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Ramakrishnan, Venkatesh. "Structural analysis of a transactivation domain cofactor complex". Doctoral thesis, [S.l. : s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976325381.
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Betney, Russell. "Mutational analysis of the human androgen receptor transactivation domain". Thesis, University of Aberdeen, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.401515.
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Mutants were created within the main activation function domain to investigate the structure and function of this region. Structural studies based on limited proteolysis and fluorescence spectroscopy experiments, indicate that though the N-terminus is not as specially structured as either the LBD or DBD, there are regions of specific folding within the activation domain. Results from in silco investigation suggest several possible regions of a helix within the AF-1 domain, and mutants designed to disrupt these regions were less folded than the wild-type protein. Protein-protein interaction studies showed that the four mutants designed to potentially disrupt function rather than structure, had reduced binding to the large subunit of TFIIF - RAP74, but had no effect on binding to the transcriptional co-activator SRC-1a. In a functional assay performed in yeast cells, these four same mutants all showed reduced activity, showing the same trend as binding to RAP74. This could be an indication that the function of the AR is dependent upon binding to the general transcription factor TFIIF. Interestingly one of the mutants that was found to show increased structure over the wild-type protein was previously shown to have a reduced interaction with RAP74. This implies that structure is important for interaction with the transcription machinery. This was confirmed by FTIR experiments which can detect changes in the proportion of secondary structure present in a protein. These data show that the proportion of a helix present in the AF-1 domain increases when it is complexed with RAP74. GST pull-down assays then demonstrated that the complex of AF-1 and RAP74 enhanced the binding of the co-activator SRC-1a. This is the first time that this cooperativity has been demonstrated with nuclear receptors and interacting proteins. Additionally, specific phosphorylation of the AF-1 domain by glycogen synthase kinase 3 also increases the level of binding with SRC-1a. These data together suggest a possible mechanism of action for the androgen receptor and its involvement in regulating transcription.
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Seth, Alpna. "Functional Analysis of the c-MYC Transactivation Domain: A Dissertation". eScholarship@UMMS, 1992. https://escholarship.umassmed.edu/gsbs_diss/315.
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Many polypeptide growth factors act by binding to cell surface receptors that have intrinsic tyrosine kinase activity. Binding of these growth factors to their cognate receptors results in the initiation of mitogenic signals which then get transduced to the interior of the cell. A critical target for extracellular signals is the nucleus. A plethora of recent evidence indicates that extracellular signals can affect nuclear gene expression by modulating transcription factor activity. In this study, I have determined that the transactivation domain of c-Myc (protein product of the c-myc proto-oncogene) is a direct target of mitogen-activated signaling pathways involving protein kinases. Further, my study demonstrates that transactivation of gene expression by c-Myc is regulated as a function of the cell cycle.
c-Myc is a sequence-specific DNA binding protein that forms leucine zipper complexes and can act as a transcription factor. Although, significant progress has been made in understanding the cellular properties of c-Myc, the precise molecular mechanism of c-Myc function in oncogenesis and in normal cell growth is not known. I have focused my attention on the property of c-Myc to function as a sequence-specific transcription factor. In my studies, I have employed a fusion protein strategy, where the transactivation domain of the transcription factor c-Myc is fused to the DNA binding domain and nuclear localization signal of the yeast transcription factor GAL4. This fusion protein was expressed together with a plasmid consisting of specific GAL4 binding sites cloned upstream of a minimal E1b promoter and a reporter gene. The activity of the c-Myc transactivation domain was measured as reporter gene activity in cell extracts. This experimental approach enabled me to directly monitor the activity of the c-Myc transactivation domain.
Results listed in Chapter II demonstrate that the transactivation domain of c-Myc at Ser-62 is a target of regulation by mitogen-stimulated signaling pathways. Furthermore, I have determined that a mitogen activated protein kinase, p41mapk, can phosphorylate the c-Myc transactivation domain at Ser-62. Phosphorylation at this site results in a marked increase in transactivation of gene expression. A point mutation at the MAP kinase phosphorylation site (Ser-62) causes a decrease in transactivation.
c-Myc expression is altered in many types of cancer cells, strongly implicating c-myc as a critical gene in cell growth control. The molecular mechanisms by which c-Myc regulates cellular proliferation are not understood. For instance, it is not clear where in the cell cycle c-Myc functions and what regulates its activity. In exponentially growing cells, the expression levels of c-Myc remain unchanged as the cells progress through the cell cycle. The function of c-Myc may therefore be regulated by a mechanism involving a post-translational modification, such as phosphorylation. Results described in chapter IV demonstrate that the level of c-Myc mediated transactivation oscillates as cells progress through the cell cycle and was greatly increased during the S to G2/M transition. Furthermore, mutation of the phosphorylation site Ser-62 in the c-Myc transactivation domain diminishes this effect, suggesting a functional role for this phosphorylation site in the cell cycle-specific regulation of c-Myc activity.
Taken together, my dissertation study reveals a molecular mechanism for the regulation of nuclear gene expression in response to mitogenic stimuli.
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Borcherds, Wade Michael. "Structure, Dynamics, and Evolution of the Intrinsically Disordered p53 Transactivation Domain". Scholar Commons, 2013. http://scholarcommons.usf.edu/etd/4640.
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in numerous disease states, including cancers and neurodegenerative diseases. All proteins are dynamic in nature, occupying a range of conformational flexibilities. This inherent flexibility is required for their function, with ordered proteins and IDPs representing the least flexible, and most flexible, respectively. As such IDPs possess little to no stable tertiary or secondary structure, they instead form broad ensembles of heterogeneous structures, which fluctuate over multiple time scales. Although IDPs often lack stable secondary structure they can assume a more stable structure in the presence of their binding partners in a coupled folding binding reaction.
The phenomenon of the dynamic behavior of IDPs is believed to confer several functional advantages but remains poorly understood. To that end the dynamic and structural properties of a family of IDPs - p53 transactivation domains (TAD) was measured and compared with the sequence divergence. Interestingly we were able to find stronger correlations between the dynamic properties and the sequence divergence than between the structure and sequence, suggesting that the dynamic properties are the primary trait being
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conserved by evolution. These correlations were strongest within clusters of the IDPs that correlated with known protein binding sites. Additionally, we show strong correlations between the several available disorder predictors and the backbone dynamics of this family of IDPs. This indicates the potential of predicting the dynamic behavior of proteins, which may be beneficial in future drug design.
The limited number of atomic models currently determined for IDPs hampers understanding of how their amino acid sequences dictate the structural ensembles they adopt. The current dearth of atomic models for IDPs makes it difficult to test the following hypotheses:
1. The structural ensembles of IDPs are dictated by local interactions.
2. The structural ensembles of IDPs will be similar above a certain sequence identity threshold.
Based on the premise that sequence determines structure, structural ensembles were determined and compared for a set of homologous IDPs. Utilizing orthologues allows for the identification of important structural features and behaviors by virtue of their conservation. A new methodology of creating ensembles was implemented that broadly samples conformational space. This allowed us to find recurring local structural features within the structural ensembles even between the more distantly related homologues that were processed. This method of ensemble creation is also the first method to show convergence of secondary structural characteristics between discrete ensembles.
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Powell, Anne Terese. "Structure and Dynamics of the p53 Transactivation Domain Binding to MDM2 and RPA70". Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4207.
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The tumor suppressor protein, p53, is mutated or dysregulated in nearly all human cancers(1). The amino terminal domains are essential for transcriptional activation in stressed cells and play a vital role in cell cycle regulation, apoptosis and senescence. The transactivation (TAD) and proline rich domains in this region are dynamic and intrinsically disordered; lacking stable secondary or tertiary structure. This region contains multiple binding sites; arguably, the most significant of these is for p53's negative regulator, the E3 ligase, MDM2. An important, but less understood interaction involving the single stranded DNA binding protein, RPA70A, is hypothesized to be involved in maintaining genome integrity(2-4). Additionally, the amino terminus contains an important single nucleotide polymorphism that has demonstrated different affinity for MDM2 and is of significant biological importance in the induction of apoptosis (5). Isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) spectroscopy were employed to investigate how the thermodynamics and the inherent flexibility of the amino terminus of p53 play a role in complex formation with the MDM2 or RPA70 proteins. Understanding the structure, dynamics, and function of p53 is paramount in the fight against cancer.
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Poosapati, Anusha. "Disorder Levels of c-Myb Transactivation Domain Regulate its Binding Affinity to the KIX Domain of CREB Binding Protein". Scholar Commons, 2017. https://scholarcommons.usf.edu/etd/7436.
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Intrinsically disordered proteins (IDPs) do not form stable tertiary structures like their ordered partners. They exist as heterogeneous ensembles that fluctuate over a time scale. Intrinsically disordered regions and proteins are found across different phyla and exert crucial biological functions. They exhibit transient secondary structures in their free state and become folded upon binding to their protein partners via a mechanism called coupled folding and binding. Some IDPs form alpha helices when bound to their protein partners. We observed a set of cancer associated IDPs where the helical binding segments of IDPs are flanked by prolines on both the sides. Helix-breaking prolines are frequently found in IDPs flanking the binding segment and are evolutionarily conserved across phyla. Two studies have shown that helix flanking prolines modulate the function of IDPs by regulating the levels of disorder in their free state and in turn regulating the binding affinities to their partners. We aimed to study if this is a common phenomenon in IDPs that exhibit similar pattern in the conservation of helix flanking prolines. We chose to test the hypothesis in c-Myb-KIX : IDP-target system in which the disordered protein exhibits high residual helicity levels in its free state.
c-Myb is a hematopoietic regulator that plays a crucial role in cancer by binding to the KIX domain of CBP. Studying the functional regulation of c-Myb by modulating the disorder levels in c-Myb and in IDPs in general provides a better understanding of the way IDPs function and can be used in therapeutic strategies as IDPs are known to be involved in regulating various cellular processes and diseases. To study the effect of conserved helix flanking prolines on the residual helicity levels of c-Myb and its binding affinity to the KIX domain of CBP, we mutated the prolines to alanines. Mutating prolines to alanines increased the helicity levels of c-Myb in its free state. This small increase in the helicity levels of a highly helical c-Myb showed almost no effect on the binding affinity between cMyb and KIX. We hypothesized that there is a helical threshold for coupled folding and binding beyond which helicity levels of the free state IDP have no effect on its binding to their ordered protein partner. To test this hypothesis, we mutated solvent exposed amino acid residues in c-Myb that reduce its overall helicity and studied its effect on the binding affinity between c-Myb and KIX. Over a broad range of reduction in helicity levels of the free state did not show an effect on the binding affinity but beyond a certain level, decrease in helicity levels showed pronounced effects on the binding affinity between c-Myb and KIX.
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Fischer, Katharina. "The mineralocorticoid receptor amino terminal transactivation domain investigation of structural plasticity and protein-protein interactions /". Thesis, Available from the University of Aberdeen Library & Historic Collections Digital Resources, 2008. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=24694.
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Thesis (Ph.D.)--Aberdeen University, 2008.Title from web page (viewed on Feb. 23, 2009). With: Natural disordered sequences in the amino terminal domain of nuclear receptors : lessons from the androgen and glucocorticoid receptors / Iain J. McEwan ... et al. Nuclear Receptor Signalling. 2007: 5. Includes bibliographical references.
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Johnson, Thomas M. "p53 transactivation domain mutant knock-in mice provide novel insight into p53 tumor suppressor function /". May be available electronically:, 2007. http://proquest.umi.com/login?COPT=REJTPTU1MTUmSU5UPTAmVkVSPTI=&clientId=12498.
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Reid, James Arthur. "Structural and functional analysis of the amino-terminal transactivation domain of the human androgen receptor". Thesis, University of Aberdeen, 2001. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU149342.
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The androgen receptor (AR) belongs to the steroid/nuclear receptor family of ligand-activated transcription factors. Most of these receptors activate transcription through two distinct regions known as activation functions (AF). The first, AF-1, is located within the variable amino-terminal domain (NTD) and the second, AF-2, within the ligand binding domain (LBD). However, the AR is unusual amongst the steroid receptor family in that an independent AF-2 function has not been demonstrated. Transactivation by the AR is therefore dependent primarily upon AF-1. Previous work in our laboratory has shown that an amino-terminal fragment of the AR (amino acids 142 to 485) is able to interact specifically with RAP74, the large subunit of general transcription factor TFIIF. Using a series of deletion constructs, two distinct AR binding sites have been identified with RAP74, one at the amino-terminus and one at the carboxyl-terminus of the protein. Functional analyses of these interactions suggest that the interaction between the AR and the carboxyl-terminus of RAP74 is the most significant with respect to transcriptional activation by the receptor. Mutational analysis of the AR-NTD identified a six amino acid motif, PSTLSL, as being involved in RAP74 binding. In addition to RAP74, an interaction has been identified between the p160 coactivator protein SRC-1a and amino acids 142-485 of the AR. Interestingly, another high related p160 coactivator protein, TIF2, showed little or no binding to this region of the receptor. Structural analysis, using circular dichroism and fluorescence spectroscopy and sensitivity to protease digestion, indicates that the AR-NTD exists in an extended conformation in aqueous solution but retains a propensity to fold into a more ordered structure in the presence of the hydrophobic solvent trifluoroethanolor the osmolyte trimethylamine-N-oxide. Significantly, RAP74 also confers protection to trypsin digestion, consistent with folding of the AR-NTD (aa 142 to 485) upon interaction with a target factor.
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Lavery, Derek Norman. "The amino-terminal transactivation domain of the human androgen receptor : protein-protein interactions and structural characteristics". Thesis, University of Aberdeen, 2007. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU490182.
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An important target protein for the AR-AF1 domain is Transcription Factor IIF (TFIIF). At initiation of transcription, TFIIF recruits additional basal transcription factors, stabilises the transcriptional complex and increases elongation efficiency. Using chromatin immunoprecipitation, it was observed that the AR occupied distinct regions of the prostate-specificantigen enhancer but did not migrate with the elongating transcriptional complex. The major subunit of TFAAF, RAP74, has previously been shown to interact with AR-AF1 by our laboratory and it was observed that AR-AF1 can interact with both terminal regions of RAP74. Now, by selectively disrupting helices that structure the globular RAP74 C-terminal domain it appears that AR-AF1 binds to a groove within this region and specific hydrophobic amino acids are important in this generation. The kinetics of RAP74/AR-AF1 interactions have not been determined using surface plasmon resonance. Interestingly, AR-AF1 interacts differently with N- and C-terminal regions of RAP74 and the overall affinities are in the nanomolar range. The structural properties of AR-AF1 were examined using both fluorescence spectroscopy and gel filtration chromatography. It was found that the transactivation domain is structurally flexible and exists in a conformation that is not random coil or globular suggesting that it may be a molten globule. AR-AF1 interacted weakly with 8-anilinonaphthalene-1-sulfonic acid, a hydrophobic probe used to characterise the molten globule folding state. Gel filtration chromatography indicates that AR-AF1 is ∼65 kDa and has a hydrodynamic radius of ∼36 Co much larger than predictions suggest. Surprisingly, by plotting these properties on "folding curves", AR-AF1 is positioned alongside molten globules.
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Carreau, Charlotte. "Propriétés œstrogéniques des phyto-œstrogènes dans une lignée de cancer du sein : implication des domaines de transactivation du récepteur aux œstrogènes alpha". Bordeaux 1, 2008. http://www.theses.fr/2008BOR13571.
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Les phyto-œstrogènes (PE), molécules d’origine végétale, possèdent des homologies structurales avec les œstrogènes leur permettant de se lier aux récepteurs spécifiques aux œstrogènes (RE) et d’agir comme des molécules pro- ou anti-œstrogéniques. Les œstrogènes et les RE sont impliqués dans de nombreux processus physiologiques et physiopathologiques, notamment le cancer du sein. Il existe deux formes de RE (66 et 46 kDa) qui diffèrent par leur partie N-terminale, où est situé le domaine de transactivation AF-1. Le deuxième domaine de transactivation AF-2, ligand dépendant, est similaire dans les deux RE. L’objet de cette étude est de trouver parmi les PE testés ceux capables de moduler spécifiquement le RE (appelés SERM) dans une lignée cancéreuse mammaire, les MCF-7, afin de mieux comprendre leur impact sur le cancer du sein. Ainsi, il apparaît que les propriétés pro-œstrogéniques de certains PE sont associées à leur capacité à mobiliser le domaine AF-1 dans l’activation transcriptionnelle du RE. A l’inverse, les composés mobilisant préférentiellement le domaine AF-2 du RE induisent une faible activation transcriptionnelle et n’ont pas d’effets œstrogéniques. C’est le cas de l’entérolactone et la naringénine, SERM potentiels qui pourraient être de bons candidats dans un cadre de nutrition préventive contre le cancer du seinPhytoestrogens (PE) are plants compounds sharing structural similarities with estrogens. These compounds, which bind to estrogen receptors (ER), may induce or inhibit estrogen action and have the potential to disrupt estrogen signalling. Estrogens and ER are involved in numerous physiological and/or pathological processes, in particular in breast cancer. The human ER (hER gene can be alternatively spliced into proteins of 66 and 46 kDa respectively, which differ on their N-terminal where is located the transactivation domain AF-1. The second transactivation domain AF-2, which is ligand-dependant, is present in both ER. To elucidate the yet unclear mechanisms of ER activation/inhibition by PE in further details and find selective estrogen receptor modulators (SERM), we performed a comprehensive analysis and potency comparison of hER transactivation by PE in a human breast cancer cell line, MCF-7. We show that estrogenic properties of some PE are linked to their capacity to induce an AF-1-dependant transactivation of hER. In contrast, the PE inducing an AF-2-dependant transactivation of hER are weak agonists and more importantly, they do not display any estrogenic properties. Such compounds, as enterolactone and naringenine, are interesting SERM, and further investigations should be performed to evaluate their potentials as candidates for a nutritional prevention of breast cancer
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Kim, KyoungHyun. "Domain analysis for estrogen receptor/Sp1-mediated transactivation and detection of estrogen receptor/Sp1 protein interactions in living cells". Texas A&M University, 2004. http://hdl.handle.net/1969.1/2666.
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Estrogen Receptor ? (ER?)/Sp1 activation of GC-rich gene promoters in breast cancer cells is dependent, in part, on the activation function 1 (AF1) of ER?. This study investigates contributions of the DNA binding domain (C) and AF2 (DEF) regions of ER? on activation of ER?/Sp1. 17Beta-estradiol (E2) and the antiestrogens 4-hydroxytamoxifen and ICI 182,780 induced reporter gene activity in MCF-7 and MDA-MB-231 cells cotransfected with human or mouse ER? (hER? or MOR), but not ER? and GC-rich constructs containing three tandem Sp1 binding sites (pSp13) or other E2-responsive GC-rich promoters. Estrogen and antiestrogen activation of hER?/Sp1 was dependent on overlapping and different regions of the C, D, E, and F domains of ER?. Antiestrogen-induced activation of hER?/Sp1 was lost using hER? mutants deleted in zinc finger 1 (amino acids (aa) 185-205), zinc finger 2 (aa 218-245), and the hinge/helix 1 (aa 265-330) domains. In contrast with antiestrogens, E2-dependent activation of hER?/Sp1 required the C-terminal F domain (aa 579-595), which contains a ?-strand structural motif. Moreover, in peptide competition experiments overexpression of NR-box peptides inhibits E2-induced luciferase activity of pERE3, which contains three tandem repeats of consensus ERE sites, whereas E2-induced hER?/Sp1 action was not inhibited by NR-box peptide expression. In contrast, overexpression of a C-terminal (aa 575-595) F domain peptide specifically blocked E2-dependent activation of hER?/Sp1, but not on activation of pERE3, suggesting that F domain interactions with nuclear cofactors are specifically required for ER?/Sp1 action.
Furthermore, direct physical interactions between hER? and Sp1 protein in vivo have been investigated by using Fluorescence Resonance Energy Transfer (FRET) microscopy and image analysis. Consistent with results from transient transfection assay, E2, 4OHT, and ICI enhanced hER?/Sp1 interactions in living cells and these interactions were also confirmed by coimmunoprecipitation. In addition, endogenous hER?/Sp1 action was evaluated by using si RNA for Sp1 and a significant decrease in ligand-induced hER?/Sp1 action was observed after decreased Sp1 expression.
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JOHN, MATTHIAS. "Analyse biochimique des domaines de dimerisation et de transactivation des proteines jun et fos". Université Louis Pasteur (Strasbourg) (1971-2008), 1996. http://www.theses.fr/1996STR13123.
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Le facteur de transcription ap-1 est compose d'homodimeres ou d'heterodimeres des proteines jun et fos qui possedent trois domaines majeurs: un domaine de dimerisation, le leucine zipper (zip), le domaine basique (b) pour la reconnaissance du site tre et un domaine de transactivation. En utilisant des peptides synthetiques, nous avons etudie l'importance de deux pairs de residus de charge oppose pour l'efficacite d'heterodimerisation des leucine zippers de jun et fos. Une etude biochimique et physico-chimique des domaines bzip entier de jun et fos fixe sur l'adn a montre qu'un site tre en solution subit un changement de conformation compatible avec une structure intermediaire entre les conformations b et a. Le facteur de transcription c-jun contient un domaine de transactivation appartenant a la famille des activateurs de transcription acide. Afin d'analyser la structure secondaire de ce domaine, j'ai synthetise un peptide correspondant aux acides amines 61 a 98 de c-jun et un deuxieme peptide avec deux phosphoserines en positions 63 et 73. En presence de trifluoroethanol, les peptides adoptent une conformation alpha-helicoidale
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Majumdar, Sonali. "Structural and functional divergence of the transcription factor Pit-1, analysis of the pou and transactivation domains". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0008/NQ28006.pdf.
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Aun, Jason Paul. "Analysis and quantitation of the cross presentation of tumor antigens using the HIV protein transduction domain transactivating regulatory protein (TAT) to alter presentation". 2009. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:1462704.
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Liu, Li Ying, i 劉麗鶯. "Point mutations in the N-terminal domain of the small delta antigen affect its transactivating activity on the replication of hepatitis delta virus". Thesis, 1996. http://ndltd.ncl.edu.tw/handle/46724860211999016230.
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碩士國立臺灣大學
生化學研究所
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Hepatitis delta virus (HDV) is a RNA virus of about 36 nm in size. The envelope of HDV particles consist of lipid bilayer membrane and the surface antigens (L, M, and S forms)of hepatitis B virus. The core struc-ture contains ribonucleoprotein complexes (RNP) of RNA genome and delta antigens (HDAgs). The delta antigens are nuclear phosphoproteins. Phosphorylation at serine residues has been detected in HDAg-transfected cells. However, the exactly phosphorylated sites and the functions of HDAg related to the phosphorylation are not known. Amino acid sequence analysis revealed that the Ser-2,-4, -123 are potential phosphorylated sites of CK-II. However, our previous studies indicated that mutations at the Ser-2 and -4 to Ala did not affect the small HDAg in supporting HDV replication. Wherease,the small HDAg with a mutation from Glu-5 to Arg could no longer support viral replication.In this study,by changing the Glu-5 to Asp or Leu, the small HDAg mutant maintained its function in transactivating the replication of HDV.The results observed with the Leu-substitution suggest that there may be other factors involved in controlling the function of small HDAg. When a plasmid encoding the small HDAg mutant Arg-5 was cotransfected with a dimeric HDV cDNA into Huh-7 cells, HDV replication was decreased in a dose-dependent manner.This indicates that the small HDAg mutant Arg-5 functions as a trans-dominant negative regulator. Biochemical studies demonstrated that the small HDAg mutant Arg-5 retained oligomerization ability and CK-II phosphorylation. Therefore, the small HDAg mutant Arg-5 may have changed in local conformation which inhibits its binding to the HDV genomic RNA or interferes the interactions between small HDAg and cellular factors that are involved in the replication of HDV.
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Lussier-Price, Mathieu. "Étude sur la reconnaissance de l'ubiquitine par les domaines de transactivation acides des activateurs de transcription". Thèse, 2014. http://hdl.handle.net/1866/11225.
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Les domaines de transactivation (TAD) acides sont présents dans plusieurs protéines oncogéniques, virales et dans des facteurs de différenciation de cellules souches. Ces domaines acides contrôlent la transcription à travers une myriade d’interactions avec divers partenaires ce qui provoque l’activation de la transcription ou leur propre élimination. Cependant, dans la dernière décennie, de plus en plus de recherches ont démontré que les TAD possédaient un sous-domaine activation/dégradation (DAD) responsable pour une fonction d'activation de la transcription dépendante de la dégradation de la protéine. Un tel phénomène peut être accompli par plusieurs moyens tels que des modifications post-traductionnelles, l’association à des cofacteurs ou la formation d’un réseau d’interaction complexe en chaînes. Or, aucune preuve concrète n’a pu clairement démontrer le fonctionnement de la dépendance paradoxale entre ces deux fonctions sur un activateur de transcription.
Le DAD, a été observé dans plusieurs facteurs de transcription incluant la protéine suppresseur de tumeur p53 et le facteur de différenciation érythrocyte EKLF. Un aspect particulier des DAD est que la composition de leur séquence d’acide aminé est fortement similaire à celle des domaines de liaison à l’ubiquitine (UBD) qui jouent un rôle clé dans le contrôle de la transcription à travers leur interaction non-covalente avec l’ubiquitine.
Ainsi, dans ce mémoire, nous avons étudié la possibilité que les TAD acides soient capables d’agir comme UBD pour réguler leur fonction paradoxale à travers des interactions non-covalentes avec l’ubiquitine. L’analyse est faite en utilisant la résonnance magnétique nucléaire (RMN) ainsi qu’avec des essais fonctionnels de dégradation. En somme, cette étude amène une plus grande compréhension des protéines impliquées dans le contrôle des TAD et caractérise le tout premier exemple de TAD capable d’interagir avec l’ubiquitine.Acidic transactivating domains have been shown to be potential targets for a number of different therapies but their dynamic nature and their ability to bind many interacting partners has made it difficult to fully understand their functioning mechanisms. What we do know about these domains is that they readily control transcription through a myriad of interactions capable of either activating specific aspects of their function or simply, signal for their own demise. Within the acidic TADs lies an unusual degradation/activation domain (DAD) capable of activating transcription at the cost of its degradation. In other words, DAD transcriptional activation is dependent on the degradation of the protein. Such a phenomenon could be explained by a wide variety of hypotheses like the play of post-translational modifications, co-factors, or maybe just a really sophisticated time scaled network of interactions. However, no concrete explanation of how this dual dependent functioning domain works has yet to surface. The DAD has been observed within acidic TADs of several transcription factors including the tumor suppressor p53 and the red blood cell differentiation factor EKLF. Interestingly though, the amino acid sequence composition of DADs share a strong similarity with several types of sequences from domains that bind ubiquitin (UBDs). These domains have been shown in the past to, in addition to their role in degradation, play a key role in regulating transcription through non-covalent interaction with ubiquitin. Hence, in this project, we investigated weather acidic TADs had the ability to function as UBDs and form non-covalent interactions with ubiquitin and also to determine the functional significance of this interaction in regards to the dual function of acidic TADs.
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Novák, Josef. "Zkoumání jaderné funkce interleukinu-1alfa". Master's thesis, 2012. http://www.nusl.cz/ntk/nusl-307796.
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Interleukin-1alpha (IL-1alpha) is a well-known proinflammatory mediator acting as a secreted molecule. However, in addition to its ability to activate its membrane-bound receptor, there is growing evidence on its noncanonical nuclear function, which classifies IL-1alpha as a "dual function cytokine". This nuclear action depends on the evolutionary conserved N-terminal domain of IL-1alpha. After proteolytic processing, the N-terminal domain of IL-1alpha translocates into nucleus. Histone acetyltransferase (HAT) complexes were previously identified as nuclear targets of IL-1alpha precursor. However, the specific protein which is responsible for the interaction between IL-1alpha and HAT complexes has not been identified yet. To dissect this interaction, the N-terminal domain of IL-1alpha was produced in yeast. Suitability of this experimental setup for testing the interaction between IL-1alpha and eukaryotic HAT complexes was evaluated in this study. IL-1alpha has been analyzed in this study using bioinformatics approaches as well. Putative amphipatic acidic helixes of IL-1alpha have been characterized. One of the potential binding partners of these domains is protein Ada2. Protein Ada2, mature IL-1alpha and IL-1alpha precursor in fusion with epitopes suitable for affinity purification were produced in...
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Chu, Ying-An, i 朱盈安. "Effect of caprine Stat5a transactivation domain mutation on β-casein promoter activity". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/97857638442712644120.
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碩士國立臺灣大學
動物科學技術學研究所
99
Signal transducers and activators of transcription 5a (Stat5a) is a member of Stat family which has the ability to sense and transmit environmental cues to regulate specific gene expression in the nucleus. During late gestation to lactation, the secretion of prolactin (PRL) stimulates the formation of alveolus and milk protein translation. Stat5a is the critical mediator in the PRL signaling pathway and regulates mammary gland development and lactation. PRL binds to prolactin receptor (PRLR) on mammary epithelial cell, leading dimerization of PRLRs and activates Janus kinase 2 (Jak2), then recruits cytoplasmic Stat5a to the Jak2. Stat5a was phosphorylated on tyrosine residue (Y694) by Jak2. Activated Stat5a forms Stat5-Stat5 dimer immediately and translocated into nucleus binds to specific DNA motif called GAS (gamma interferon activation site) element on milk protein promoter and initiate gene transcription. Since the pivotal role of Stat5a on lactation of mammary gland, the milk protein production difference between species may cause by the diversity of Stat5a sequence. In order to find the unique sites of STAT5a sequence among species, this study compared STAT5a cDNAs of caprine, bovine, pig, human, mouse, and rat and pick the variational sites to do mutation. Then study on the effects of different Stat5a mutation on β-casein promoter activity. The caprine STAT5a (cSTAT5a) cDNA was cloned from primary caprine mammary gland. The similarity of amino acid sequences among caprine, bovine, pig, human, mouse and rat is about 95%. Among species mentioned, caprine and bovine have most similar amino acid sequences, 98.2%. Stat5a protein possesses 7 functional domains, each has different structures and functions. Major variation was found in transactivation domain. It is interesting that both caprine and bovine have proline residue at 780 amino acid (P780) while other species are serine (S780) and were confirmed would be phosphorylated by previous study. In order to confirm proline residue in bovine is not due to individual difference, genomic DNA sequences from 40 Holstein cows were analyzed and found out all of them contained the same P780 amino acid sequences like caprine. In order to investigate the effects of cStat5a on milk protein transcription, using the immortalized caprine mammary epithelial cells (CMC) as experimental model. Treated CMC with Jak2 or MEK1 inhibitor to see the PRL signaling pathway on CMC. The results showed that β-casein promoter activity decreased under Jak2 inhibitor treatment and increased when treated with MEK1 inhibitor. Phosphorylation of Y694 also decreased under Jak2 inhibitor treatment. Then CMC was co-transfected β-casein promoter drove luciferase report gene and exogenous cStat5a. Five different mutants of cStat5a: Y694A, P780D, P780S, 774/778/780A and 774/778/780D were constructed. The different effects of mutated cStat5a on β-casein promoter activity were determined by exogenous expression of different mutated cStat5a in CMC and luminescence system was applied to quantitate the β-casein promoter activity. The result showed that after insulin-hydrocortisone-PRL (IHP) induction for 12 hours, P780S cStat5a had the highest induction ratio than wild type (WT) cStat5a, and Y694A cStat5a had the lowest β-casein promoter activity. Other mutations of cStat5a kept the same β-casein promoter activity with WT. Taken together, this study indicated that Jak2/Stat5a is the major downstream singling pathway of PRL. And there were some factors in MEK1 singling pathway which can regulate β-casein promoter activity. On the other hand, both of the amino acid P780 and Y694 in caprine and bovine are important for transcription activation of Stat5a in CMC. Future work is to focus on investigating the role of P780 in controlling the activity of caprine β-casein promoter and understanding why caprine and bovine which should have more activation state of Stat5a did not go the same way of evolution.
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Ramakrishnan, Venkatesh [Verfasser]. "Structural analysis of a transactivation domain cofactor complex / vorgelegt von Venkatesh Ramakrishnan". 2005. http://d-nb.info/976325381/34.
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Weaver, Amanda Mae. "Relevance of phosphotyrosines in the transactivation domain of STAT5b implications for STAT5b in breast cancer /". 2008. http://wwwlib.umi.com/dissertations/fullcit/3300273.
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Morin, Geneviève. "Identification et caractérisation d'un domaine de transactivation dans l’hélicase E1 des papillomavirus humains". Thèse, 2010. http://hdl.handle.net/1866/4129.
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Les papillomavirus sont des virus à ADN qui infectent la peau et les muqueuses. Ils
causent des verrues et peuvent aussi mener au développement de cancers, dont le cancer du col de l’utérus. La réplication de leur génome nécessite deux protéines virales : l’hélicase E1 et le facteur de transcription E2, qui recrute E1 à l’origine de réplication virale. Pour faciliter l’étude de la réplication du génome viral, un essai quantitatif et à haut débit basé sur l’expression de la luciférase a été développé. Parallèlement, un domaine de
transactivation a été identifié dans la région régulatrice N-terminale de la protéine E1. La caractérisation de ce domaine a montré que son intégrité est importante pour la réplication de l’ADN. Cette étude suggère que le domaine de transactivation de E1 est une région protéique intrinsèquement désordonnée qui permet la régulation de la réplication du génome viral par son interaction avec diverses protéines.Papillomaviruses are small DNA viruses that infect skin and mucosa. They cause warts and can also lead to the development of cancers, including cervical cancer. Replication of their genome requires two viral proteins: the E1 helicase and the E2 transcription factor, which recruits E1 to the viral origin of replication. To facilitate the study of viral genome replication, a quantitative and high-throughput assay based on luciferase expression has been developed. In parallel, a transactivation domain has been identified in the N-terminal regulatory region of the E1 protein. Characterization of this domain showed that its integrity is important for DNA replication. This study suggests that the E1 transactivation domain is an intrinsically unstructured protein region that allows regulation of viral genome replication by its interaction with diverse proteins.
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Jou, Han-Lin, i 周翰林. "Expression and purification of recombinant transactivation domain of hepatitis B virus (HBV) X protein in Escherichia coli". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/33473364677241661962.
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碩士高雄醫學大學
生物科技學系碩士班
98
It was estimated that more than 350 million individuals were infected with chronic hepatitis B virus (HBV). Especially, half of the world’s population lives in an area with high HBV prevalence. The HBV will eventually develop severe liver diseases, including liver cirrhosis, and hepatocellular carcinoma (HCC), one of the most common forms of human cancer. But its involvement in the development of HCC is still unknown. HBV is a prototype of Hepadnaviridae, a small hepatotropic DNA viruse with partially double-stranded DNA genome. The genome structure of HBV is a 3.2-kb DNA. Natural host for HBV is humans. The ORFs correspond to core protein, envelope protein (surface antigen), polymerase (pol protein), and HBx protein. Our group is interested in transactivation domain of Hepatitis B Virus x protein (HBx). The HBx protein is a multifunctional protein. It has been suggested to affect viral replication by modulating a wide variety of cellular processes, including transcriptional transactivation, apoptosis, phosphorylation or acetylation, as well as interaction with damaged DNA binding proteins. HBx is encoded by the smallest HBV ORFs and consists of 154 amino acids, with a molecular weight of about 17.5 kDa. The HBx gene is conserved among all mammalian hepadnaviruses. But the X protein lacks homology with known proteins. And lacks three-dimensional structure determined by from nuclear magnetic resonance (NMR) or X-ray crystallography. Unfortunately, the other reports showed that anti-HBV drugs such as interferon alpha and nucleoside analogs have adverse reactions or effect only in the short time, Therefore, the development of novel antiviral strategies is important. The information of the three-dimensional structure of the HBx protein will provide an alternative to understand the molecular mechanism between HBV infection and HCC. Our group has constructed the recombinant HBx gene and expressed the fusioned protein. The fusioned transactivation domain of the HBx protein was obtained in a large amount from supernatant of lysate, but not from inclusion body, and purified with Ni-NTA by AKTA prime plus (GE). In the future work enzyme digestion will be proceeded by digestion enzyme and the HBx protein will be purified by Ni-NTA and AKTA purifier.
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Roblin, Steven. "The aryl hydrocarbon receptor: Impact of structural variation of the transactivation domain and interaction with a transcriptional repressor". 2008. http://link.library.utoronto.ca/eir/EIRdetail.cfm?Resources__ID=742618&T=F.
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Pektas, Serap. "O2 Activation and Allosteric Zn(Ii) Binding on Hif-Prolyl Hydroxylase-2 (Phd2)". 2013. https://scholarworks.umass.edu/open_access_dissertations/828.
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Oxygen homeostasis is essential to the life of aerobes, which is regulated in humans by Hypoxia Inducible Factor-1α (HIF-1α). Under hypoxic conditions, HIF-1α transactivates over a hundred genes related angiogenesis, erythropoiesis, etc. HIF-1α level and function is regulated by four HIF hydroxylase enzymes: three isoforms of prolyl hydroxylase domain (PHD1, PHD2 and PHD3) and factor inhibiting HIF-1α (FIH). PHD2 is the focus of this research. PHD2 is a non-heme Fe(II) 2-oxoglutarate dependent dioxygenase, which controls HIF-1α levels by hydroxylating two proline residues within the ODD domain of HIF-1α, then the hydroxylated prolines are recognized by pVHL, which targets HIF-1α for proteasomal degradation. Under hypoxic conditions PHD2 cannot hydroxylate HIF-1α and its level rises in cells. The aims of this research include understanding how PHD2 chooses its substrate, how the O2 activation occurs, and how certain transition metals inhibit PHD2.
Our results revealed that electrostatics play a role in substrate selectivity of PHD2 by provoking a change in the opening and closing rate of β2β3 loop for NODD and CODD substrates. Mutational studies of second coordination sphere residues combined with kinetic studies indicated that decarboxylation of 2OG is the slow step in the chemical mechanism. The removal of a hydrogen-bond by the Thr387aAla mutation revealed a rate 15 times faster than WT-PHD2 by making O2 a better nucleophile. Our results indicate that this hydrogen bonding is essential for proper O2 activation.
Previous reports show that certain metals increase HIF-1α levels by inhibiting PHD2. However there are conflicts about how this inhibition occurs, either through metal replacement from the active site or metals binding to a different site causing inhibition. Our competitive and non-competitive kinetic assays showed different inhibition profiles. Under competitive conditions Zn2+, Co2+, Mn2+, and Cu2+ can bind to the enzyme active site and lead to inhibition but under non-competitive conditions Zn2+, Co2+, and Mn2+ partially inhibit PHD2 suggesting that these metals cannot displace the Fe2+ from the active site. XAS experiments with Zn2+ and Fe3+ indicate that Zn2+ binds to the surface of PHD2 in a six-coordinate manner composed of two Cys201, 208, His205, Tyr197 and two water ligands.
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R, Chabot Philippe. "Caractérisation structurale et fonctionnelle des interactions impliquant TFIIH et les domaines de transactivation viraux". Thèse, 2014. http://hdl.handle.net/1866/11003.
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Le facteur de transcription IIH (TFIIH) joue un rôle crucial dans la transcription et
dans la réparation de l’ADN. La sous-unité Tfb1/p62 (levure et humain) de TFIIH interagit
avec de nombreux facteurs de transcription (p53, NFκB, TFIIEα) et de réparation
(Rad2/XPG and Rad4/XPC) (1). La majorité des interactions avec Tfb1/p62 requiert le
domaine d’homologie à la Pleckstrin (PH) localisé dans la région N-terminal de la protéine
(2, 3). Ce domaine PH forme des complexes avec des domaines de transactivation acide
provenant de protéines cibles impliquées dans la transcription et la réparation de l’ADN.
De récentes études ont montré que Tfb1/p62 est une cible pour les protéines virales
telles que la protéine VP16 du virus de l’herpès simplex (HSV) de type 1, la protéine E1 du
virus du papillome humain (VPH) et la protéine EBNA-2 du virus Epstein-Barr (EBV) (4,
5). Ces protéines virales interagissent avec la sous-unité Tfb1/p62 par un domaine de
transactivation acide suggérant une interaction similaire à ce qui est observé chez les
facteurs de transcription humains comme p53.
Ce mémoire présente une caractérisation structurelle et fonctionnelle du complexe
formé par la protéine virale EBNA2 et la protéine humaine Tfb1/p62. L’analyse est faite en
utilisant le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire
(RMN) et une expérience de transactivation chez la levure. Cette étude amène une plus
grande compréhension des protéines impliquées dans les maladies comme le lymphome de
Burkitt et le lymphome de Hodgkin qui sont souvent associées à l’infection à l’EBV (revue
dans (6)) et caractérise une cible potentielle pour un antiviral.The general transcription factor IIH (TFIIH) plays crucial roles in both transcription and DNA repair. Tfb1/p62 (yeast and human), one of the ten/eleven subunits of TFIIH, has been shown to interact with several important transcription (p53, NFκB, TFIIEα) and repair factors (Rad2/XPG and Rad4/XPC) (1). Most of the interactions with Tfb1/p62 require the Pleckstrin homology (PH) domain located at the amino-terminal end of the protein (2, 3). This PH domain in particular forms complexes with highly acidic domains from target proteins involved in both transcriptional activation and DNA repair. Recent studies has shown that the Tfb1/p62 subunit of TFIIH is also targeted by a number of viral proteins including the Herpes Simplex virus (HSV) protein VP16, the Human papillomavirus (HPV) protein HPV E1 and the Epstein-Barr virus (EBV) protein EBNA-2 (4, 5). These viral proteins interact with the Tfb1/p62 subunit via acidic domain which suggests that they are forming similar interactions as the one observed with human transcription and repair factors. This thesis provides a structural and functional characterization of the complex formed by the viral proteins EBNA2 and the human protein Tfb1/p62 subunit of TFIIH. The analysis is done using isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy and a yeast activation assay. This study brings a greater understanding of proteins implicated in diseases such as the Burkitt’s lymphoma directly linked to an EBV infection (review in (6)) and shows a viable target for antiviral drug.
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Mas, Caroline. "Études structurales d’interactions protéine/protéine impliquées dans l’érythropoïèse". Thèse, 2010. http://hdl.handle.net/1866/4220.
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Le développement hématopoïétique est régulé par l’action combinée de facteurs de transcription lignée spécifiques et de la machinerie transcriptionnelle de base, permettant ainsi l’expression de gènes en temps et lieu appropriés. Les travaux présentés dans cette thèse portent sur l’étude structurale et fonctionnelle d’interactions décisives pour la régulation de l’expression de gènes et impliquant des domaines de transactivation (TAD). En effet, les interactions faisant intervenir les TAD d’activateurs permettent de réguler l’activation de la transcription de façon spécifique.
La première étude présentée dans cette thèse relate l'identification et la caractérisation d'une nouvelle interaction entre deux facteurs de transcription : le facteur hématopoïétique GATA-1 et la protéine suppresseur de tumeur p53. En combinant des études in vitro par titrage calorimétrique en condition isotherme (ITC) et par spectroscopie RMN et des études in vivo, nous avons identifié et caractérisé cette nouvelle interaction. Il s'avère que le TAD de p53 et le domaine de liaison à l’ADN de GATA-1 sont les domaines minimaux requis pour la formation de ce complexe. L'inhibition de la voie p53 par GATA-1 s’est avérée être la conséquence majeure de cette interaction, permettant ainsi le maintien en vie des précurseurs érythrocytaires via l’inhibition de l’apoptose.
Un deuxième type d’interaction a fait l’objet d’études : l’interaction entre divers TAD et la machinerie transcriptionnelle de base, plus spécifiquement avec le Facteur général de Transcription IIH (TFIIH). La structure des complexes constitués par la sous-unité Tfb1/p62 du facteur TFIIH en interaction avec le TAD viral de VP16 d’une part, et avec le TAD humain du facteur érythrocytaire « Erythroid Krüppel-like factor» (EKLF) d’autre part, ont été résolues par spectroscopie RMN. La structure du complexe Tfb1/VP16 a révélée que le mode de liaison de VP16 à Tfb1 est similaire au mode de liaison du TAD de p53 avec le même partenaire. En effet, les TAD de VP16 et de p53 forment tous deux une hélice α de 9 résidus en interaction avec Tfb1. En dépit de partager avec p53 et VP16 le même site de liaison sur Tfb1/p62, la structure RMN du complexe EKLF/Tfb1 démontre que le mode d’interaction de ce TAD se distingue du mode de liaison canonique des activeurs transcriptionnels. Etonnamment, EKLF adopte un mécanisme de liaison semblable au mécanisme de liaison du facteur général de transcription TFIIEα avec p62, leurs conformations demeurent étendues en interaction avec Tfb1/p62. En se basant sur nos données structurales, nous avons identifié un résidu dans le TAD d'EKLF décisif pour la formation du complexe EKLF/p62 : le Trp73. La mutation de cet acide aminé perturbe son interaction avec Tfb1PH/p62PH et réduit significativement l'activité transcriptionnelle d'EKLF dans les érythrocytes.Hematopoietic development is regulated through a combinatorial interplay between lineage-specific activators and the general transcription machinery that enables cell-specific patterns of gene expression. This thesis reports structural and functional studies of interactions involving the transcativation domains (TAD) of activators proteins and their role in hematopoietic development. Interactions between the TAD of activators and their partners play an important role in the transcriptional regulation of all genes including those regulating hematopoiesis. The first section reports the identification and characterization of a novel interaction between the erythroid transcription factor GATA-1 and the tumor suppressor protein p53. Using a combination of isothermal titration calorimetry (ITC), NMR spectroscopy and in vivo studies, we identified and characterized the direct interaction between these two important transcription factors in an attempt to determine the role of this interaction in erythroid development. Based on our results, the TAD of p53 directly interacts with the DNA-binding domain of GATA-1 in a cell-type specific manner. Through this interaction, GATA-1 inhibits activation of select p53-regulated genes and we postulate that the inhibition of p53-dependent apoptotic pathways is essential for survival of erythroid precursor cells. In the second section, we report on the interactions between two acidic TADs and the general transcription factor IIH (TFIIH). The structure of the complexes formed by the Tfb1/p62 subunit of TFIIH (Tfb1PH/p62PH) and the acidic TAD of Herpes Simplex viral protein 16 (VP16) and the Erythroid Krüppel-like factor (EKLF) were determined by NMR spectroscopy. The structure of the Tfb1PH/VP16 complex demonstrated that a viral TAD has the ability to mimic the actions of the TAD from the human p53 with Tfb1PH/p62PH. The TADs of both VP16 and p53 adopt a 9-residue α-helix in complex with Tfb1PH/p62PH. Interestingly, the NMR structure of the EKLF/Tfb1PH complex demonstrated that despite sharing a common binding site with p53 and VP16 on Tfb1PH, the EKLF/Tfb1PH binding interface is distinctly different from the binding interfaces we previously observed with p53/Tfb1PH and VP16/Tfb1PH complexes. Surprisingly, EKLF adopted a similar binding mechanism as the general transcription factor TFIIEα in interaction with p62PH as both interact in an extended conformation. Moreover, based on our structural data, we have identified Trp73 as a key residue within the TAD of EKLF that is required for the formation of the EKLF/Tfb1PH complex. Mutations of Trp73 disrupted the binding to Tfb1PH/p62PH and significantly reduced the transcriptional activity of EKLF in red blood cells.
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Idrissa, Moussa Mohamed. "Études structurales d’interactions protéine/protéine impliquées dans la leucopoïèse". Thèse, 2017. http://hdl.handle.net/1866/18660.
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La génération des cellules hématopoïétiques, aussi connue sous le nom d'hématopoïèse, est contrôlée par l’activité conjuguée de facteurs de transcription lignée-spécifiques permettant l’expression, en temps et lieu, de gènes spécifiques nécessaires pour le développement cellulaire. Dans le cadre de notre étude, nous avons étudié les facteurs de transcription KLF2 et KLF4 qui jouent des rôles cruciaux dans la formation des lymphocytes B et T. KLF2 et KLF4 activent la transcription de gènes spécifiques via leur interaction avec le co-activateur (CBP). Leurs interactions avec CBP requièrent le domaine de transactivation (TAD) qui est localisé dans la région N-terminal des facteurs KLF2 et KLF4.
Des études préalables ont montré que des domaines TAD sont aussi présents chez la protéine suppresseur de tumeur p53 et que ces domaines sont requis pour les interactions entre la protéine p53 et le co-activateur CBP. Récemment, plusieurs structures des TADs de p53 en complexe avec les domaines TAZ2 et KIX de CBP ont permis de démontrer que ces TADs sont de nature acide et contiennent un motif ΦΧΧΦΦ crucial pour la formation des interactions. De plus, il s’avère que ces TADs sont similaires aux TADs de KLF2 et KLF4.
L’étude présentée dans ce mémoire relate la caractérisation structurelle et fonctionnelle des interactions formées par les facteurs de transcription KLF2 et KLF4 avec leur partenaire d'interaction, CBP, pour activer la transcription de gènes spécifiques. Nos analyses ont été faites en utilisant différentes techniques telles que le titrage calorimétrique isotherme (ITC), la résonance magnétique nucléaire (RMN) ainsi que des expériences de transactivation chez la levure. Notre étude permet une meilleure compréhension des rôles opposés mais complémentaires qu'ont les protéines KLF2 et KLF4 au cours du développement et de la différentiation des lymphocytes B et T en plus de fournir les détails mécanistiques à la base de leurs interactions. Ces informations seront potentiellement utiles pour le développement d'outils à des fins thérapeutiques dans le cadre des leucémies, notamment.Hematopoietic development is regulated through a combinatorial interplay between lineage-specific activators and the general transcription factors that enables cell-specific patterns of gene expression. In this study, the transcription factors KLF2 and KLF4 play crucial roles in lymphocytes B and T development by activating transcription of specific genes through interactions with the co-activator (CBP). These interactions involve the transactivation domains (TAD) localized in the N-terminal region of KLF2 and KLF4 factors. Previous studies have shown that TADs are also found in the tumor suppressor protein p53 and these TADs are responsible for the interactions between the p53 protein and the coactivator CBP. Recently, several structures of p53TADs in complex with the TAZ2 and KIX domains of CBP have shown that these TADs are acidic and possess a ΦΧΧΦΦ motif crucial for the formation of the interaction. Interestingly, these TADs are similar to the ones found on KLF2 and KLF4. This thesis provides a structural and functional characterization of the interactions formed by the transcription factors KLF2 and KLF4, which have opposing roles, and competes for the same interacting partner CBP to activate transcription. The analysis is done using isothermal titration calorimetry (ITC), nuclear magnetic resonance (NMR) spectroscopy and a yeast activation assay. This study brings a greater understanding on the opposing roles yet complementary of KLF2 and KLF4 proteins involved in B and T lymphocytes specific lineages selection and also provides information for potential therapeutic research regarding disease such as leukemia.