Dissertations / Theses: 'Homodimers'

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Burnell, David. "Multi-scale modelling of allostery in protein homodimers." Thesis, Durham University, 2015. http://etheses.dur.ac.uk/11055/.

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Allostery is a form of signalling within biomolecules such that ligand binding to a protein affects its activity at a second site. Allostery was described by early models to be driven by structural changes in the protein. However, more recently there has been increasing evidence that dynamics can contribute to or even drive allostery. The protein studied in this thesis, the Catabolite Activator Protein (CAP), is an allosteric protein homodimer that has been shown to exhibit negatively cooperative binding of the ligand cyclic Adenosine Monophosphate (cAMP) to each of its monomers. Interestingly, CAP is a protein whose allostery is believed to be driven by dynamics rather than a conformational change. In this thesis, a number of coarse grained models are employed to investigate this dynamic allostery in CAP. One family of models, termed Super Coarse Grained (SCG) models explore the global properties of the dynamics of the CAP dimer that cause it to exhibit negatively cooperative allostery. It is shown through these models that changes in protein interactions can provide a basis for changing cooperativity. A second family of coarse grained models called Elastic Network Models (ENM) are studied. These are used to show that adjusting the interactions between specific residues can affect cooperative binding of cAMP to CAP. A number of atomistic approaches are also used to study the cAMP-CAP system, including Molecular Dynamics (MD) and Normal Mode Analysis (NMA). The efficacy of using such approaches for studying the thermodynamics of the allostery in CAP is investigated. The motion observed within the protein is also studied closely to identify potential allosteric pathways. X-ray crystallography and Isothermal Titration Calorimetry (ITC) are finally used to investigate how accurately computational methods can describe the cooperative binding of cAMP to CAP. They are also used to try and determine whether the allostery in CAP can be manipulated experimentally without any observed changes to its structure.

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Sanders, David A. R. 1968. "Thioredoxin homodimers: Effects of mutation and oxidation on structure and dimer formation." Diss., The University of Arizona, 1998. http://hdl.handle.net/10150/282851.

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Thioredoxins are a family of small redox proteins that mediate a number of cytosolic processes in the cells of all organisms. Human thioredoxin has a number of additional functions, including the apparent stimulation of NF-κB and AP-1 transcriptional activation. It can also be exported out of the cell, where it apparently has additional functions including the ability to stimulate cell growth. The crystal structure of human thioredoxin revealed that the protein can form a homodimer. The dimer contains a disulfide bond between the Cys 73 residues of each monomer, and a novel hydrogen bond between the Asp 60 residue of each monomer that is responsible for the pH dependent behaviour of dimerization. This work was undertaken with two goals. First, three mutant protein with changes in the dimer interface, Asp58 → Ser (D58S), Ala66 → Arg (A66R) and Trp31 → Ala (W31A) were isolated and characterized. The ability of the mutant proteins to form dimers, their activities with respect to the reduction of insulin disulfides and their interaction with thioredoxin reductase were examined. The three mutations affected thioredoxins ability to form dimers: the W31A and A66R mutant proteins formed dimers less well, and the D58S mutant protein lost the pH dependence for dimer formation. Thioredoxin with a D58S mutation behaved very similarly to wild type in the activity assays, while A66R showed a 6-fold increase in K(m) and W31A showed large changes to both K(m) and V(max). From these results I suggest that the dimer interface plays a role in defining the binding site for thioredoxin reductase. The second goal was to examine the role that oxidation of the active site plays in dimerization. Oxidation, which results in structural changes to the dimer interface, also caused thioredoxin to form dimers much less readily. The reduction in dimer formation offers a possible mechanism through which thioredoxin could play a role in signaling oxidative stress. The findings detailed here begin to describe the role that the dimeric form of thioredoxin could play in physiological situations. The structural bases for the changes in dimerization are not yet fully characterized, as the monomeric form of thioredoxin has not yet been crystallized.

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3

Harish, S. "Transcriptional Regulation By Nuclear Receptor Homodimers Binding To The Direct Repeat Motif DR1 : Investigations In An in vitro Transcription System Derived From Rat Liver Nuclear Extracts." Thesis, Indian Institute of Science, 2000. http://hdl.handle.net/2005/164.

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Nuclear receptors (NRs) are important transcription factors involved in the regulation of a variety of physiological processes such as embryonic development, cell differentiation and homeostasis (for review, see Mangelsdorf et al., 1995 TenBaum and Baniahrned, 1997). In contrast to membrane bound receptors, they bind small lipophilic ligands and function in the nucleus as ligand-modulated transcription factors. The ligands for nuclear receptors include steroids (glucocorticoids, progestins, mineralocorticoids, androgens and estrogens), vitamin D3, retinoids, thyroid hormone, prostaglandins, farnesoids etc. Several other nuclear receptors are classified as orphan receptors for which no ligand has yet been identified. More than 300 nuclear receptors have now been identified and together these proteins comprise the single largest family of metazoan transcription factors, the nuclear receptor superfamily. Recently, a unified nomenclature has been evolved (nuclear receptor nomenclature committee, 1999), a summary of which is presented in Table 1.

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4

Silva, Wagner André Vieira da. "Uso da estratégia drogas gêmeas para a síntese de novos homodimeros de adutos de morita-bayllis- hilman potenciais candidatos a fármacos antiparasitários." Universidade Federal da Paraíba, 2016. http://tede.biblioteca.ufpb.br:8080/handle/tede/9022.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES
This work was performed in order to synthesize and bioavaliar the activity of new adducts Morita-Baylis-Hillman (AMBH) as potential drug candidates. The AMBH were synthesized from the twin drug approach (approach twin drugs) and bioavaliados against Leishmania promastigote form donovanii, a kind of visceral leishmaniasis and more severe disease, which has a drug used for the treatment accompanied by high toxicity. As Michael acceptor to be used in Morita-Baylis-Hillman (MRBH) was synthesized diacrylate ethylene glycol (50) from the esterification reaction between ethylene glycol (65) and acrylic acid (66). The first MRBH was investigated between two equivalent 2-nitrobenzaldehyde (57) and one equivalent of diacrylate 50, in acetonitrile as solvent in the presence of DABCO, yielding two products: an adduct 67 and an adduct homodimeric 42. In investigations of experimental parameters the MRBH, DMF, DABCO and room temperature proved to be the most favorable conditions for the formation of adducts homodimeric, these being obtained in yields of 35-94% and reaction times between 24 and 20 days, isolated by liquid / liquid and via flash chromatography. Homodimers and other bioavaliados AMBH results were satisfactory to excellent IC50 for homodimeric adducts (IC50 126.20 to 0,50M). All homodimeric AMBH had higher bioactivity the corresponding AMBH, showing the success of the twin drugs approach against promastigote species of leishmania donovanii, reaching the impressive result, in the case of 49 homodimer be 393.1 times more active than the corresponding AMBH 56, being 1.24 more active than the anfoterinica B, and no reported toxicity exposure in red blood cells of human blood (iS> 400 against iS = 18.73 amphotericin B). These results show that 49 homodimer is a promising molecule in the search for new drug candidates.
Este trabalho foi realizado com o objetivo de sintetizar e bioavaliar a atividade de novos Adutos de Morita-Baylis-Hillman (AMBH) como potenciais candidatos a fármacos. Os AMBH foram sintetizados a partir da abordagem drogas gêmeas (twin drugs approach) e bioavaliados contra a forma promastigota Leishmania donovanii, uma espécie da forma visceral e mais grave da doença, a qual possui o fármaco utilizado para o tratamento acompanhado de grande toxicidade. Como aceptor de Michael para ser utilizado na reação de Morita-Baylis-Hillman (RMBH), foi sintetizado o diacrilato do etileno glicol (50) a partir da reação de esterificação entre o etileno glicol (65) e o ácido acrílico (66). A primeira RMBH investigada foi entre dois equivalentes 2-nitrobenzaldeído (57) e um equivalente do diacrilato 50, em acetonitrila como solvente na presença de DABCO, obtendo-se dois produtos: um aduto 67 e um aduto homodimérico 42. Nas investigações dos parâmetros experimentais da RMBH, o DMF, o DABCO e a temperatura ambiente mostraram ser as condições mais favoráveis para a formação dos adutos homodiméricos, sendo esses obtidos com rendimentos entre 35-94% e em tempos reacionais entre 24h e 20 dias, isolados por extração líquido/líquido e via cromatográfia flash. Os homodímeros e os demais AMBH bioavaliados tiveram resultados de satisfatórios a excelentes de CI50 para os adutos homodiméricos (CI50 126,20 a 0,50M). Todos os AMBH homodiméricos tiveram bioatividade superior aos AMBH correspondentes, evidenciando o sucesso da abordagem de drogas gêmeas contra a espécie promastigota da leishmania donovanii, chegando ao impressionante resultado, no caso do homodímero 49, ser 393,1 vezes mais ativo que o AMBH correspondente 56, sendo 1.24 mais ativo que a anfoterinica B, além de não apresentar toxicidade na exposição em glóbulos vermelhos do sangue humano (IS > 400, contra IS = 18,73 da anfotericina B). Estes resultados evidenciam que homodímero 49 é uma molécula promissora na busca de novos candidatos a fármacos.

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5

Clement, Ella Chow. "Design and Syntheses of Potential Drugs Based on GABA(A) Receptor Pharmacophores." Diss., Virginia Tech, 2005. http://hdl.handle.net/10919/28271.

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Numerous previous studies of GABAAR ligands have suggested that GABAAR agonists must be zwitterionic and feature an intercharge separation similar to that of GABA (approx. 4.7-6.0 Ã ). We have demonstrated that monomeric, homodimeric and heterodimeric non-zwitterionic GABA amides are partial, full, or superagonists at the murine GABAA receptor (GABAAR). The agonism of these GABA amides is comparable to that of THIP, as shown by in vitro assay results. The assay data indicate that the agonism of GABA amides is tether length-dependent. Optimum agonism is achieved with a tether length of four methylenes in GABA amide dimers and in GABA amides bearing pendant amide or amino groups. We have further investigated the structure-activity relationship for GABA amides on the GABAAR by performing structural modifications to both the superagonist 2c and the agonist 6c. Synergism and [3H]muscimol binding experiments show that 2c binds to the same sites as GABA. Structural modification of 2c demonstrated that partial rigidification of the tether eliminated agonism and caused ligands to behave as weak competitive antagonists. We have also investigated the agonism of four ZAPA derivatives in 36Cl- uptake functional assay. Two of them are found to be as potent as GABA. In our studies of 1,4-benzodiazepines, our goal was to synthesize three different subtypes of quaternary 1,4-benzodiazepines by use of the memory of chirality (MOC) strategy. Disappointingly, most of the deprotonation/alkylations failed, due to various reasons. The failure of the reactions of (S)-alanine-derived tetrahydro-1,4-benzodiazepin-3-ones was probably due to either the unexpected side reactions or the steric hindrance of enolate alkylation. In the case of tetrahydro-1,4-benzodiazepin-2-ones, computational studies suggested that steric hindrance by both the benzo ring and N4-allyl group might retard deprotonation at C3 by bulky bases like KHMDS or LDA. Finally, (S)-serine-derived 1,4-benzodiazepin-2-ones and their elimination products (ï ¡-methylene benzodiazepines) were prepared. These proved unreactive towards deprotonation/alkylations and conjugate additions, respectively. The low reactivity of the ï ¡-methylene benzodiazepines towards nucleophiles was attributed to highly delocalized LUMOs that failed to direct nucleophiles to the ï ¢-carbons.
Ph. D.

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6

Connerney, Jeannette J. "Balance between Formation of Twist1 Homodimer and Heterodimer Regulate Suture Fusion." Fogler Library, University of Maine, 2007. http://www.library.umaine.edu/theses/pdf/ConnerneyJJ2007.pdf.

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7

Negron, Christopher. "Computational design of orthogonal antiparallel homodimeric coiled coils." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/93805.

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Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Living cells integrate a vast array of protein-protein interactions (PPIs) to govern cellular functions. For instance, PPIs are critical to biosynthesis, nanostructural assembly, and in processing environmental stimuli through cell-signaling pathways. As fields such as synthetic biology and protein engineering mature they seek to mimic and expand the functions found in living systems that integrate PPIs. A critical feature to many PPIs that are integrated together to perform a complex function is orthogonality, i.e. PPIs that do not cross interact with each other. The engineering of orthogonal PPIs is thus an alluring problem. Since it not only tests our understanding of molecular specificity by having to stabilize and destabilize interactions simultaneously. The results of the design process can also have interesting applications in synthetic biology or bionanotechnology. The coiled coil, a rope-like structure made of helices, is a PPI ubiquitously found in biological systems and is an attractive fold for engineering orthogonal PPIs. Though the coiled coil is well studied, destabilization of undesired interactions still remains challenging. In this thesis I will discuss strategies for obtaining orthogonal PPIs, and describe the current sequence-to-structure relationships known about coiled coils. I will then introduce the computational multistate design framework, CLASSY, and explain how I applied it to the computational design of six orthogonal antiparallel homodimeric coiled coils. Five of these designed sequences were experimentally tested, of which only three of the sequences adopted the target antiparallel homodimer topology. All three of these sequences, as well as a previously designed antiparallel homodimer, were tested for cross reactivity in a pairwise manner. None of these sequences appeared to cross react. The sequences that failed to adopt the antiparallel topology highlight the need for improving our computational design framework. In the final chapter I will discuss strategies to improve our models, and applications for orthogonal antiparallel coiled coils.
by Christopher Negron.
Ph. D.

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8

Shin, Jong M. "Role of C121A in mGluR2 homodimeric expression and function." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5576.

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The group II metabotropic glutamate receptors are known for their involvement in various psychiatric disorders. The mGluR2 in particular is linked with etiology of schizophrenia especially in the context of crosstalk with 5-HT2A. Thus, the mGluR2 has attracted attentions for its potential therapeutic applications. Despite numerous physiological evidences on the actions of mGluR2, its mechanism is still unclear to this day. It is partially due to the lack of understanding in characteristics of mGluR2 homodimer which is its functionally active form. Therefore, the characterization of dimeric interaction serves as a foundation to advanced understanding of the role of mGluR2. On that note, the role of the conserved cysteine residue (C121) in the ligand binding domain of mGluR2 has been evaluated in this study as they are known to play a critical part in homodimer formation. Collectively, C121 has been shown to affect the dimerization, subcellular localization, and pharmacokinetics of mGluR2. Lastly, the effect of mGluR2 on mouse behavior was examined in a partial effort to elucidate its role in crosstalk with 5-HT2A.

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9

Mcgeagh, John David. "Conformation and cooperativity in homodimeric enzymes investigated by molecular dynamics simulations." Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.549446.

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10

Lin, Yi-Jan. "Solution structure of the 30 kDa homodimeric sud protein from Wolinella succinogenes." [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968620817.

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Ali, Ambereen. "Human leukotriene C4 synthase : a unique homodimeric and phisphoregulated glutathione S-transferase." Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41520.

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Human Leukotriene C$ sb4$ synthase is a membrane-bound glutathione S-transferase that catalyzes the first committed step leading to the biosynthesis of the pro-inflammatory mediators, the cysteinyl leukotrienes (LTC$ sb4,$ LTD$ sb4$ and LTE$ sb4$). Using chromatographic separation and photoaffinity labelling techniques, LTC$ sb4$ synthase was found to be a unique enzyme distinct from all other known glutathione S-transferases. An 18 kDa membrane polypeptide was specifically labelled in the microsomal membranes of myelocytic cell lines containing LTC$ sb4$ synthase activity (U937 and THP-1), with a photoaffinity derivative of LTC$ sb4$ (azido (I$ sp{125}$) -LTC$ sb4$) and was identified as a candidate for being LTC$ sb4$ synthase or a subunit thereof. LTC$ sb4$ synthase was subsequently purified to homogeneity from THP-1 cells and the purified preparation contained only one polypeptide which had a molecular mass of 18 kDa. By gel filtration chromatography, the native molecular mass of LTC$ sb4$ synthase was determined to be approximately $39 pm3$ kDa. It was therefore concluded that LTC$ sb4$ synthase is enzymatically active as a homodimer. The sequence of the N-terminal 35 amino acids of purified human LTC$ sb4$ synthase was determined and was found to be unique, composed primarily of hydrophobic amino acids and containing a protein kinase C (PKC) consensus sequence. Further analysis of the potential phosphoregulation of LTC$ sb4$ synthase in neutrophilic and eosinophilic HL-60 cells demonstrated that cysteinyl leukotriene biosynthesis, but not non-cysteinyl leukotriene biosynthesis, was specifically attenuated by phorbol ester-mediated activation of PKC. In eosinophilic HL-60 and THP-1 cells this decrease in cysteinyl leukotriene production was demonstrated to be due to non-competitive inhibition of LTC$ sb4$ synthase activity. Concomitant with the PKC-mediated decrease in cysteinyl leukotriene biosynthesis, an increase in prostanoid biosynthesis occurred. Based

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Lockbaum, Gordon J. "Molecular Mechanisms of Resistance and Structure-Based Drug Design in Homodimeric Viral Proteases." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1072.

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Drug resistance is a global health threat costing society billions of dollars and impacting millions of lives each year. Current drug design strategies are inadequate because they focus on disrupting target activity and not restricting the evolutionary pathways to resistance. Improved strategies would exploit the structural and dynamic changes in the enzyme–inhibitor system integrating data from many inhibitors and variants. Using HIV-1 protease as a model system, I aimed to elucidate the underlying resistance mechanisms, characterize conserved protease-inhibitor interactions, and generate more robust inhibitors by applying these insights. For primary mechanisms of resistance, comparing interactions at the protease–inhibitor interface showed how specific modifications affected potency. For mutations distal to the active site, molecular dynamics simulations were necessary to elucidate how changes propagated to reduce inhibitor binding. These insights informed inhibitor design to improve potency against highly resistant variants by optimizing hydrogen bonding. A series of hybrid inhibitors was also designed that showed excellent potency by combining key moieties of multiple FDA-approved inhibitors. I characterized the structural basis for alterations in binding affinity in HIV-1 protease both from mutations and inhibitors. I applied these strategies to HTLV-1 protease, a potential drug target. I identified the HIV-1 inhibitor darunavir as a viable scaffold and evaluated analogues, leading to a low-nanomolar compound with potential for optimization. Hopefully, insights from this thesis will lead to the development of potent HTLV-1 protease inhibitors. More broadly, these inhibitor design strategies are applicable to other rapidly evolving targets, thereby reducing drug resistance rates in the future.

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Isbert, Simone [Verfasser]. "Cellular localization and mechanism of amyloid precursor protein (APP) homodimer formation in an oxidizing environment / Simone Isbert." Mainz : Universitätsbibliothek Mainz, 2012. http://d-nb.info/1020164611/34.

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Chen, Andrew Ph D. Massachusetts Institute of Technology. "Discovery and characterization of a small molecule that modulates c-Myc mediated transcription via max homodimer stabilization." Thesis, Massachusetts Institute of Technology, 2019. https://hdl.handle.net/1721.1/123060.

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Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2019
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 190-200).
The transcription factor Myc is a basic helix-loop-helix leucine zipper (bHLHLZ) protein with crucial roles in regulating normal cellular processes, but its transcriptional activity is deregulated in a majority of human cancers. Myc transcriptional activity is dependent on dimerization with its obligate partner Max, another bHLHLZ transcription factor. Max also forms homodimers as well as heterodimers with other proteins including the Mxd family of proteins, creating a dynamic network of protein-protein interactions to regulate transcriptional programs. Despite the significance of this network, the arsenal of chemical probes to interrogate these proteins in cancer biology remains limited. Here, we utilized small molecule microarrays and luciferase-based reporter assays to identify compounds that bind Max and modulate Myc transcriptional activity. We discovered the small molecule KI-MS2-008, which stabilizes the Max homodimer while reducing Myc protein and Myc-regulated transcript levels. KI-MS2-008 also decreases viable cancer cell growth in a Myc-dependent manner and suppresses tumor growth in mouse models of Myc-driven cancers. In a cancer cell line model treated with KI-MS2-008, the equilibrium of protein-protein interactions shifts toward a transcriptionally repressed state over time by recruiting Mxd4 and other repressive machinery to Max. This study establishes that perturbing Max dimerization with small molecules is a tractable approach to targeting Myc activity in cancer.
by Andrew Chen.
Ph. D.
Ph.D. Massachusetts Institute of Technology, Department of Biological Engineering

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15

Yuan, Xiaohui. "Characterization of the ligand-binding specificity and transcriptional properties of estrogen receptor homodimeric/heterodimeric complexes." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3036871.

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16

Kukuk, Laura [Verfasser], and Bernd [Gutachter] König. "High-resolution structure of the SAM domain homodimer of the murine adapter protein SLY1 / Laura Kukuk ; Gutachter: Bernd König." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2018. http://d-nb.info/1164763040/34.

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Benleulmi-Chaachoua, Abla. "Etude des partenaires protéiques associés aux homodimères et aux hétérodimères des récepteurs couplés aux protéines G." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA11T018/document.

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La mélatonine est une neuro-hormone secrétée par la glande pinéale pour réguler les rythmes circadiens, le sommeil, la physiologie de la rétine, la reproduction saisonnière et diverses fonctions neuronales. La mélatonine exerce ses fonctions en se liant à deux récepteurs membranaires appelés MT1 et MT2 qui appartiennent à la famille des récepteurs couplés aux protéines G (RCPG). Les RCPG sont connus pour former des homo- et hétérodimères mais la pertinence physiologique de ces complexes reste à démontrer. Plusieurs études montrent que la fonction de ces complexes ne se limite pas à la régulation des protéines G hétérotrimériques, mais inclue également la régulation d'autres protéines comme les transporteurs et les canaux ioniques. Dans ce travail, nous rapportons la formation d'hétérodimères MT1/MT2 dans les photorécepteurs de la rétine de souris et nous montrons que l’augmentation de la sensibilité de ces cellules à la lumière par la mélatonine requiert l'activation de la voie Gq/PLC/PKC qui est spécifique de l’hétéromère. Cette étude confirme alors la pertinence physiologique de l’hétérodimérisation des récepteurs de la mélatonine.Nous avons ensuite cherché à identifier de nouveaux partenaires de MT1 et MT2 en effectuant plusieurs cribles protéomiques et génétiques et un interactome de 378 protéines a pu être construit. L'analyse bioinformatique a révélé la présence de plusieurs protéines présynaptiques (canaux calciques voltage-dépendants Cav2.2, SNAP25, Synapsin et Munc-18) dans l'interactome MT1. Parmi ces partenaires, nous avons montré dans les cellules CHO que le récepteur MT1 interagit avec la protéine Cav2.2 et inhibe l’entrée du calcium d'une manière indépendante de la stimulation par l’agoniste, ce qui suggère un rôle régulateur de MT1 dans la libération des neurotransmetteurs.Un autre partenaire caractérisé est le transporteur de la dopamine DAT. L'interaction physique de DAT avec les récepteurs de la mélatonine diminue l’expression de DAT à la surface cellulaire et diminue l'absorption de la dopamine dans les cellules HEK293. La pertinence physiologique de ces observations a été appuyée par l’augmentation de la recapture de la dopamine dans les synaptosomes du striatum de souris knock-out pour les récepteurs de la mélatonine. En conclusion, ce rapport montre que la construction des interactomes des RCPG offre de nouvelles perspectives pour la découverte de nouvelles fonctions de ces récepteurs, comme les fonctions rétiniennes et neuronales des récepteurs de la mélatonine dans notre étude. La formation de complexes RCPG/RCPG, RCPG/canaux ioniques et RCPG/transporteurs peut avoir un effet fonctionnel réciproque au niveau de l’activité du récepteur et de ces partenaires, mettant ainsi en évidence de nouveaux mécanismes moléculaires de cross-talk cellulaire
Melatonin is a neurohormone secreated by the pineal gland in a circadian manner. This hormone is involved in the regulation of circadian rhythms, sleep, retinal physiology, seasonal reproduction and various neuronal functions. Melatonin exerts its effects through two G protein-coupled receptors (GPCR) called MT1 and MT2. GPCRs are known to form homo- and heterodimers, but the physiological relevance of these complexes remains a matter of debate. An increasing number of reports show that the function of these GPCR complexes is not restricted to the regulation of heterotrimeric G proteins but include also the regulation of other proteins like transporters and ion channels. Here, we report the formation of MT1/MT2 heterodimers in mouse retinal rod photoreceptors and show that the enhancing effect of melatonin on light sensitivity in these cells requires the activation of the heteromer-specific Gq/PLC/PKC signaling pathway. This study demonstrates the physiological relevance of GPCR heterodimerization.We next searched for new MT1 and MT2 interacting proteins in an unbiased manner by performing several proteomic and genetic screens. An interactome of 378 proteins was built. Bioinformatic analysis revealed the presence of several presynaptic proteins (voltage-gated calcium channel Cav2.2, SNAP25, Synapsin and Munc-18) in the MT1 interactome. Presynaptic localization of MT1 and spatial proximity with presynaptic proteins was confirmed in mouse and rat brains. Among these potential partners, we show that MT1 physically interacts with Cav2.2 in CHO cell line and inhibits Cav2.2-promoted Ca2+ entry in an agonist-independent manner suggesting a regulatory role of MT1 in neurotransmitter release.Another proteins identified in the screens was the dopamine transporter DAT. Physical interaction of DAT with melatonin receptors decreased DAT cell surface expression and diminished dopamine uptake in HEK293 cell. Supporting this result we found using the in vivo model of melatonin receptors knockout mice a respective increase of dopamine uptake in synaptosomal preparations of the striatum of supporting the physiological relevance of these GPCR/transporter complexes. In conclusion, this report shows that GPCR interactome building provides new insights into receptor function, like retinal and neuronal functions of melatonin receptors in our case. Formation of GPCR/GPCR, GPCR/ion channel and GPCR/transporter complexes may have a reciprocal functional impact, on the activity of the receptor and interacting partners thus elucidating new molecular mechanisms cellular cross-talk

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Nguyen, Andreas [Verfasser], and Gerhard [Akademischer Betreuer] Klebe. "Neue Ansätze zur Entwicklung von Modulatoren der homodimeren tRNA-Guanin-Transglycosylase / Andreas Nguyen ; Betreuer: Gerhard Klebe." Marburg : Philipps-Universität Marburg, 2021. http://d-nb.info/1236692063/34.

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Stephanou, Augoustinos S. "Biophysical study of the DNA charge mimicry displayed by the T7 Ocr protein." Thesis, University of Edinburgh, 2010. http://hdl.handle.net/1842/4348.

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The homodimeric Ocr protein of bacteriophage T7 is a molecular mimic of a bent double-stranded DNA molecule ~24 bp in length. As such, Ocr is a highly effective competitive inhibitor of the bacterial Type I restriction modification (R/M) system. Thus, Ocr facilitates phage infection of the bacterial cell to proceed unhindered by the action of the R/M defense system. The main aim of this work was to understand the basis of the DNA mimicry displayed by Ocr. The surface of the protein is replete with acidic residues, most or all of which mimic the phosphate backbone of DNA. Aspartate and glutamate residues on the surface of Ocr were either mutated or chemically modified in order to investigate their contribution to the tight binding between Ocr and the EcoKI Type I R/M enzyme. Single or double mutations of Ocr had no discernable effect on binding to EcoKI or its methyltransferase component (M.EcoKI). Chemical modification was then used to specifically modify the carboxyl moieties of Ocr, thereby neutralizing the negative charges on the protein surface. Ocr samples modified to varying degrees were analysed to establish the extent of derivatisation prior to extensive biophysical characterisation to assess the impact of these changes in terms of binding to the EcoKI R/M system. The results of this analysis revealed that the electrostatic mimicry of Ocr increases the binding affinity for its target enzyme by at least ~800-fold. In addition, based on the known 3-D structure of the protein, a set of multiple mutations were introduced into Ocr aimed at eliminating patches of negative charge from the protein surface. Specifically, between 5 and 17 acidic residues were targeted for mutation (Asp and Glu to Asn and Gln, respectively). Analysis of the in vivo activity of the mutant Ocr along with biophysical characterisation of the purified proteins was then performed. Results from these studies identified regions of the Ocr protein that were critical in forming a tight association with the EcoKI R/M system. Furthermore by comparing the relative contribution of different groups of acidic residues to the free energy of binding, the actual mechanism by which Ocr mimics the charge distribution of DNA has been delineated.

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Klingler, Andrea M. "Novel Insight into the Role of LXRa in Metabolic Regulation viaDNA Binding as a Heterodimer with PPARa and as a Homodimer." Wright State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=wright1472486254.

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Fallatah, Hafsah M. "Regulation of COX-2 expression by the BCL-3:NF-KappaB homodimeric complex : implications for colorectal carcinogenesis." Thesis, University of Bristol, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.685143.

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Cyclooxygenase-2 (COX-2) is an enzyme that plays a key role in the synthesis of prostaglandin (PGE2). COX-2 has been widely investigated in cancer progression studies generally and in colorectal cancer (CRC) specifically, not only because it is regulated by various signalling pathways, but also because it is one of the main players in the inflammation/cancer link. The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) is among the transcription factors that have been repOlied to regulate the transcriptional activity of the COX-2 gene. Constitutive activity of NF-KB clearly results in the over-expression observed of COX-2. Although there are two KB sites localised at the promoter region of COX-2, the definitive mechanism by which COX-2 is regulated by NF-KB needs more elucidation. Several modulators have been repOlied that control NF-KB activity in order to maintain the selectivity or specificity of responses to the trigger. B-cell lymphoma-3 (BCL-3) is an oncogene that is up-regulated by inflammatory cytokines, such as tumour necrosis factor-a (TNF-a) and interleukin-l ~ (IL-l ~). It functions as a co-activator protein that binds favourably to the homodimer' subunits of NF-KB, namely pSO/pSO or pS2/pS2. Those two dimers are found to have repressor roles in the regulation of NF-KB target genes. However, when they bind to BCL-3, their action switches to an activator role. Therefore, understanding the exact role ofBCL-3: NF-KB homodimers complex in the context ofCOX-2 regulation by NF-KB signalling is crucial. Initially, I investigated the expression of BCL-3 and COX-2 in a range of different adenoma and carcinoma cells. I chose HCA 7/P and HT29 cells to be included in subsequent studies. In studying the effect of BCL-3 suppression in the level of COX-2 protein and its activity, potential regulation of COX-2 and PGE2 by BCL-3 has been revealed. This regulation has also been observed after the treatment of cells with TNF-a. Interestingly, the mRNA level of COX-2 has also been regulated by BCL-3 suppression, especially at 48 hours of BCL-3 silencing. Dual-Luciferase assay results of COX-2 gene also support this finding. Transfecting the cells with mutant BCL-3 (which cannot bind the homodimer pSO/pSO) has shown no change to the promoter activity of COX-2 as compared with cells transfected with wild-type BCL-3 protein; this suggests an addition to requirement amount of pSO to regulate COX-2 by BCL-3. Results obtained from Chromatin Immunoprecipitation (ChIP) experiments emphasised the presence of both BCL-3 and pSO in the predicted KB site of the COX-2 promoter. In vivo, there was a marked increase of both BCL-3 and COX-2 expression in carcinoma tissue as compared to healthy normal tissue, indicating the important role they play in the progression from adenoma to carcinoma. Because BCL-3 has been shown to regulate the level of PGE2, it was important to investigate whether this had . any consequence on the role played by PGE2 in the proliferation and sternness of the adenoma cell RG/C2. It was also significant to study the effect of Nonsteroidal anti-inflammatory drugs (NSAIDs) on the function of BCL-3 in regards to the COX-2/PGE2 signalling pathway. Treating cells with aspirin results in a decrease in the protein level of BCL-3 and COX-2, which strengthens the notion of aspirin chemoprevention 's role in tumour regression. This study is proposing, for the first time, the potential use of BCL-3: COX-2/PGE2 as a therapeutic target of CRC prevention and treatment.

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Ence, Chloe Christine. "Organic Synthesis using Bimetallic Catalysis." BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/8397.

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Bimetallic Catalysis is an emerging field of study that uses two metals to cooperatively perform organic transformations. These metals can serve to activate or bind substrates in order to increase the rate and selectivity of reactions. This work first describes the synthesis and utilization of six new chiral, titanium-containing phosphinoamide ligands. These Lewis acidic ligands withdraw electron density from an active palladium center to induce chirality and increase the rate of allylic amination of hindered, secondary N-alkyl amines. X-ray quality crystals were grown for each ligand and completed the allylic amination of hindered secondary amines in minutes whereas other non-titanium-containing ligands produced trace product. Although enantioselectivity was low initially, through a dynamic kinetic resolution enantioselectivity was increased over time, reaching 53% enantioselectivity. The second type of bimetallic catalysis discussed is dinuclear Pd(II) and Pd(I) catalysis. These dimers were built on a 2-phosphinoamide ligand scaffold and present interesting molecular structure and unique reactivity. These dimers were found to perform tandem arylketone coupling to produce disubstituted naphthalene products under oxidative conditions. It is proposed that the Pd(II) dimer undergoes oxidative addition to produce a Pd(III) dimer which subsequently produces an aryl-ketone intermediate. This process is made possible by the cooperativity of the two palladium centers which enable the formation of a Pd(III) dimer, circumventing the need for the high energy Pd(IV) oxidation state. Oxidative conditions then allows coupling and cyclization of a second ketone to form the naphthalene product.

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Southern, Samantha. "A novel interaction between BAG-1 and the p50-p50 homodimeric NF-KB complex : implications in colorectal carcinogenesis." Thesis, University of Bristol, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.529825.

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Fäh, Christoph. "Zyklische Difluorketone : neue Bausteine für Inhibitoren der Aspartylproteasen Plasmepsin I-IV und für die Darstellung auf C=O...C=O-Wechselwirkungen basierter Homodimere /." [S.l.] : [s.n.], 2009. http://e-collection.ethbib.ethz.ch/show?type=diss&nr=18261.

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25

Chen, Yujie, and 陈宇杰. "Structural and functional studies of human APPL1-APPL2 BAR-PH heterodimer, APPL2 BAR-PH homodimer, and lanthionine synthetase component C-like protein 2." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hdl.handle.net/10722/197138.

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APPL BAR-PH heterodimer and APPL2 BAR-PH homodimer The APPL (Adaptor protein containing PH domain, PTB domain and Leucine zipper) family are adaptor proteins with only two isoforms, APPL1 and APPL2. They bind to early endosomes with a small GTPase, Rab5, and mediate the interactions between various receptors and downstream signaling components, thus functioning in many signaling pathways evoked by adiponectin, insulin, FSH, EGF, and so on. However, evidences have shown APPL1 and APPL2 should perform some opposite functions, which cannot be simply explained by the functional differences attributed to their PTB domains. We hypothesize that the heterodimerization of APPL1 and APPL2’s BAR domains may account for their opposing functions. The crystal structure of APPL BAR-PH heterodimer was solved to resolution 2.8 Å. Its overall structure exhibits crescent shape with a larger curvature radius of 76 Å, compared to 55 Å of the APPL1 BAR-PH homodimer. And the crystal structure APPL2 BAR-PH homodimer was solve to resolution 3.3 Å. The overall structure of APPL2 BAR-PH homodimer is very similar to that of APPL BAR-PH heterodimer, but shows greater difference in curvature to the APPL1 BAR-PH homodimer structure. The concave side of APPL BAR-PH heterodimer and APPL2 BAR-PH homodimer all possess less positive charge than the APPL1 BAR-PH homodimer. Structural analysis reveals that leucine patches at the dimer interface may account for the formation of dimeric curve with certain curvature. Consequently, APPL2 BAR is able to enforce the curvature reduction to APPL1 BAR upon heterodimerization. In conclusion, the alterations of curvature and electrostasis are responsible for the modulation of endosome binding specificity and can elucidate the opposite roles of APPL1 and APPL2. LanCL2 LanCL2 is a member of Lanthionine synthetase component C-like family. In human, LanCL2 binds to lanthionine derivatives and glutathione, participating in keeping intracellular reducing state. By binding to absiscic acid (ABA), LanCL2 is indispensible for the ABA-stimulated adipogenesis, insulin release, glucose homeostasis, and inflammatory response. It is also implicated in anticancer drug resistance. All these functions underscore the importance of LanCL2 in the diseases like diabetes, inflammation, and cancer. The crystal structure of LanCL2 was solved to resolution 1.8 Å. The overall structure displays canonical double-layer α-barrel. The major differences from LanCL1 lay in the loops on the barrel top, which are implicated in various substrate bindings. A zinc-coordinating pocket was found among the loops, with conserved amino acid residues of distinct conformation. The electrostatic surface shows remarkable differences compared to that of LanCL1, suggesting that it may contribute to distinct substrate binding profile. Future implications APPL proteins and LanCL proteins are all involved in the regulation of metabolism, such as glucose uptake, fatty acid oxidation, and insulin secretion, and play roles in diseases like obesity and type 2 diabete. Structural and functional studies of these proteins can provide insights into the molecular mechanisms and clues for related therapeutic approaches.
published_or_final_version
Physiology
Doctoral
Doctor of Philosophy

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Gachard-Bouty, Laëtitia. "Utilisation de la métathèse des alcènes pour la synthèse de porphyrines O- et C-glycosylées éthyleniques : Application à la photothérapie dynamique." Limoges, 2003. http://aurore.unilim.fr/theses/nxfile/default/c10866ac-9681-46ce-b476-0a4567702eb4/blobholder:0/2003LIMO0050.pdf.

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En raison de leur caractère photosensible, les porphyrines ont retenu l'attention des chercheurs pour une application en photothérapie dynamique, notamment, pour le traitement de certains cancers. Les porphyrines glycosylées sont, dans ce domaine, particulièrement prometteuses car elles peuvent permettre de vectoriser le photosensibilisateur vers les cellules tumorales enrichies en récepteurs glucidiques. Au cours de ce travail, nous avons, dans un premier temps, réalisé la synthèse de porphyrines glycosylées éthyléniques appartenant à la famille des méso-tétraarylporphyrines. Une stratégie en deux étapes qui consiste à dimériser le précurseur glucidique allylique puis à faire réagir l'homodimère obtenu avec les mono para- et ortho-allyloxyphényltritolylporphyrines a été appliquée. La dihydroxylation asymétrique de ces composés a été ensuite effectuée afin d'en améliorer le caractère amphiphile. Les unités glucidiques fixées sont de deux types. La première série de porphyrines synthétisées appartient à la famille des porphyrines O-glycosylées. La seconde série est constituée de porphyrines C-glycosylées qui se révèlent particulièrement intéressantes en raison de leur aptitude à résister à l'hydrolyse enzymatique. Ces composés ont été caractérisés par spectroscopie UV-visible et spectrométrie de masse. Une étude détaillée de RMN (1H et 13C) a été effectuée sur les composés acétylés ou benzoylés au niveau de la partie glucidique. Enfin, les différents produits issus de cette étude ont été déprotégés et ont fait l'objet de tests biologiques in vitro sur des cellules K562 sous forme libre ou encapsulés par incorporation dans des liposomes
In recent years, photodynamic therapy has received increasing attention as a new modality for selective treatment of solid tumors. Glycosylated porphyrins are especially promising compounds since it is possible that the sugar moiety might lead the conjugate to a cell surface target through specific binding to its receptor. In the present work, we describe, in a first time, the synthesis, via olefin metathesis, of new ethylenic glycoconjugated porphyrins which are derivatives of 5,10,15,20-tetraarylporphyrins. We applied a two-step procedure in which allylic saccharides were first homodimerized prior to the cross-metathesis reaction with mono para- and ortho-allyloxyphenyltritolylporphyrins. The asymetric dihydroxylations of these compounds were then performed in order to increase the amphiphilic properties of the resulting porphyrins. The first synthetised compounds are O-glycosylated porphyrins and the second series, C-glycosylated porphyrins. These last ones are of special interest because of their capacity to resist to enzymatic hydrolysis. The products were characterised by UV-visible and NMR spectroscopy as well as by mass spectrometry. Finally, all the compounds were deacetylated or debenzoylated and in vitro biological tests were carried out so as to evaluate the activity in photodynamic therapy of the free or encapsulated into liposomes products

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Immekus, Florian Peter Philip [Verfasser], and Gerhard [Akademischer Betreuer] Klebe. "lin-Benzopurines as Inhibitors of tRNA-Guanine Transglycosylase: Perturbance of Homodimer Formation, Import of Water Clusters and Determinants of Crystallographical Disorder / Florian Peter Philip Immekus. Betreuer: Gerhard Klebe." Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/1032314567/34.

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Abdallah, Bassim Violla. "Structural and functional analysis on GacS homodimeric histidine kinase reveals a non-canonical autokinase activity and new insights into the heterodimer partnership with RetS." Thesis, Aix-Marseille, 2020. http://www.theses.fr/2020AIXM0256.

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Pseudomonas aeruginosa (PA) est un pathogène qui infecte particulièrement les patients atteints de la mucoviscidose. PA provoque des infections aiguës et chroniques et alterne entre des modes de vie planctonique et sédentaire. Cette transition est principalement régulée par les systèmes à deux composants (TCS). Durant ma thèse, je me suis focalisée sur le TCS GacS/GacA, impliqué dans un réseau de signalisation multikinase. GacS est une histidine kinase (HK) et GacA est son régulateur de réponse (RR). La région cytoplasmique de GacS est composée des domaines HAMP, S-Hélice, H1, D1 et H2. Des tests fonctionnels ont montré que la région HAMP/S-Hélice est nécessaire pour la transduction du signal et contribue à son activité. L’analyse structurale a montré une boucle de fixation de l’ATP structurée contrairement à ce qui est observé dans d'autres HKs. De plus, un nouveau domaine (ND) a été identifié et s’est révélé essentiel pour assurer une activité autokinase. Nous avons proposé que le ND contribue à des changements conformationnels pour placer GacS dans une configuration active. Par ailleurs, RetS HK inhibe GacS par des interactions directes. Des tests de spectrométrie de masse native et des tests d’interaction ont montré que le domaine HAMP est nécessaire pour assurer l'interaction entre ces HKs. De plus, des anticorps à domaine unique contre GacS nous ont permis d’identifier des sites potentiels d'inhibition de la voie GacS/GacA et de la formation de biofilm. Dans cette étude, nous avons dévoilé une activité autokinase non canonique de GacS et de nouvelles perspectives sur son interaction avec RetS qui contribuent au processus décisionnel pendant l'infection par PA
Pseudomonas aeruginosa (PA) is a pathogen that particularly infects patients with cystic fibrosis. PA causes acute and chronic infections and can switch from a planktonic to a sedentary lifestyle. This transition is mainly coordinated by Two-Component Systems (TCS). During my thesis, I focused on GacS/GacA TCS, a master regulator of the acute-to-chronic transition. In fact, GacS is a membrane-bound histidine kinase (HK) and GacA is the response regulator (RR). GacS cytoplasmic region is composed by the HAMP, S-Helix coiled-coil region. This helical part is followed by the H1, the D1 and H2 domains. GacS/GacA TCS is involved in a multikinase regulatory network and its activity is modulated by three HKs. Mutagenesis and functional assays showed that the HAMP and S-Helix orchestrate the signal transduction prior to its autokinase activity. In addition, GacS presented a folded ATP lid in contrast to what is observed in other HKs. Furthermore, we unveiled a new domain (ND) in GacS sequence. Functional assays showed that the ND domain is essential to insure a full autokinase activity of GacS. We proposed that this domain contributes in conformational rearrangements in order to shift GacS to its active state. RetS HK is known to inhibit GacS/GacA via direct interactions. Native mass spectrometry and microscale thermophoresis assays showed that the HAMP domain is essential for this interaction. Furthermore, nanobodies generated against the cytoplasmic region of GacS revealed potential sites of inhibition of GacS activity. In this study, we unveiled a non-canonical autokinase activity in GacS and new insights into its interaction with RetS that underlie the decision-making of PA

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29

Rumpel, Sigrun. "Protein NMR studies of two systems involved in bacterial pathogenicity." Doctoral thesis, [S.l.] : [s.n.], 2006. http://webdoc.sub.gwdg.de/diss/2006/rumpel.

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Prokopyeva, Elena. "Měření viability buněk." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2012. http://www.nusl.cz/ntk/nusl-219530.

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This master's thesis deals with the ways of cell viability determination and its measuring. The first part of this thesis has a brief introduction into the theory of cell viability, also the methods of cell viability measurement and different types of fluorescent probes are described. The thesis further includes a brief description of the image processing methods, as well as the fluorescent microscope and applied filters are described. In the practical part the system implemented at a graphical programming environment LabVIEW used for the cell viability evaluation is described, functions of applied units are explained. The program is checked on model data.

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Andric, Vedrana. "Study of the mechanisms of sexual differentiation in the fission yeast Schizosaccharomyces pombe Formation of S. pombe Erh1 homodimer mediates gametogenic gene silencing and meiosis progression A scaffold lncRNA shapes the mitosis to meiosis switch." Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASL056.

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Chez la levure fissipare Schizosaccharomyces pombe, un sous-ensemble de gènes méiotiques est transcrit de manière constitutive au cours de la mitose. Afin d’éviter l'expression prématurée du programme méiotique et l'initiation de la différenciation sexuelle, les cellules ont développé un système de dégradation de l'ARN qui élimine sélectivement les transcrits méiotiques correspondants. Ce processus nécessite la protéine de liaison à l'ARN Mmi1 (à domaine YTH), qui reconnaît en cis les molécules d'ARN (motifs UNAAAC) et les cible pour la dégradation par l'exosome nucléaire. Au début de la méiose, Mmi1 est séquestrée au sein d’une particule ribonucléoprotéique composée de la protéine de liaison à l'ARN Mei2 et du long ARN non-codant (lncRNA) meiRNA, permettant ainsi l'expression des gènes méiotiques et le déroulement de la méiose. Mon travail de thèse a consisté à étudier les mécanismes par lesquels Mmi1 assure la dégradation des transcrits méiotiques et qui régulent son activité au cours des cycles mitotiques et méiotiques. Pendant la croissance végétative, Mmi1 s'associe étroitement à la protéine conservée Erh1 pour former le complexe hétérotétramérique Erh1-Mmi1 (EMC) qui est essentiel pour la dégradation des transcrits méiotiques. Par des approches de biologie structurale et de biochimie, nous avons montré qu'Erh1 s'assemble en homodimère in vitro et in vivo, en accord avec des analyses récentes. Des mutations qui empêchent l'homodimérisation d'Erh1 mais préservent son interaction avec Mmi1 entraînent l'accumulation de transcrits méiotiques en raison d'un défaut de liaison de Mmi1 à ses cibles ARN. L'homodimérisation d’Erh1 est également nécessaire pour séquestrer Mmi1 dans le complexe Mei2-meiRNA et assurer la progression de la méiose. Ainsi, l'assemblage d’EMC est essentiel pour la reconnaissance et la dégradation des transcrits méiotiques par Mmi1 dans les cellules mitotiques et contribue à l'inactivation de cette dernière au début de la méiose. Des travaux antérieurs ont montré que, pendant la croissance végétative, Mmi1 recrute le complexe Ccr4-Not pour ubiquitinyler et limiter l’accumulation de son propre inhibiteur Mei2, maintenant ainsi son activité dans la dégradation des ARNs méiotiques. Nous avons identifié un lncRNA, différent de meiRNA et appelé mamRNA (Mmi1- and Mei2-associated RNA), qui sert de plateforme à Mmi1 pour cibler Mei2 vers le complexe Ccr4-Not. Réciproquement, lorsque cette régulation négative de Mei2 est défectueuse, mamRNA est nécessaire pour l'inactivation de Mmi1 par les niveaux élevés de Mei2. Des expériences d’hybridation in situ par fluorescence en molécules uniques (smFISH) ont également montré que mamRNA est localisé dans un corps nucléaire contenant Mmi1, suggérant que le contrôle mutuel de Mmi1 et Mei2 est confiné dans l’espace. mamRNA peut également relayer meiRNA pour inhiber Mmi1 et favoriser la progression de la méiose. mamRNA apparait donc comme un régulateur critique des activités de Mmi1 et Mei2 pour ajuster la dégradation des ARNs méiotiques et modeler la transition de la mitose vers la méiose
In the fission yeast S. pombe, a subset of meiosis-specific genes is constitutively transcribed during the mitotic cell cycle. To prevent untimely expression of the meiotic program and premature initiation of sexual differentiation, cells have evolved an RNA degradation system that selectively eliminates the corresponding meiotic transcripts. This process requires the YTH-family RNA-binding protein Mmi1, which recognizes cis-elements within RNA molecules (UNAAAC motifs) and targets them for degradation by the nuclear exosome. At the onset of meiosis, Mmi1 is sequestered in a ribonucleoparticle composed of the RNA-binding protein Mei2 and the long non-coding RNA (lncRNA) meiRNA, thereby allowing expression of meiotic genes and meiosis progression. My PhD work consisted in studying the mechanisms by which Mmi1 promotes the degradation of meiotic transcripts and how its activity is regulated during both the mitotic and meiotic cell cycles. During vegetative growth, Mmi1 tightly associates with the evolutionarily conserved Erh1 protein to form the heterotetrameric Erh1-Mmi1 complex (EMC) that is essential for the degradation of meiotic transcripts. Using biochemical and structural approaches, we have shown that Erh1 assembles as a homodimer in vitro and in vivo, consistent with recent analyses. Mutations that disrupt Erh1 homodimerization but preserve interaction with Mmi1 result in the accumulation of meiotic transcripts due to inefficient binding of Mmi1 to its RNA targets. Erh1 homodimerization is also required for Mmi1 luring by the Mei2-meiRNA complex and meiosis progression. Thus, EMC assembly is essential for the recognition and degradation of meiotic transcripts by Mmi1 in mitotic cells and contributes to Mmi1 inactivation at meiosis onset. Previous work showed that, during vegetative growth, Mmi1 recruits the conserved Ccr4-Not complex to ubiquitinylate and downregulate a pool of its own inhibitor Mei2, thereby maintaining its activity in meiotic RNA degradation. We have identified a lncRNA, different from meiRNA and termed mamRNA (Mmi1- and Mei2-associated RNA), to which Mmi1 associates to target Mei2 to the Ccr4-Not complex. Conversely, when Mei2 downregulation is impaired, mamRNA is necessary for Mmi1 inactivation by increased Mei2 levels. Single molecule RNA FISH experiments also indicated that mamRNA localizes to a nuclear body enriched in Mmi1, suggesting that the mutual control of Mmi1 and Mei2 is spatially confined. mamRNA can also take over meiRNA to inhibit Mmi1 and promote meiosis progression. Therefore, mamRNA emerges as a critical regulator of Mmi1 and Mei2 activities to fine tune meiotic RNA degradation and shape the mitosis to meiosis transition

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32

Chotikasemsri, Pongsathorn. "Computational Prediction of the Agregated Structure of Denatured Lysozyme." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/120.

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Mis-folded proteins and their associated aggregates are a contributing factor in some human diseases. In this study we used the protein lysozyme as a model to define aggregation structures under denaturing conditions. Sasahara et al. (2007), Frare et al. (2009, 2006), and Rubin et al. (2008) observed conditions where heat denatured lysozyme formed fibril structures that were observed to be 8-17 nanometers in diameter under the electron microscope. Even though the crystal structure of lysozyme is known, the denatured form of this protein is still unknown. Therefore, we used Rosetta++ protein folding and blind docking software to create in silico models of the protein at denaturing temperatures and subsequently docked them into aggregates. Here we compare those structures and select forms consistent with the fibril structure from the previous papers. The next step is to be able to use the predicted models of the fibrilar forms of denatured lysozyme to help us understand the exact conformation of fibril structures. This will let us confirm the docking interactions during the fibril aggregation process. The ultimate goal is to use the validated denatured structures to model interactions with heat shock proteins during the dis-aggregation process.

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De, Poorter Cédric. "Mécanismes d'activation et interactions fonctionnelles hétérologues des récepteurs aux chimiokines." Doctoral thesis, Universite Libre de Bruxelles, 2012. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209589.

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Mécanismes d’activation et conséquences fonctionnelles de la dimérisation des récepteurs aux chimiokines

Les chimiokines sont de petites protéines qui régulent la migration des cellules immunitaires. Elles exercent leur action en se liant à des récepteurs appartenant à la famille des récepteurs couplés aux protéines G (RCPG) dont la fonction est intimement liée à la régulation des cellules immunitaires. Notre laboratoire étudie depuis plusieurs années les relations reliant la structure et la fonction des récepteurs aux chimiokines. Ces dernières années, un nouveau concept est venu révolutionner le mode de fonctionnement des RCPGs. En effet, des travaux ont montré que la plupart des RCPGs sont capables de former des dimères. Le but de cette thèse de doctorat est d’étudier de manière systématique la dimérisation des récepteurs aux chimiokines et d’analyser les conséquences fonctionnelles de la dimérisation.

Dimérisation des récepteurs humains aux chimiokines et conséquences fonctionnelles

En utilisant une technique biophysique basée sur un transfert d’énergie de luminescence (BRET) nous avons montré au cours de ce travail de thèse que les récepteurs CCR1, CCR2, CCR5, CCR7 et CXCR4 sont capables de former des homodimères et des hétérodimères. De plus, une dimérisation entre ChemR23, dont le ligand naturel, la chémérine, est structurellement différent des chimiokines, et les récepteurs CCR7 et CXCR4, a également été identifiée.

D’un point de vue fonctionnel, des expériences réalisées au laboratoire dans le cadre d’un autre travail de thèse ont identifié une forme de compétition croisée entre CCR2, CCR5 et CXCR4 où la liaison de ligands (agonistes ou antagonistes) spécifiques de l'un des deux récepteurs inhibe la liaison des ligands spécifiques de l’autre. Ces effets ont été démontrés sur des cellules recombinantes mais aussi sur des cellules immunes et dans un modèle in vivo. (El-Asmar, 2005; Springael, 2006; Sohy, 2007; Sohy, 2009). Au cours de ce travail, nous nous sommes dans un premier temps focalisés sur les

hétéromères de ChemR23 avec CXCR4 et CCR7 et nous avons ensuite étudié plus en profondeur les hétéromères de CCR7. Concernant la dimérisation de ChemR23 avec les récepteurs aux chimiokines CCR7 et CXCR4, nous avons pu mettre en évidence une coopérativité négative de liaison entre les agonistes des récepteurs comme cela avait pu être démontré pour CCR2/CCR5/CXCR4. Par contre, nous n’avons observé aucun effet de compétition hétérologue ou d’inhibition fonctionnelle croisée de l’AMD3100 sur ChemR23 quand il est coexprimé avec CXCR4. De manière additionnelle, nous avons pu observer cette compétition croisée sur des cellules dendritiques murines immatures, démontrant l’existence des effets de l’hétérodimérisation lorsque les récepteurs sont exprimés à un niveau physiologique. Lors de l’étude approfondie des hétéromères de CCR7, nous avons montré que les conséquences fonctionnelles de l’hétérodimérisation de CCR7 sont différentes suivant le récepteur avec lequel il interagit. Pour l’hétérodimère CCR7/CCR2, nous avons identifié une forme de compétition croisée, où la liaison de ligands spécifiques de l'un des deux récepteurs inhibe la liaison des ligands spécifiques de l’autre, rejoignant les effets mis en évidence pour les hétéromères CCR2/CCR5/CXCR4. Ensuite, nous avons montré pour l’hétérodimère CCR7/CCR5 que les ligands de CCR7 sont capables d’inhiber la liaison des ligands spécifiques sur CCR5 mais que l’inverse n’est pas vrai. Enfin, pour l’hétérodimère CCR7/CXCR4, nous n’avons pas pu mettre en évidence d’inhibition croisée, que ce soit dans un sens ou dans l’autre. D’autre part, un effet inhibiteur de CCR7 a également été identifié pour les hétéromères CCR7/CCR5 et CCR7/CXCR4. Nous avons pu montrer que l’expression de CCR7 exerce un effet négatif sur la réponse fonctionnelle de certains récepteurs hétérologues comme CCR5 et CXCR4 mais pas CCR2 ou ChemR23.

L’ensemble de ces données permet de mieux comprendre les interactions entre récepteurs et pourrait mener à l’identification de nouvelles cibles pour les programmes de recherche de molécules thérapeutiques, qui, jusqu’à présent, ciblaient presque exclusivement un seul et unique récepteur.

Etude du mécanisme d’activation du récepteur CCR5 et étude de la relation entre activité constitutive et dimérisation.

De nombreux travaux ont été menés ces dernières années afin de mieux comprendre les mécanismes moléculaires à la base de l’activation des récepteurs couplés aux protéines G (RCPG). Il apparaît que les RCPGs peuvent se trouver dans plusieurs états conformationnels, dont certains sont favorisés par la présence d’agonistes ou d’antagonistes, ou encore d’anticorps reconnaissant des épitopes conformationnels. Certaines mutations peuvent également induire la stabilisation de certaines conformations, actives ou inactives. Pour les RCPGs appartenant à la famille de la rhodopsine, il en a résulté un modèle selon lequel les récepteurs sont maintenus dans une conformation inactive par un ensemble d’interactions ioniques impliquant l’arginine (R3.50) d’un motif DRY conservé, présent à l’extrémité cytosolique du troisième segment transmembranaire. Les interactions responsables de ce qu’on appelle le « DRY-lock » feraient intervenir notamment l’aspartate (D3.49) adjacent de l’arginine et un aspartate ou glutamate (D/E6.30) localisé au sein de l’hélice 6. Selon ce modèle, la liaison d’un agoniste, ainsi que certaines mutations, favoriseraient la rupture de ces interactions ioniques, et une conformation permettant aux récepteurs de se coupler plus efficacement aux protéines G. Des résultats du laboratoire indiquent cependant que ce modèle ne serait pas transposable complètement au récepteur CCR5.

CCR5 possède intrinsèquement une activité constitutive en absence d'agoniste. Cette activité peut être mise en évidence par l'action d'un des antagonistes de CCR5, le TAK-779, qui s'est révélé posséder une activité de type agoniste inverse. D'autre part, CCR5 possède au sein de l'hélice 6 une arginine en position 6.30 et non pas un glutamate ou un aspartate. Une arginine à cette position ne peut donc contribuer au maintien d’une conformation inactive par interaction avec R3.50 .Dans le but de tester le modèle de « DRY-lock » sur CCR5 et de mieux comprendre les interactions moléculaires impliquées dans l’activation du récepteur, plusieurs récepteurs mutants ont été construits au laboratoire. Tout d’abord, l’arginine 3.50 du motif DRY a été mutée en Asn (R3.50N) afin de rompre les interactions ioniques de ce résidu. L’aspartate 3.49 a été muté en Asn (D3.49N) ou en Val (D3.49V), afin de neutraliser une des interactions du « DRY-lock » (Lagane, 2005). L’arginine 6.30 a été mutée d’une part en Asp (R6.30D) ou en Glu (R6.30E), afin de rétablir une possibilité d’interaction avec R3.50, d’autre part en Ala (R6.30A) et en Gln (R6.30Q) afin de mieux cerner le rôle de la charge de l’arginine. Afin de tester l’hypothèse d’interaction entre le résidu 6.30 et le résidu 2.40, l’aspartate 2 .40 a été mutée en Ala (D2.40A) ou en Arg (D2.40R) et des récepteurs présentant les deux mutations ont également construits (D2.40A/R6.30A et D2.40R/R6.30D). L’ensemble des résultats obtenus par l’analyse de ces mutants a permis de montrer que la nature des interactions entre l’extrémité cytosolique des hélices 3 et 6 influence l’activité du récepteur CCR5 (Springael, 2007). Une interaction forte conduit à une forme de récepteur inactif alors qu’une interaction faible s’accompagne d’une augmentation d’activité constitutive. Cette propriété de CCR5 serait donc partagée avec d’autres récepteurs appartenant à la famille de la rhodopsine. Cependant les interactions inter-hélice stabilisant ces conformations seraient différentes pour CCR5. D’autre part, l’étude de la position 2.40 laisse supposer l'importance du résidu aspartate 2.40 dans le maintien d'une conformation permettant l'activité constitutive du récepteur. Nous avons également testé s’il existait une corrélation entre activité constitutive et capacité du récepteur CCR5 à former des dimères, mais les résultats ne nous ont pas permis de mettre en évidence une quelconque relation entre activité et dimérisation.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

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Jakimenko, Ana. "Localization and dimerization of the ABC half transporter rAbcb6 as compared to rAbcb7." Doctoral thesis, [S.l.] : [s.n.], 2006. http://webdoc.sub.gwdg.de/diss/2006/jakimenko.

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Chang, Ya-Jen, and 張雅珍. "Synthesis and Comparison Physiological Activities of Homodimers and Heterodimer Comprising Different Splice Forms of Vascular Endothelical Growth Factor." Thesis, 2004. http://ndltd.ncl.edu.tw/handle/26851502614141487909.

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Yu-Chen, Lu, and 盧郁蓁. "Preparation of HLA-B27 heavy chain homodimer." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/3kqs3h.

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碩士
國立中正大學
生命科學系分子生物研究所
107
Ankylosing spondylitis (AS) is strongly associated with the expression of human leukocytic antigen-B27 (HLA-B27). HLA-B27 heavy chain (HC) has a propensity to fold slowly, resulting in the accumulation of misfolded HLA-B27 HC in the ER, triggering the unfolded protein response, and forming a homodimer, (B27-HC)2. (B27-HC)2 can be displayed on the cell surface. (B27-HC)2 is the ligand of KIR3DL2 that is displayed on the cell surface of nature killer (NK) cells and Th17 cells, especially in AS patients. (B27-HC)2 binds to and activates KIR3DL2, resulting in expansion and survival of NK cells and activation of Th17 cells to up-regulation of IL-17 secretion, respectively. The consequences from both activated pathways will trigger the pro-inflammatory reactions, making the major contributions to one of the AS pathogeneses. Therefore, blockage of the interaction between (B27-HC)2 and KIR3DL2 is a therapeutic target to suppress the development of AS. The goal of this proposal was to prepare the recombinant (B27-HC)2 for further study to use phage display to search the molecules that specifically block the interaction between (B27-HC)2 and KIR3DL2. In addition, I also confirmed that HLA-B27 transgenic mice (B6(Cg)-Tg(B2M, HLA B2705)563 Trg/DcrJ, C57BL/6J-Tg(HLA-B27)30-4Trg/DcrJ) display (B27-HC)2 in their splenocytes.

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El, Salti Saly. "L'homodimérisation du CD40 et son implication indirecte dans l'asthme allergique." Thèse, 2018. http://hdl.handle.net/1866/20520.

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Chen, Tai-Chi, and 陳泰吉. "Investigation of Cooperative Interaction between the Homodimer Catalytic Subunits of Xanthine Oxidase." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/20411189589807391343.

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Jundi, Malek. "Le rôle du CD40 homodimère dans la réponse immunitaire." Thèse, 2013. http://hdl.handle.net/1866/10869.

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Le CD40 est une glycoprotéine transmembranaire de type I, appartenant à la famille des TNFRs, exprimée à la surface des cellules immunitaires, hématopoïétiques, vasculaires, épithéliales, et d’autres types de cellules, y compris les cellules tumorales. Le CD40 ne possédant pas de domaine kinase, pour induire un signal il interagit directement ou indirectement avec des protéines adaptatrices telles que les TRAFs et les JAKs. L’interaction du CD40 avec son principal ligand, le CD154, joue un rôle primordial dans la régulation de la réponse immunitaire et le maintien de l’homéostasie. L’activation du CD40 à la surface des cellules B augmente leur capacité de présentation d’antigène, en plus d’induire la prolifération, la commutation isotypique et l’apoptose. Les patients souffrant de mutations au niveau du gène codant pour le CD40 ou de son ligand sont immunosupprimés et sensibles à des infections opportunistes. Des études ont montré que le CD40 comme d’autres membres de la famille des TNFRs est capable de former des homodimères. Plus récemment, on a montré que la formation du CD40 homodimère est le résultat de son engagement sur les cellules B. En plus, cette homodimérisation du CD40 est importante pour la phosphorylation de l’Akt. L’interaction CD40/CD154 peut avoir un rôle direct dans l’immunothérapie par l’induction de l’apoptose de certaines cellules cancéreuses ou un rôle indirect en activant les cellules présentatrices d’antigènes (CPA) afin d'augmenter l’efficacité de l’activation des cellules T cytotoxiques. Nos résultats montrent que l’induction de la mort cellulaire par le CD40 requiert la perméabilisation du lysosome, la libération de la cathepsine B, la présence de ROS et une interaction avec le TRAF6, cette mort cellulaire programmée est plus importante en présence de la forme monomérique du CD40, muté au niveau de la cystéine 238. Par ailleurs, l’homodimérisation du CD40 requerrait sa translocation vers les radeaux lipidiques et nécessiterait la présence des ROS. Cette homodimérisation du CD40 semble être importante pour l’activation des cellules B par le biais de l’induction de l’expression du CD23, CD69 et CD80. De plus, nos résultats montrent pour la première fois une implication du CD40 homodimère dans l’induction du CD23 par le biais du TLR4. Nos résultats soulignent l’importance du CD40 homodimère dans certaines voies de signalisation. Ainsi, ils mettent en évidence le rôle de la Cys-238 dans la coopération entre des récepteurs de la réponse immunitaire innée et adaptative. Toutes ces données permettraient une meilleure compréhension de certaines voies de signalisation impliquées dans plusieurs maladies auto-immunes et faisant objet de plusieurs essais thérapeutiques.
CD40 is a type I transmembrane glycoprotein belonging to the TNFRs family, which is expressed on the surface of immune, hematopoietic cells, vascular, epithelial, and other cell types, including a wide range of tumour cells. CD40 does not have a kinase domain. Thus, to induce a signal, CD40 interacts directly or indirectly with adapter proteins such as TRAFs and Jaks. The interaction of CD40 with its main ligand, CD154, plays an important role in regulating the immune response and homeostasis. The activation of CD40 on the surface of B cells increases its ability to promote antigen presentation, in addition to inducing proliferation, isotype switching, and apoptosis. Patients affected by mutations in the gene encoding the CD40 or its ligand are immunosuppressed and susceptible to opportunistic infections. Studies have shown that CD40, as other members of the family of TNFRs is capable of forming homodimers. More recently, it was shown that the formation of the CD40 homodimer is the result of the engagement of CD40 on B cells by CD154. In addition, the homodimerization of CD40 is important for the phosphorylation of Akt. The CD40/CD154 interaction can have a direct role in immunotherapy by inducing apoptosis of some cancer cells or an indirect role in activating antigen-presenting cells (APCs), thereby increasing the effectiveness of activation of cytotoxic T cells. Our results show that the induction of cell death by CD40 requires permeabilization of the lysosome, the release of cathepsin B, the presence of ROS and interaction with TRAF6, this programmed cell death is greater in the presence of the monomeric form of CD40, due to a mutation at the level of the cysteine 238. Moreover, the homodimerization of CD40 requires its translocation to lipid rafts and the presence of ROS. This homodimerization is necessary for the CD40 B-cell activation via the induction of expression of CD23, CD69 and CD80. In addition, our results show for the first time the involvement of the CD40 homodimer in the induction of CD23 expression via TLR4. Our results emphasize the importance of CD40 homodimer in signaling pathways and highlight the role of Cys-238 in the cooperation between receptors of the innate and adaptive immune response. All together our results will allow a better understanding of CD40 signaling pathways involved in several autoimmune diseases, which give a rise to a better therapeutic trial design.

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Chien-Cheng and 王建程. "The Yeast Peroxisomal ABC Transporter Pxa2p, a Human ALDP Homolog, Forms a Homodimer." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/50019880119076341200.

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碩士
中山醫學大學
生化暨生物科技研究所
97
Peroxisomal beta-oxidation play an important role in mammalian and yeast. The ABC transporter (ATP-binding cassette transporter) of peroxisomal membrane has been identified that is closely related to human ALD (Adrenoleukodystrophy). So far four ABC transporter have been detected in mammalian peroxisomes that contains four ABC transporters named ALDP (Adrenoleukodystrophy protein), ALDRP (ALD-related protein), PMP70 (The 70-kDa peroxisomal membrane protein) and PMP69 (The 69-kDa peroxisomal membrane protein). Saccharomyces cerevisiae contains two peroxisomal half-ABC transporters named Pxa1p (peroxisomal ABC transporter1 protein) and Pxa2p (peroxisomal ABC transporter2 protein). The ALDP and PMP70 are homolog Pxa1p and Pxa2p. ALDP are located in the peroxisome, where function as homo- and/or heterodimers in the regulation of very long chain fatty acid transport. The yeast Pxa1p and Pxa2p dimerize to form a functional transporter involved in very long chain fatty acid oxidation in the peroxisome. The formation of PMP70 assembles as dimeric or oligomeric forms on peroxisomal membranes implies that Pxa2p may form a homodimers. We used IPTG to induce His-Pxa2pC1-HA protein. Then, we purified His-Pxa2pC1-HA proteins sequentially by 8 M urea denaturation, dialysis and nicole’s column purification. We analysed the molecular size of this purified His-Pxa2pC1-HA protein by FPLC. The His-Pxa2pC1-HA proteins are expressed at low levels and insoluble. Then, we prepare His-Pxa2pC2-HA by the same way, but dialyse with Triton-X 100 to increase its solubility and skip nicole’s column purification step. We analysed the molecular size of this His-Pxa2pC2-HA protein by FPLC. However, the molecular size determination was interferenced by Triton-X 100. In yeast, by using coimmunoprecipitation assays of differentially tagged full-length Pxa2p, we demonstrated that Pxa2p can form a homodimer or homo-oligomer.

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Jia-Chi and 許家齊. "The Yeast Peroxisomal ABC Transporter Pxa1p, a Human ALDP Homolog, Forms a Homodimer." Thesis, 2009. http://ndltd.ncl.edu.tw/handle/55310088494297685093.

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碩士
中山醫學大學
生化暨生物科技研究所
97
ATP-binding cassette (ABC) transporters are members of a superfamily of membrane proteins involved in the transport of a variety of molecules across biological membranes. So far, four ABC transporters have been detected in mammalian peroxisomes, one of which, named ALDP, is specifically involved in the

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Hsieh, Wan-Ting, and 謝宛廷. "Excited-State Double Proton Transfer: the Concerted (Homodimer) versus Stepwise (Heterodimer) Reaction on the 7-Azaindole Analogues." Thesis, 2007. http://ndltd.ncl.edu.tw/handle/75828365330468029586.

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碩士
國立臺灣大學
化學研究所
95
A four fused-ring system 11-propyl-6H-indolo[2,3-b]quinoline (6HIQ) is strategically designed and synthesized; it possesses a central moiety of 7-azaindole (7AI) and undergoes excited-state double proton transfer (ESDPT). Despite the concerted ESDPT in the 6HIQ dimer, femtosecond dynamics unveils a stepwise ESDPT process in the 6HIQ/7AI heterodimer complex, in which 6HIQ delivers the pyrrolyl proton to 7AI in less than 150 fs, followed by the transfer of pyrrolyl proton from 7AI to the pyridinyl nitrogen of 6HIQ in ~1.5 ± 0.3 ps.

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Hsieh, Wan-Ting. "Excited-State Double Proton Transfer: the Concerted (Homodimer) versus Stepwise (Heterodimer) Reaction on the 7-Azaindole Analogues." 2007. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2506200711241400.

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Musso, Juliana. "Efecto del Péptido Beta Amiloide Fibrilar sobre la Homodimerización de la proteína precursora de Beta AMILOIDE." Bachelor's thesis, 2017. http://hdl.handle.net/11086/5527.

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Tesina (Grado en Ciencias Biológicas)--Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Lugar de Trabajo: Instituto de Investigación Médica Mercedes y Martín Ferreyra. INIMEC-CONICET-U.N.C. 2017. 31 h.; ils. col.; tabls.; grafs. Contiene Referencias Bibliográficas. Abstract en español e inglés.
La Enfermedad de Alzheimer (EA) es una de las patologías de mayor interés e importancia médica en la actualidad. Numerosos estudios científicos se han desarrollado con el fin de encontrar técnicas de diagnóstico temprano y terapias eficientes que modulen el desarrollo de la enfermedad, sin embargo, tal meta aún no se ha alcanzado. Varios estudios sugieren que APP, además de ser la única fuente de Aβ, actuaría como un receptor de sus formas agregadas, de manera tal que muchos de sus efectos neurotóxicos están ligados a la interacción Aβ-APP. Así, el Aβ fibrilar se une a APP y activa la proteína Go y al complejo βγ. El interés de este trabajo, es demostrar que la multimerización de APP inducida por Aβ agregado, es un evento clave para la activación de la proteína Go y su cascada de señalización aguas abajo, que concluiría en neurotoxicidad. Para ello, se caracterizó la expresión de los plámidos CAMKII-APP-GFP y CAMKII-APP-Cherry, mediante transfecciones en cultivos hipocampales primarios. Se estudiaron tres variables: edad de las neuronas, concentración de los plásmidos utilizados y tiempo de expresión de los mismos, para establecer las condiciones en la que se logre una mayor eficiencia en la transfección. Con estas condiciones establecidas, posteriormente se analizó el efecto de Aβ fibrilar sobre la homodimerizacón de APP. Se cotransfectaron los plásmidos CAMKII-APP-GFP y CAMKII-APP-Ch en cultivos hipocampales primarios control y tratados con Aβ fibrilar, y se comparó el grado de homodimerización de APP entre ambos tratamientos, a través de la técnica de FRET en su variante fotoblanqueo del aceptor. Los resultados muestran que existe FRET entre APP-GFP y APP-Ch en los cultivos hipocampales primarios tratados con vehículo, lo cual indica interacción física entre ambas proteínas. Sin embargo, no se observaron diferencias significativas en la eficiencia media de FRET (Em), entre el grupo tratado con vehículo (control) y el tratado con Aβ. Esto podría sugerir que, en nuestras condiciones experimentales, el APP exógeno se expresa basalmente dimerizado desde la vía secretoria; o bien, que el evento de dimerización no es modulable mediante el tratamiento con Aβ.
Fil: Musso, Juliana. Universidad Nacional de Córdoba Facultad de Ciencias Exactas, Físicas y Naturales; Argentina.

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Sahtout, Naheda Mohamad Fayez. "Analyzing the Homodimeric and Heteromeric Nature of the Osmosensory Transporter ProP from Escherichia coli." Thesis, 2013. http://hdl.handle.net/10214/7410.

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In this study, the homodimeric and heteromeric nature of ProP, an H+/solute symporter, from E. coli was analyzed. The measured initial rates of proline uptake via ProP-His6 and His6-ProP indicated that as the growth medium osmolality increased, the assay medium osmolality required for half maximal transport activity (Π½/RT) increased and the maximal uptake rate (Amax) decreased. The oligomeric state of ProP, as determined by Blue Native PAGE, showed that both monomeric and dimeric forms of the transporter were present in wild type and cardiolipin deficient bacteria expressing ProP, ProP-His6 or His6-ProP, after culturing in low or high growth medium osmolality. The BACTH System was used to confirm the homodimeric ProP-ProP interaction and to verify the heteromeric interaction between ProP and YdhP. Initial rates of proline uptake via ProP and Western blots indicated that replacement of the ydhP locus with a kanamycin cassette had no effect on ProP function or expression.

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"Structure and Function of the Homodimeric Reaction Center, and Hydrogen Production, in Heliobacterium modesticaldum." Doctoral diss., 2017. http://hdl.handle.net/2286/R.I.46187.

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abstract: The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center. The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the primary electron acceptor, a chlorophyll a derivative, is reduced in ~25 ps, and forward electron transfer occurs directly to a 4Fe-4S cluster in ~650 ps without the requirement for a quinone intermediate. A 2.2-angstrom resolution X-ray crystal structure of the homodimeric heliobacterial reaction center is solved, which is the first ever homodimeric reaction center structure to be solved, and is discussed as it pertains to the structure-function relationship in energy and electron transfer. The structure has a transmembrane helix arrangement similar to that of Photosystem I, but differences in antenna and electron transfer cofactor positions explain variations in biophysical comparisons. The structure is then compared with other reaction centers to infer evolutionary hypotheses suggesting that the ancestor to all modern reaction centers could reduce mobile quinones, and that Photosystem I added lower energy cofactors to its electron transfer chain to avoid the formation of singlet oxygen. In the second part of this dissertation, hydrogen production rates of H. modesticaldum are quantified in multiple conditions. Hydrogen production only occurs in cells grown without ammonia, and is further increased by removal of N2. These results are used to propose a scheme that summarizes the hydrogen-production metabolism of H. modesticaldum, in which electrons from pyruvate oxidation are shuttled through an electron transport pathway including the reaction center, ultimately reducing nitrogenase. In conjunction, electron microscopy images of H. modesticaldum are shown, which confirm that extended membrane systems are not exhibited by heliobacteria.
Dissertation/Thesis
Doctoral Dissertation Biochemistry 2017

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Lin, Yi-Jan [Verfasser]. "Solution structure of the 30 kDa homodimeric sud protein from Wolinella succinogenes / vorgelegt von Yi-Jan Lin." 2003. http://d-nb.info/968620817/34.

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Tzeng, Shiau-Ru, and 曾筱茹. "Effect of adrenoleukodystrophy (ALD) - associated point mutation W679R and L684P of ALDP protein on its homodimeric interaction." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/15466276844434998762.

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碩士
中山醫學大學
生化暨生物科技研究所
103
Adrenoleukodystrophy, ALD, is a common disease resulted from abnormal metabolism of the very long chain fatty acids (VLCFA; ≥ C22) in peroxisome. The cause of the disease is the mutation of ABCD1 gene on X chromosome, leading to defective beta-oxidation of VLCFA and eventually causing a neuro-degenerative disease ALD. ABCD1 gene product is a kind of ABC half-transporter protein, located at peroxisomal membrane, and involved in the transport of VLCFA. Because this protein is related to ALD so closely, it is named Adrenoleukodystrophy Protein (ALDP). There are two peroxisomal ABC half-transporters, Pxa1p and Pxa2p, in yeast. They can form a heterodimer and are orthologous to human ALDP. All these proteins gain energy by ATP hydrolysis and assist the transport of fatty acid through peroxisomal membrane to proceed beta-oxidation. Previous studies published by Chuang et. al. found that the c-terminus at the end of NBD of Pxa2p is required for Pxa1p- Pxa2p interaction. He made a series of deletions in the CT of Pxa2p and showed that the central part of the CT (designated CT2) of Pxa2p was most important for the interaction between Pxa1_NBD and Pxa2_NBD. The unpublished data produced by Lin et. al. showed that the CT2 region of ALDP also played a similar role in protein-interaction. He constructed CT2-deleted mutation plasmids, pACT2-ALDP-ΔCT2 and pEG202-ALDP-ΔCT2, for yeast two-hybrid assay. The results of yeast two-hybrid assay showed that CT2 region of ALDP was critical for the homo-dimeric interaction of ALDP. Also, he found a point-mutation in CT2 region affected this interaction. My studies focus the other two point mutations, W679R and L684P, located in CT2 of ALDP which were identified from ALD disease. By SOE (spliced by overlapping extension) PCR method, we individually introduced the point-mutation, W679R or L684P, into ALDP gene inserted in yeast-two hybrid plasmid. The results of yeast two-hybrid assay demonstrated that both W679R and L684P affected the homo-interaction of ALDP. This may eventually lead to the formation of X-ALD disease.

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CHUANG, CHENG-YI, and 莊秉毅. "The roles of yeast peroxisomal half abc transporter pxa1p and pxa2p carboxyl-terminal region in Pxa1p-Pxa2p heterodimer and Pxa1p-Pxa1p homodimer formation." Thesis, 2015. http://ndltd.ncl.edu.tw/handle/52817405223862435548.

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博士
中山醫學大學
生化暨生物科技研究所
103
The peroxisome is a single membrane-bound organelle in eukaryotic cells involved in lipid metabolism, including β-oxidation of fatty acids. The human genetic disorder X-linked adrenoleukodystrophy (X-ALD) is caused by mutations in the ABCD1 gene (encoding ALDP, a peroxisomal half ATP-binding cassette (ABC) transporter). This disease is characterized by defective peroxisomal β-oxidation and a large accumulation of very long-chain fatty acids in brain white matter, adrenal cortex, and testis. ALDP forms a homodimer proposed to be the functional transporter, whereas the peroxisomal transporter in yeast is a heterodimer comprising two half ABC transporters, Pxa1p and Pxa2p, both orthologs of human ALDP. While the carboxyl-terminal domain of ALDP is engaged in dimerization, it remains unknown whether the same region is involved in the interaction between Pxa1p and Pxa2p. Using a yeast two-hybrid assay, we found that the carboxyl-terminal region (CT) of Pxa2p, but not of Pxa1p, is required for their interaction. Further analysis indicated that the central part of the CT (designated CT2) of Pxa2p was indispensable for its interaction with the carboxyl terminally truncated Pxa1_NBD. An interaction between the CT of Pxa2p and Pxa1_NBD was not detected, but could be identified in the presence of Pxa2_NBD-CT1. A single mutation of two conserved residues (aligned with X-ALD-associated mutations at the same positions in ALDP) in the CT2 of the Pxa2p impaired its interaction with Pxa1p, resulting in a mutant protein that exhibited a proteinase K digestion profile different from that of the wild-type protein. Functional analysis of these mutant proteins on oleate plates indicated that they were defective in transporter function. In addition, using a yeast two-hybrid and co-immunoprecipitation assay, we found that Pxa1p could form homodimer and its carboxyl-terminal region was required for this homodimer interaction. By analyzing the CT fragment deletion and CT-point mutations of Pxa1p, we found that a conserved residues E868 was critical in its self-interaction. By oleate plate assay, we showed that the growth of yeast strains was suppressed due to the overexpression of Pxa1p. We also proved that Pxa1p homodimer formed through its carboxyl-terminal region was required for the interference to endogenous Pxa1p/Pxa2p heterodimer function and cell growth, indicating a negative regulation role of Pxa1p in Pxa1p/Pxa2p transporter function. The CT of Pxa2p and Pxa1p is involved in its interaction with Pxa1p and in transporter function. This concept may be applied to human ALDP studies, helping to establish the pathological mechanism for CT-related X-ALD disease.

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Lin, Cheng Wei, and 林丞偉. "Study of the effect of adrenoleukodystrophy (ALD) - associated point mutations, G677D and T693M, of ALDP protein on its homodimeric interaction." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/06356489916289246025.

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碩士
中山醫學大學
生化暨生物科技研究所
102
In th past studies,it was showed that Pxa1p and Pxa2p proteins were belonged to half ABC transporter on yeast peroxisome and could form a heterodimer,which plays an essential role in long chain fatty acid metabolism.They are homologus to human ALDP,ALDRP,and PMP70. Besides,our submitting results for publication have indicated that the NBD of Pxa2p could interact with the NBD of Pxa1p by the aid of its C-terminal CT2 region.So, we want to study whether the CT2 of ALDP, Homologus to yeast Pxa2p, plays a role in ALDP homodimeric interaction. In the beginning of this study, I used the previously constructed plasmid by our laboratory, containing only the NBD-CT (residue 361 to 745) and not containing the transmembrane region (residue1 to 360) of ALDP protein. Based on the CT sequence homology between ALDP and Pxa2p, I divided the CT region of ALDP into the CT1 sequence (residue 631 to 676 ) and the CT2 sequence (residue 677 to 745 ). First of all, I builded two plasmids with the CT2 deletion in the NBD_CT, pACT2-ALDP-ΔCT2 and pEG202-ALDP-ΔCT2, and used the yeast two hybrid assay to analysize the protein interaction. I found that the CT2 of ALDP played an essential role in ALDP homodimeric interaction. By searching X-ALD database website and I found that two point mutations, G677D and T693M, in the CT2 region of ALDP were associated with ALD disease. Then, I bulided the G677D and T693M mutation in ALDP plasmids by SOE method. From the result on the yeast two hybrid analysis, I found that G677D might affect ALDP homo-interaction and eventually leads to X-ALD. Although the T693M mutation had no obvious effects on protein interaction, it may affect other aspects of protein properties, such as ATP-binding and hydrolysis, the process of substrate transport across the membrane, the interaction between the substrate and ALDP, ect. Because these results mentioned above were generated from genetic method, I performed the immunoprecipitation method to verify the effect of point-mutations on the ALDP homodimeric interaction. This part of the experiment failed and was probablely due to the inability of the anti-HA antibody to precipitate Gal4 AD fusion protein.

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Dissertations / Theses on the topic 'Homodimers'

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