Дисертації з теми "Homodimers"
Оформте джерело за APA, MLA, Chicago, Harvard та іншими стилями
Ознайомтеся з топ-50 дисертацій для дослідження на тему "Homodimers".
Біля кожної праці в переліку літератури доступна кнопка «Додати до бібліографії». Скористайтеся нею – і ми автоматично оформимо бібліографічне посилання на обрану працю в потрібному вам стилі цитування: APA, MLA, «Гарвард», «Чикаго», «Ванкувер» тощо.
Також ви можете завантажити повний текст наукової публікації у форматі «.pdf» та прочитати онлайн анотацію до роботи, якщо відповідні параметри наявні в метаданих.
Переглядайте дисертації для різних дисциплін та оформлюйте правильно вашу бібліографію.
Burnell, David. "Multi-scale modelling of allostery in protein homodimers." Thesis, Durham University, 2015. http://etheses.dur.ac.uk/11055/.
Повний текст джерелаSanders, 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.
Повний текст джерелаHarish, 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.
Повний текст джерелаSilva, 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.
Повний текст джерелаMade available in DSpace on 2017-06-27T14:23:29Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 5840129 bytes, checksum: 587ce0612688b158f82e9c8c528f38e7 (MD5) Previous issue date: 2016-08-15
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 0,50M). 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 0,50M). 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.
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.
Повний текст джерелаPh. D.
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.
Повний текст джерелаNegron, Christopher. "Computational design of orthogonal antiparallel homodimeric coiled coils." Thesis, Massachusetts Institute of Technology, 2014. http://hdl.handle.net/1721.1/93805.
Повний текст джерелаCataloged 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.
Shin, Jong M. "Role of C121A in mGluR2 homodimeric expression and function." VCU Scholars Compass, 2018. https://scholarscompass.vcu.edu/etd/5576.
Повний текст джерелаMcgeagh, 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.
Повний текст джерелаLin, 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.
Повний текст джерелаAli, 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.
Повний текст джерелаLockbaum, 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.
Повний текст джерелаIsbert, 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.
Повний текст джерелаChen, 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.
Повний текст джерелаCataloged 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
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.
Повний текст джерелаKukuk, 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.
Повний текст джерелаBenleulmi-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.
Повний текст джерелаMelatonin 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
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.
Повний текст джерелаStephanou, 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.
Повний текст джерелаKlingler, 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.
Повний текст джерелаFallatah, 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.
Повний текст джерелаEnce, Chloe Christine. "Organic Synthesis using Bimetallic Catalysis." BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/8397.
Повний текст джерелаSouthern, 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.
Повний текст джерелаFäh, 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.
Повний текст джерелаChen, 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.
Повний текст джерелаpublished_or_final_version
Physiology
Doctoral
Doctor of Philosophy
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.
Повний текст джерелаIn 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
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.
Повний текст джерелаAbdallah, 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.
Повний текст джерелаPseudomonas 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
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.
Повний текст джерелаProkopyeva, 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.
Повний текст джерелаAndric, 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.
Повний текст джерелаIn 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
Chotikasemsri, Pongsathorn. "Computational Prediction of the Agregated Structure of Denatured Lysozyme." TopSCHOLAR®, 2009. http://digitalcommons.wku.edu/theses/120.
Повний текст джерелаDe, 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.
Повний текст джерелаLes 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
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.
Повний текст джерелаChang, 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.
Повний текст джерелаYu-Chen, Lu, and 盧郁蓁. "Preparation of HLA-B27 heavy chain homodimer." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/3kqs3h.
Повний текст джерела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.
El, Salti Saly. "L'homodimérisation du CD40 et son implication indirecte dans l'asthme allergique." Thèse, 2018. http://hdl.handle.net/1866/20520.
Повний текст джерелаChen, 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.
Повний текст джерелаJundi, Malek. "Le rôle du CD40 homodimère dans la réponse immunitaire." Thèse, 2013. http://hdl.handle.net/1866/10869.
Повний текст джерелаCD40 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.
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.
Повний текст джерела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.
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.
Повний текст джерела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
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.
Повний текст джерела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.
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.
Повний текст джерелаMusso, 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.
Повний текст джерелаLa 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.
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.
Повний текст джерела"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.
Повний текст джерелаDissertation/Thesis
Doctoral Dissertation Biochemistry 2017
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.
Повний текст джерелаTzeng, 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.
Повний текст джерела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.
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.
Повний текст джерела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.
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.
Повний текст джерела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.