. Importantly, this function requires FKBP25’s catalytic activity, identifying for the first time a functional requirement for cis-trans prolyl isomerization by FKBP25.
Collectively, this work identifies FBKP25 as a multifunctional protein that is required for the maintenance of genomic stability. The knowledge gained contributes to the exploration of PPIs as important drug targets.Graduate
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Stein, Walter von [Verfasser]. "Charakterisierung von PTEN, FKBP59 und CG4420, Interaktionspartnern des PDZ-Domänen-Proteins Bazooka aus Drosophila melanogaster / vorgelegt von Walter von Stein". 2005. http://d-nb.info/981590128/34.
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