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1
Howard, Megan Wilder. "Coronavirus mediated membrane fusion /." Connect to full text via ProQuest. Limited to UCD Anschutz Medical Campus, 2008. http://proquest.umi.com/pqdweb?did=1552538711&sid=1&Fmt=6&clientId=18952&RQT=309&VName=PQD.
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Thesis (Ph.D. in Microbiology) -- University of Colorado Denver, 2008.Typescript. Includes bibliographical references (leaves 161-183). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;
2
Mair, Caroline. "Membrane fusion mediated by the influenza virus hemagglutinin." Doctoral thesis, Humboldt-Universität zu Berlin, Lebenswissenschaftliche Fakultät, 2015. http://dx.doi.org/10.18452/17217.
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Der Eintritt von Influenza A Viren in Wirtszellen erfolgt anhand des Hämagglutinin (HA) Proteins. Neueste Entwicklungen zielen darauf ab, die fusionsinduzierende Konformations-änderung des HA und damit die Freisetzung des viralen Genoms in die Wirtszelle zu inhibieren. Der Fusionsprozess ist pH-abhängig da nur bei einem niedrigen pH-Wert (~5.0-6.0) die Protonierung bestimmter Reste innerhalb des HA eine Konformationsänderung, und somit die Membranfusion, auslöst. Die Identifizierung von konservierten, titrierbaren Resten und die Aufklärung der Strukturveränderungen im HA ermöglichen eine gezielte Entwicklung neuer antiviraler Medikamente. In dieser Arbeit wurden bestimmte Histidine im HA mittels umfassender experimenteller und theoretischer Methoden als potentielle pH-Sensoren untersucht. Dabei konnte das Histidin an Position 184 als wichtiger Schalter der pH-induzierten Konformationsänderung identifiziert werden. Außerdem bewirkte der Austausch des geladenen Rests an Position 216 in der Nähe des His184 eine Veränderung der pH-Abhängigkeit des H5 HA aufgrund der Beeinflussung des pKa-Werts des His184. Da die Mutation R216E im HA des hochpathogenen H5N1 Virus in allen Isolaten während der Vogelvirenseuche im Jahr 2003/04 detektiert wurde, deutet das Ergebnis daraufhin, dass diese Mutation zur Entstehung des hochvirulenten Vogelvirus und dessen Adaptierung an den Menschen beigetragen hat. In diesem Zusammenhang wurde auch der Einfluss der pH-Abhängigkeit des HA auf die Fusion und Infektiosität von Viren in lebenden Zellen getestet. Eine destabilisierende Mutation im HA eines rekombinanten WSN-H3 Virus reduzierte dessen Infektions- und Replikationseffizienz in MDCK-Zellen, was auf den endosomalen pH-Wert dieser Zellen zurückgeführt werden konnte. Die Messung der Virus-Endosom-Fusionskinetik in lebenden Zellen machte außerdem die Bedeutung der pH-Abhängigkeit des HA für den Zeitpunkt der Membranfusion und dessen Einfluss auf die Effizienz der Virusinfektion deutlich.The entry of influenza A virus into host cells is established by the hemagglutinin (HA) protein. New antiviral strategies aim to inhibit the fusion inducing conformational change of HA and thereby liberation of the viral genome into the cell. This process is strictly pH dependent since the conformational change of HA initiating the fusion of membranes only occurs upon protonation of yet unknown residues within HA at low pH (~5.0-6.0). The identification of conserved titrable residues and better understanding of the sequential structural rearrangements within HA may facilitate the development of new broad-spectrum antivirals. In the present work His184 and His110 were characterized as potential pH sensors by a comprehensive mutational and computational analysis. The results suggest that His184, but not His110, is an important regulator of HA conformational change at low pH. Furthermore, an exchange of charge at position 216 in vicinity to His184 was shown to alter the pH dependence of conformational change and of fusion in correlation to the known pKa dependence of histidines on neighboring residues. The result advocates that the mutation R216E, which emerged in the highly pathogenic H5 HA in 2003-2004, contributed to an altered acid stability of H5 HA via its effect on His184 and thus to the adaptation of avian H5N1 viruses to the human host. Therefore, the role of an altered acid stability of HA for viral fusion and infectivity in living cells was assessed. Recombinant viruses containing a destabilizing mutation in the HA protein were found to have a reduced infectivity and replication efficiency in MDCK cells compared to the respective wild type. Studying virus-endosome fusion kinetics in these cells we could resolve a significant difference in the timing of fusion induction suggesting that the time-point of fusion is a critical determinant of viral infection efficiency which depends on the endosomal acidification as well as on the acid stability of HA.
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Liu, Tina Yu. "Mechanism of endoplasmic reticulum membrane fusion mediated by the Atlastin GTPase." Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13064987.
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How organelles acquire their unique shapes is a fundamental question of cell biology. The peripheral endoplasmic reticulum (ER) consists of a vast network of membrane sheets and tubules, the formation of which requires homotypic membrane fusion. Previous studies suggest that the dynamin-like GTPase, atlastin (ATL), mediates ER fusion, but the mechanism by which this occurs is unclear. In this study, I investigate 1) the role of dimerization and conformational changes in the N-terminal domain of ATL, 2) how the C-terminal amphipathic helix and the transmembrane domain of ATL cooperate with the N-terminal domain, and 3) the formation of cis and trans ATL dimers in the fusion mechanism.
ATL has a cytosolic N-terminal domain, consisting of a GTPase domain and three-helix bundle (3HB), followed by two transmembrane segments (TMs) and a cytosolic C-terminal tail (CT). Crystal structures of ATL and biochemical experiments suggest that nucleotide-dependent dimerization between ATL molecules sitting in different membranes can tether the membranes together. A subsequent conformational change triggered by GTP hydrolysis could pull the membranes toward one another for fusion. This mechanism is supported by in vitro membrane tethering and fusion assays using vesicles containing full-length Drosophila ATL.
The CT and TMs of ATL are also required for efficient membrane fusion. A synthetic peptide corresponding to a conserved amphipathic helix in the CT can act in trans to restore the fusion activity of a tailless ATL mutant. We characterize CT mutants to show that the C-terminal helix promotes fusion by perturbing the lipid bilayer. The TMs of ATL also mediate nucleotide-independent oligomerization, which may allow ATL molecules in the same membrane to synchronously undergo the conformational change leading to fusion.
Lastly, we show that continuous GTP hydrolysis is required for membrane tethering, occasionally resulting in fusion. The N-terminal cytosolic domain mediates trans dimer formation between ATL molecules on different membranes. GTP binding induces dimerization through the GTPase domains and 3HBs. We propose that GTP hydrolysis and phosphate release are required not just to drive fusion, but also to dissociate cis dimers that form on the same membrane, thus allowing ATL molecules to form trans dimers.
4
Stone-Hulslander, Judith. "Mechanisms of Newcastle Disease Virus-Mediated Membrane Fusion: A Dissertation." eScholarship@UMMS, 1999. https://escholarship.umassmed.edu/gsbs_diss/131.
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For many paramyxoviruses, including Newcastle disease virus (NDV), syncytia formation requires the expression of both surface glycoproteins (HN and F) in the same cell, and evidence suggests that fusion involves a specific interaction between the HN and F proteins (23, 73). Because a potential interaction in paramyxovirus infected cells has never been clearly demonstrated, such an interaction was explored in Chapter 2 using coimmunoprecipitation and crosslinking. Both HN and F proteins could be precipitated with heterologous antisera after a five minute radioactive pulse as well as after a two hour chase in non-radioactive media, but at low levels. Chemical crosslinking increased detection of complexes containing HN and F proteins at the cell surface. After crosslinking, intermediate as well as high molecular weight species containing both proteins were precipitated with monospecific antisera. Precipitation of proteins with anti-HN after crosslinking resulted in the detection of complexes which electrophoresed in the stacker region of the gel, from 160-300 kD, at 150 kD and at 74 kD. Precipitates obtained with anti-F after crosslinking contained species which migrated in the stacker region of the gel, between 160-300 kD, at 120 kD and at 66 kD. The 3-4 discrete complexes ranging in size from 160-300 kD contained both HN and F proteins when precipitated with either HN or F antisera. That crosslinking of complexes containing both HN and F proteins was not simply a function of overexpression of viral glycoproteins at the cell surface was addressed by demonstrating crosslinking at early time points post infection, when levels of viral surface glycoproteins are low. Use of cells infected with an avirulent strain of NDV showed that chemically crosslinked HN and F proteins were precipitated independent of cleavage of F0. Furthermore, under conditions that maximized HN protein binding to its receptor, there was no change in the percentages of HN and F0 proteins precipitated with heterologous antisera, but a decrease in F1protein precipitated was observed upon attachment. These data argue that the HN and F proteins interact in the RER. Upon attachment of the HN protein to its receptor, the HN protein undergoes a conformational change which causes a subsequent change in the associated F protein, releasing the hydrophobic fusion peptide into the target membrane and initiating fusion.
Chapter 3 explores the stalk region of the NDV HN protein, which has been implicated in both fusion promotion and virus specificity of that activity. The NDV F protein contains two heptad repeat motifs which have been shown by site-directed mutagenesis to be critical for fusion (7, 51, 57). Heptad repeat motifs mediate protein-protein interactions by enabling the formation of coiled-coils. Upon analysis of the stalk region of the NDV HN protein, we identified two heptad repeats. Secondary structure analysis of these repeats suggested the potential for these regions to form alpha-helices. To investigate the importance of this sequence motif for fusion promotion, we mutated the hydrophobic "a" position amino acids of each heptad repeat to alanine or methionine. In addition, hydrophobic amino acids in other positions were also changed to alanine. Every mutant protein retained levels of attachment activity that was greater than or equal to the wild-type protein and bound to conformation-specific monoclonal as well as polyclonal antisera. Neuraminidase activity was variably affected. Every mutation, however, showed a dramatic decrease in fusion promotion activity. The phenotypes of these mutant proteins indicate that individual amino acids within the heptad repeat region of the stalk domain of the HN protein are important for the fusion promotion activity of the protein. These data are consistent with the idea that the HN protein associates with the F protein via specific interactions between the heptad repeat regions of both proteins.
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Atfield, Rachel Sarah. "Herpes simplex virus glycoprotein-mediated membrane fusion." Thesis, University of Cambridge, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.615860.
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Chen, Yong. "Structural and functional studies on SNAREs-mediated membrane fusion." [Ames, Iowa : Iowa State University], 2006.
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Lu, Xiaobing. "Studies of intermediates and regulation in SNARE-mediated membrane fusion." [Ames, Iowa : Iowa State University], 2008.
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8
Abdulreda, Midhat H. "Investigation of Snare-Mediated Membrane Fusion Mechanism Using Atomic Force Microscope Spectroscopy." Scholarly Repository, 2007. http://scholarlyrepository.miami.edu/oa_dissertations/55.
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Membrane fusion is essential for survival in eukaryotic cells. Many physiological processes such as endocytosis and exocytosis are mediated by membrane fusion, which is driven by highly specialized and conserved family of proteins. Neuronal soluble Nethylmaleimide- sensitive factor attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane during neurotransmitter release; however, the mechanism for SNARE-mediated membrane fusion remains to be established. In the current work, we aimed at investigating this mechanism using atomic force microscope (AFM) spectroscopy. We established an AFM lipid bilayer system, which proved effective in detecting fusion of bilayers and measuring compression forces required to generate fusion. It also revealed that SNARE-mediated membrane fusion proceeds through an intermediate hemifused state. Using this system, we revealed the energy landscape for membrane fusion using a dynamic force approach. We carried out compression force measurements at different compression rates and a significant reduction in the force was observed when SNAREs were present in the bilayers. The results also indicated that a single energy barrier governed membrane fusion in our experimental system. The energy barrier is characterized by its width and height, which determine the slope of the activation potential. With SNAREs in the opposing (trans) bilayers, the width of the barrier increased > 2 fold, which is interpreted as an increase in the compressibility of the membranes and subsequently a greater ease in their deformation and fusion under compression. Moreover, specific perturbations to the SNARE interaction interfered with the observed facilitation of membrane fusion, which indicated the involvement of SNAREs in the observed fusion facilitation and increase in the fusion rate. Furthermore, dissociation kinetics analysis of the SNARE interaction revealed a strong binding force during trans SNARE-complex formation, and a correlation between the strength of the SNARE interaction and the degree of fusion facilitation was established. In conclusion, the present findings provide support for a mechanism for SNAREmediated membrane fusion, where trans-interaction between SNAREs provides close apposition of the membranes and reduces fusion energy requirements by locally destabilizing the bilayers, in which the SNAREs are anchored, through pulling on or tilting of their transmembrane segments.
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Webb, Stacy. "Viral Fusion Protein TM-TM Interactions: Modulators of Protein Function and Potential Antiviral Targets." UKnowledge, 2017. http://uknowledge.uky.edu/biochem_etds/30.
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Enveloped viruses, such as HIV, influenza, and Ebola, utilize surface glycoproteins to bind and fuse with a target cell membrane. This fusion event is necessary for release of viral genomic material so the virus can ultimately reproduce and spread. The recently emerged Hendra virus (HeV) is a negative-sense, single-stranded RNA paramyxovirus that presents a considerable threat to human health as there are currently no human vaccines or antivirals available. The HeV utilizes two surface glycoproteins, the fusion protein (F) and the attachment protein (G), to drive membrane fusion. Through this process, the F protein undergoes an irreversible conformational change, transitioning from a meta-stable pre-fusion conformation to a more thermodynamically stable post-fusion structure. Understanding the elements which control stability of the pre-fusion state and triggering to the post-fusion conformation is important for understanding F protein function. Studies that replace or mutate the TM domain of the F protein of several viruses implicated the TM domain in the fusion process, but the structural and molecular details in fusion remain unclear. Previously, analytical ultracentrifugation was used to demonstrate that isolated TM domains of HeV F protein associate in a monomer-trimer equilibrium. To determine factors driving this association, we analyzed the sequence of several paramyxovirus F protein TM domains and found a heptad repeat of β-branched residues. Analysis of the HeV F TM domain specifically revealed a heptad repeat leucine-isoleucine zipper motif (LIZ). Replacement of the LIZ with alanine resulted in dramatically reduced TM-TM association. Mutation of the LIZ in the whole protein resulted in decreased protein expression and pre-fusion conformation. To further understand the role of the TM domain, the TM domain was targeted as a potential modulator of F protein stability and function. Exogenous HeV F TM constructs were co-expressed with the full length F protein in Vero cells to analyze the effects on protein expression. Co-expression of the exogenous HeV F TM constructs dramatically reduced the expression of HeV F. However, the co-expression of exogenous HeV F TM constructs with a different paramyxovirus F protein, PIV5 F, did not strongly affect PIV5 F expression levels, suggesting that the interaction of the exogenous TM constructs is specific. Fusion assays revealed that HeV F TM constructs dramatically reduced HeV F, but not PIV5 F fusion activity. We hypothesize that the short exogenous HeV TM constructs associate with the TM domain from full-length HeV F, resulting in pre-mature triggering or protein misfolding. The work presented here demonstrates that specific elements in the TM domain contribute to TM association and pre-fusion protein stability. Furthermore, targeting these interactions may be a viable approach for antiviral development against this important pathogen.
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Kuwana, Tomomi. "Characterisation of a lysosomal protein that interfrers with membrane fusion assays." Thesis, University of Cambridge, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337875.
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Martinović, Vladan. "Protein complex formation and membrane remodelling in clathrin-mediated endocytosis." Thesis, University of Cambridge, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.709269.
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Mühlenbrock, Peter [Verfasser]. "SNARE-mediated membrane fusion on pore-spanning membranes – several fusion pathways analyzed by single-vesicle content release / Peter Mühlenbrock." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2021. http://d-nb.info/1225121612/34.
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Hoffman, Mary M. "Mechanism of MDR protein mediated multidrug resistance /." Access full-text from WCMC, 1997. http://proquest.umi.com/pqdweb?did=733008491&sid=6&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Darsow, Tamara. "Molecular mechanisms for protein transport and regulation of membrane fusion in the vacuolar protein sorting pathway /." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2000. http://wwwlib.umi.com/cr/ucsd/fullcit?p3023449.
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Kuhlmann, Jan Wilhelm. "Modulation of lateral membrane tension and SNARE-mediated single vesicle fusion on pore spanning membranes." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://hdl.handle.net/11858/00-1735-0000-0023-3F13-E.
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Suntoke, Tara R. "HIV entry : a biophysical and mutational analysis of gp41-mediated membrane fusion and its inhibition." Thesis, Massachusetts Institute of Technology, 2005. http://hdl.handle.net/1721.1/31181.
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Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2005.Includes bibliographical references.
The experiments described in this thesis were designed to elucidate the manner in which the HIV-1 envelope protein (Env) initiates infection of host cells, and to develop inhibitors of viral entry. Env comprises two non-covalently attached subunits, gpl20 and gp41, that associate as a trimer on the virion surface. Once gp 120 contacts the target cell, gp41 undergoes extensive conformational changes to mediate fusion of viral and cellular membranes. First, a short hydrophobic stretch of residues at the gp4 1 N-terminus insert into the target membrane, anchoring the protein in both viral and cellular membranes. This 'prehairpin' intermediate structure exposes an N-terminal a-helical coiled coil that is the target of promising antiviral peptides and small molecules. A previously unstudied region of N-terminal trimer was stabilized by fusion to a trimeric scaffold peptide and biophysically characterized (Chapter 2). This hybrid peptide itself potently inhibited HIV fusion, and the basis for this inhibition was assessed by studying mutant molecules. Efforts to use this peptide as an immunogen to elicit anti-gp41 antibodies are also outlined. Similar design strategies may be useful in developing N-terminal peptide inhibitors and HIV vaccine candidates, and in screening for antiviral molecules that bind to this region. As fusion progresses, the prehairpin intermediate resolves into a hairpin structure. This critical transition involves interaction of the N-terminal coiled coil with the gp41 C-terminal region. Evidence suggests that this N-C association provides the energy necessary to promote juxtaposition and merging of viral and cellular membranes.
(cont.) This hypothesis was tested using a biophysical and cell biological approach (Chapter 3), in which residues essential for this transition were mutated and analyzed. These studies confirm the hypothesis and highlight the importance of specific hydrophobic and polar contacts between the N- and C- terminal gp41 regions. This work contributes to a detailed understanding of the gp41 fusion machinery; furthermore, it shows that such knowledge can be used to design effective viral entry inhibitors. Finally, Chapter 4 places this work in the context of a broad overview of current drug and vaccine developments, and addresses some of the significant challenges that confront HIV researchers.
by Tara R. Suntoke.
Ph.D.
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White, Paul. "Bacterial protein import mediated by an iron transporter." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:20298bb4-0998-4dad-9dfa-dd9e52854dec.
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Multidrug resistant bacteria (MDR) have the potential to push back society to the pre-antibiotic era. Although discovered before penicillin, the inexorable rise in antibiotic resistance has revitalised interest in bacteriocins as treatments for bacterial infections. Bacteriocins are protein antibiotics principal to competition amongst pathogens and commensals, but the mechanisms by which they translocate across the Gram-negative cell envelope are poorly understood. The work presented in this thesis demonstrates how the endonuclease bacteriocin pyocin S2 (pyoS2) exploits the iron transporter FpvAI to translocate across the outer membrane (OM) of Pseudomonas aeruginosa. FpvAI is a 22-strand β-barrel and virulence factor in P. aeruginosa that transports iron into the cell in the form of a small siderophore, ferripyoverdine (Fe-Pvd). Uptake of Fe-Pvd requires the proton motive force (PMF), which is transduced to the ligand-bound receptor by TonB1 and its partner proteins ExbB-ExbD in the inner membrane (IM). The crystal structure of the high affinity complex (Kd = 240 pM) formed between the N-terminal domain of pyoS2 (pyoS2NTD) and FpvAI is presented, which shows pyoS2NTD mimics Fe-Pvd, and induces the same conformational changes in the receptor. Fluorescently-labelled pyoS2NTD was actively imported into P. aeruginosa PAO1 cells and this import was dependent on the PMF, TonB1 and a TonB1-box motif at the N-terminus of pyoS2NTD. Finally, photo-activated crosslinking of stalled translocation intermediates demonstrated pyoS2NTD translocates through the FpvAI β-barrel lumen by a process analogous to that of Fe-Pvd. Following binding to FpvAI, translocation begins by the unfolding of a force-labile portion of the plug domain, opening a narrow channel through FpvAI. This enables pyoS2 to deliver its own TonB1-box to the periplasm where contact with TonB1 activates its import through the same channel, most likely as an unfolded polypeptide. Hence, this study demonstrates that bacteria possess a rudimentary protein import system that exploits energised nutrient transporters in the OM.
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Troke, Philip J. F. "The leukaemogenic fusion protein MOZ-TIF2 inhibits nuclear receptor-mediated transcription and mislocalises CBP." Thesis, University of Leicester, 2003. http://hdl.handle.net/2381/29687.
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Chromosomal rearrangements associated with the M4/M5 subtype of Acute Myeloid Leukaemia include fusions of the gene encoding the histone acetyltransferase (HAT) MOZ with genes encoding the transcription factor coactivators TIF2, CBP or p300. The physiological roles of MOZ in vivo remain to be established. However, TIF2 functions to recruit the HATs CBP/p300 to ligand-bound Nuclear Receptors (NR), thus leading to transcription activation. Studies investigating the role of NRs and their ligands in haematopoiesis suggest that they aid the regulation of myeloid cell differentiation. The research described in this thesis therefore investigated the effect of the MOZ-TIF2 fusion protein on NR-mediated transcription. Expression of MOZ-TIF2 resulted in an inhibition of ligand-dependent transcription by the Retinoid X Receptor a, Retinoic Acid Receptor a and Estrogen Receptor a in the C0S1 cell line. Further studies showed that MOZ-TIF2 also inhibited transcription mediated by endogenous NRs in the haematopoietic cell line U937. Investigation of protein interactions demonstrated that MOZ-TIF2 bound CBP through its Activation Domain 1 (AD1) and that deletion of this region resulted in the inability of the protein to inhibit NR transcription. Immunofluorescence analysis showed that MOZ-TIF2 formed a mesh-like nuclear pattern in contrast to the speckled nuclear localisations of MOZ, TIF2 and CBP. In addition, in cells expressing MOZ-TIF2, but not the AD1 deletion mutant, endogenous CBP displayed a diffuse staining pattern. Thus, the experiments described here show that MOZ-TIF2 inhibits NR-mediated transcription and mislocalises CBP, and that both of these activities are dependent upon its AD1. Further experiments investigating the effect of MOZ-TIF2 on p53 and AML1 -mediated transcription have indicated that MOZ-TIF2 also has the ability to affect the function of transcription factors in addition to NRs. Thus, expression of the MOZ-TIF2 fusion protein may result in the formation of leukaemia through the misregulation of normal haematopoietic transcription.
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Schwamborn, Miriam. "Establishment of a fluorescence assay for characterization of protein-mediated vesicle fusion and acidification." Doctoral thesis, Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2017. http://hdl.handle.net/11858/00-1735-0000-0023-3E83-7.
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Fass, Deborah 1970. "The protein structures underlying receptor binding and membrane fusion of ecotropic murine leukemia viruses." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/10385.
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Salsman, Scott J. "Redox regulation of protein tyrosine phosphatases in cell membrane receptor-mediated signal transduction." Oklahoma City : [s.n.], 2005.
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Witkowska, Agata [Verfasser], Reinhard [Akademischer Betreuer] Jahn, Reinhard [Gutachter] Jahn, and Andreas [Gutachter] Janshoff. "Study of SNARE-mediated membrane fusion with a novel single vesicle fusion assay / Agata Witkowska ; Gutachter: Reinhard Jahn, Andreas Janshoff ; Betreuer: Reinhard Jahn." Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2018. http://d-nb.info/1161183191/34.
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Ryan, Marnie A. "The Role of Membrane Remodeling in Surfactant Protein B (SP-B) Function." University of Cincinnati / OhioLINK, 2005. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1127263783.
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Joshi, Supriya. "Role of membrane fusion protein Ykt6 in regulating epithelial cell-cell and cell-matrix adhesions." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3350.
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Intercellular junctions and cell-matrix adhesions play important roles in the maintenance of epithelial integrity. Assembly and remodeling of the plasma membrane complexes are regulated by membrane trafficking and fusion. This thesis is aimed to elucidate the roles of an important membrane fusion protein, Ykt6, in the regulation of epithelial cell adhesion and migration. For the first time, we show that Ykt6 is essential for assembly of adherens junctions and tight junctions in human prostate epithelial cells. We also observed that Ykt6 negatively regulates both collective epithelial cell migration and cell invasion into Matrigel. The effects of YKT6 on epithelial junctions involves expressional regulation of key junctional proteins, E-cadherin and claudin-4, whereas its effects on cell motility can be explained by antagonizing functions of junctional adhesion molecule-A. Overall, this study identifies YKT6 as a novel regulator of epithelial cell adhesions and motility.
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Ling, Rebecca. "Construction of a fusion protein for anchoring the inflammatory receptor NLRP3 to the cell membrane." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17482.
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The innate immune system are a cooperation of many components – receptors being one of them. Both membrane-bound and cytosolic receptors play a large role in the defence system against pathogens and danger. NLRP3 is a receptor which assembles a protein complex called inflammasome in response to cytosolic stress and is responsible for many autoimmune diseases if it malfunctions. The activation of the NLRP3 inflammasome leads to secretion of inflammatory cytokines and in many cases to programmed cell death. The structure, function and activation of the NLRP3 inflammasome is still not fully understood and the urge to understand the mechanisms behind are important for future medical improvements. The aim was to anchor the NLRP3 inflammasome by the cell membrane - By Overlap PCR, the NLRP3 cDNA was fused extracellular and trans-membrane parts of the TLR4 cDNA to anchor the NLRP3 to the membrane and in turn analyse the inflammasome with LPI™ technology. Multiple primers and a TLR4 nucleotide were designed and the NLRP3 was amplified with specific overhangs by PCR. The fusion protein was successfully linked together by Overlap PCR but not confirmed by sequencing. The gene fusion demands high quality primers for amplification and further evaluation must be made to the details of the laboratory. To anchor the protein complex to the cell membrane, continue to be of full importance and can be an asset in many structural studies and biopharmaceuticals trials.
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Bokvist, Marcus. "Membrane mediated aggregation of amyloid-β protein : a potential key event in Alzheimer's disease." Doctoral thesis, Umeå universitet, Kemi, 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-969.
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The pathogenesis of Alzheimer’s disease (AD), the most common senile dementia, is a complex process. A crucial event in AD is the aggregation of amyloid-β protein (Aβ), a cleavage product from the Amyloid Precursor Protein (APP). Aβ40, a common component in amyloid plaques found in patients, aggregates in vitro at concentrations, much higher than the one found in vivo. But in the presence of charged lipid membranes, aggregations occurs at much lower concentration in vitro compared to the membrane-free case. This can be understood due to the ability of Aβ to get electrostatically attracted to target membranes with a pronounced surface potential. This electrostatically driven process accumulates peptide at the membrane surface at concentrations high enough for aggregation while the bulk concentration still remains below threshold. Here, we elucidated the molecular nature of this Aβ-membrane process and its consequences for Aβ misfolding by Circular Dichroism Spectroscopy, Differential Scanning Calorimetry and Nuclear Magnetic Resonance Spectroscopy. First, we revealed by NMR that Aβ40 peptide does indeed interact electrostatically with membranes of negative and positive surface potential. Surprisingly, it even binds to nominal neutral membranes if these contain lipids of opposite charge. Combined NMR and CD studies also revealed that the peptide might be shielded from aggregation when incorporated into the membrane. Moreover, CD studies of Aβ40 added to charged membranes showed that both positively and negatively membranes induce aggregation albeit at different kinetics and finally that macromolecular crowding can both speed up and slow down aggregation of Aβ.
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Huber, Scott David. "On Protein Recruitment Dynamics in Clathrin-Mediated Endocytosis and its Relation to Membrane Tension." The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1545921340769193.
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Williamson, Shawn T. "Trafficking of integral membrane proteins of the inner nuclear membrane can be mediated by the ''sorting motif'' of autographa californica nucleopolyhedrovirus odv-e66." Texas A&M University, 2005. http://hdl.handle.net/1969.1/4353.
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The amino-terminal 33 amino acids of the baculovirus integral membrane
protein, ODV-E66, are sufficient for localization of fusion proteins to viralinduced
intranuclear microvesicles (MV) and occlusion derived virus envelopes
during infection, and has been termed the sorting motif (SM). When abundantly
expressed, SM-fusions are also detected in the inner nuclear membrane (INM),
outer nuclear membrane and endoplasmic reticulum of infected cells, suggesting
proteins with the SM use the same trafficking pathway as cellular INM proteins
to traffic to nuclear membranes. This study identifies the essential
characteristics required for sorting of the SM to the INM of uninfected cells, and
the MV and ODV envelopes of infected cells. These features are an 18 amino
acid transmembrane sequence that lacks polar and charged amino acids (a.a.)
with a cluster of charged a.a. spaced 5-11 residues from the end of the
transmembrane sequence. A comparison of the a.a. sequence of these SM
features with cellular INM proteins shows the features are conserved.
The model of INM protein sorting and localization predicts the only known
sorting event during INM protein trafficking is immobilization/retention in the INM. This study uses confocal microscopy and fluorescence recovery after
photobleaching to compare the localization and mobility of lamin B receptor
(LBR) fusions (which contain SM-like sequences) to a viral SM fusion when
expressed in either mammalian or insect cells. The results show that
immobilization is not necessarily required for accumulation of proteins in the
INM. Furthermore, the results from infected cells show that an active sorting
event, likely independent of immobilization, can distinguish the viral SM from
cellular sequences similar to the SM.
The results of this study show that sorting of proteins to the INM can be
mediated by the viral SM or INM protein SM-like sequences that can function
either independent of, or in addition to, immobilization. These data combined
with recent reports suggest that in addition to diffusion:retention a signal
mediated mechanism for sorting and localization to the INM can occur.
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Wasiak, Sylwia. "Characterization of protein-protein interactions involved in clathrin-mediated budding at the plasma membrane and the trans-Golgi network." Thesis, McGill University, 2004. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=85656.
Full textAbstract:
Clathrin-mediated membrane budding mainly occurs at the cell surface, where it drives the formation of endocytic vesicles necessary for the internalization of membrane proteins, and at the trans-Golgi network (TGN), where it mediates the trafficking of cargo proteins from the TGN to the endosomal/lysosomal system. The general objective of my doctoral research was to study adaptor and accessory proteins that play structural and regulatory roles in the formation of clathrin-coated vesicles (CCVs). This was achieved principally through the characterization of two protein complexes: a first, which functions at the cell surface and a second, which regulates vesicle formation at the TGN/endosome.First, we discovered that the endocytic protein PACSIN 1 is a binding partner for the signaling molecule mSos1. Further analysis of this interaction revealed that both proteins form a complex in vivo, that they co-localize at actin-rich sites, and that their interaction is regulated by phosphorylation. These data strengthen the link between endocytosis, signaling and actin cytoskeleton dynamics and provide the basis for further investigation of the role of this protein complex.
Second, we used subcellular proteomics to identify novel components of brain-derived CCVs. Among these was an ENTH domain-containing protein, which we named enthoprotin. Further work demonstrated that enthoprotin localizes to CCVs, interacts with clathrin adaptors AP-1 and GGA2 at the TGN, and stimulates clathrin assembly in vitro. Altogether, our data suggest a role for enthoprotin in clathrin coat assembly on internal membranes. Analysis of the enthoprotin protein sequence led to the discovery of two peptide motifs responsible for interactions with AP-1 and GGA2. We used alanine-scan mutagenesis and nuclear magnetic resonance to biochemically and structurally characterize the high-affinity site responsible for the interaction between enthoprotin and the TGN adaptors. This experimental approach, coupled to alignments, allowed us to deduce an AP-1/GGA-binding consensus motif that can be used towards the identification of novel TGN adaptor-binding partners. Altogether, my doctoral research contributed to furthering knowledge of the mechanisms that underlie clathrin-mediated membrane budding.
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Chang, Andres. "EARLY EVENTS OF HUMAN METAPNEUMOVIRUS INFECTION." UKnowledge, 2012. http://uknowledge.uky.edu/biochem_etds/5.
Full textAbstract:
Human metapneumovirus (HMPV) is a worldwide respiratory pathogen that belongs to the paramyxovirus family of enveloped viruses and affects primarily the pediatric, geriatric, and immunocompromised populations. Despite its prevalence and importance to human health, no therapies are available against this pathogen. For paramyxoviruses, it is believed that infection starts by attachment of the virus to the surface of the cell through the viral attachment protein followed by fusion between the viral and cellular membranes, a process mediated by the fusion (F) protein at the plasma membrane and at neutral pH. Previous work showed that HMPV infection can occur in the absence of the attachment protein and membrane fusion triggered by the F protein can be promoted by low pH. The work presented here are significant advances in our understanding of the entry process of HMPV. We confirmed that the F protein has receptorbinding functions and identified the cellular binding partner to be heparan sulfate proteoglycans (HSPGs). Additionally, we provide evidence that electrostatic interactions at two different regions play important roles for the proper folding, stability, and low pH triggering of the HMPV F protein. We confirmed the hypothesis that protonation of H435 is important for HMPV F triggering and provide additional evidence that the entry of HMPV may be occurring through endocytosis. Therefore, we hypothesize that HMPV entry occurs through endocytosis after viral binding to HSPGs through the F protein and membrane fusion occurs in an acidified compartment.
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Huyton, Trevor. "Structural studies of p97, A AAA ATPase involved in membrane fusion and ubiquitin dependent protein pathways." Thesis, Imperial College London, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414829.
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Haward, Fiona. "Investigation of the physiological roles of SRSF1-mediated translation." Thesis, University of Edinburgh, 2018. http://hdl.handle.net/1842/31188.
Full textAbstract:
The serine/arginine-rich (SR-) family proteins constitute a diverse group of pre-mRNA splicing factors that are essential for viability. They can be characterised based on the presence of one or two RRMs and an RS domain. A subset, of which SRSF1 is the prototype, is capable of nucleocytoplasmic shuttling; a process governed by continual cyclic phosphorylation of the RS domain. In contrast, SRSF2, another member of the SR family, is unable to shuttle due to the presence of a nuclear retention sequence (NRS) at the C-terminus of its RS domain. When this NRS is fused to SRSF1, it prevents nucleocytoplasmic shuttling of the SRSF1-NRS fusion protein. In addition to its nuclear roles, SRSF1 is directly associated with the translation machinery and can activate mRNA translation of target transcripts via an mTOR-dependent mechanism. The specific mRNA translational targets that SRSF1 serves to regulate encode numerous factors including RNA processing factors and cell-cycle proteins. The aim of this work is to study the physiological relevance of SRSF1 cytoplasmic functions, as previous data have relied on overexpression systems. CRISPR/Cas9 editing was used to knock-in the NRS naturally present in SRSF2 at the SRSF1 genomic locus, creating an SRSF1-NRS fusion protein. After numerous attempts, it was only possible to obtain a single viable homozygous clone in mouse embryonic stem cells (mESCs), despite being able to successfully tag the genomic SRSF1 locus. This strongly suggests that the ablation of SRSF1 shuttling ability is highly selected against in mESCs. To assess the physiological importance of SRSF1 nucleocytoplasmic shuttling during development, a mouse model for SRSF1-NRS was also developed. SRSF1-NRS homozygous mice are born at correct Mendelian ratios, but are small in size and present with severe hydrocephalus. Finally, proteomics was used to identify interactors of endogenous cytoplasmic SRSF1 and those that bind the NRS of SRSF2 to gain insights into the mechanism of nuclear retention for non-shuttling SR proteins. In summary, this work analyses the physiological relevance of cytoplasmic SRSF1 function and the consequences of the SRSF1-NRS allele in mouse development.
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Kalli, Antreas C. "Computational studies of talin-mediated integrin activation." Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:ed4652dd-af20-4550-8c45-2b5f9c443ff6.
Full textAbstract:
Integrins are large heterodimeric (αβ) cell surface receptors that play a key role in the formation of focal adhesion complexes and are involved in various signal transduction pathways. They are ‘activated’ to a high affinity state by the formation of an intracellular complex between the membrane, the integrin β-subunit tail and talin, a process known as ‘inside-out activation’. The head domain of talin, a FERM domain homologue, plays a vital role in the formation of this complex. Recent studies also suggest that kindlins act in synergy with talin to induce integrin activation. Despite much available structural and functional data, details of how talin activates integrins remain elusive. In this thesis a multiscale simulation approach (using a combination of coarse-grained and atomistic molecular dynamics simulations) together with NMR experiments were employed to study talin-mediated integrin inside-out activation. A number of novel insights emerged from these studies including: (i) the crucial role of negatively charged lipids in talin/membrane association; (ii) a novel V-shape conformation of the talin head domain which optimizes its interactions with negatively charged lipids; (iii) that interactions of talin with negatively charged moieties in the membrane orient talin to optimize interactions with the β cytoplasmic tail; (iv) that binding of talin to the β cytoplasmic tail promotes rearrangement of the integrin TM helices and weakens the integrin α/β association; and (v) that an increase in the tilt angle of the β integrin TM helix initiates a scissoring movement of the two integrin TM helices. These results, combined with experimental data, provide new insights into the mechanism of integrin inside-out activation. The same multiscale approach was used to demonstrate the crucial role of the Gx3G motif in the packing of the integrin transmembrane helices. Using recent structural data the integrin/talin complex was modelled and inserted in bilayers which resemble the biological plasma membrane. The results demonstrate the dynamic nature of the integrin receptor and suggest that the integrin/talin complex alters the lipid organization and motion in the outer and inner bilayer leaflets in an asymmetric way and that diffusion of lipids in the vicinity of the protein is slowed down. The work in this thesis demonstrates that multiscale simulations have considerable strengths when applied to investigations of structure/function relationships in membrane proteins.
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Wan, Jun. "Elucidation of the JNK pathway mediated by Epstein-Barr virus encoded latent membrane protein 1 /." View abstract or full-text, 2004. http://library.ust.hk/cgi/db/thesis.pl?BICH%202004%20WAN.
Full textAbstract:
Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2004.Includes bibliographical references (leaves 105-128). Also available in electronic version. Access restricted to campus users.
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Wang, Mingyang [Verfasser]. "Attachment of stearic acid to the Hemagglutinin-Esterase-Fusion (HEF) protein of influenza C virus affects membrane fusion and virus replication / Mingyang Wang." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1077211996/34.
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Nour, El Din Suzanne [Verfasser]. "Azurin (P28) fusion protein mediated photodynamic therapy in the treatment of malignant tumors / Suzanne Nour El Din." Ulm : Universität Ulm, 2017. http://d-nb.info/1140118137/34.
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Wu, Liming. "Elucidation of the NF-kB pathway mediated by Epstein - Barr virus-encoded latent membrane protein 1 (LMP1) /." View abstract or full-text, 2005. http://library.ust.hk/cgi/db/thesis.pl?BICH%202005%20WU.
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Dashan, Li. "Factors affecting the membrane fusion-inducing capacity of the spike protein of avian infectious bronchitis coronavirus (IBV)." Thesis, Royal Veterinary College (University of London), 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.522192.
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Tam, John. "Expression of the membrane fusion protein of measles virus in insect and mammalian cells using recombinant viruses." Thesis, McGill University, 1993. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=69521.
Full textAbstract:
The fusion (F) protein of measles virus is responsible for viral penetration at the plasma membrane and syncytia formation. In order to facilitate future structure/function studies, the F protein was expressed in insect cells using recombinant baculovirus, and in mammalian cells using both vaccinia and adenovirus recombinants. The baculovirus system exhibited the highest levels of recombinant protein expression, but the majority of the product was found to be in the form of detergent-insoluble precursors. Replacing the signal peptide of the F protein with insect-derived sequences did not enhance posttranslational processing. Also, expression of truncated, anchor-minus forms of the F protein did not result in improved solubility or secretion. The vaccinia virus recombinant VF exhibited higher levels of expression than measles virus-infected cells. However, the majority of the F protein was expressed as insoluble precursors. The biologically active portion, represented by the proteolytically cleaved F protein, was found to be soluble. The vP455 vaccinia virus recombinant and the AdF adenovirus recombinant expressed similar levels of processed F as in measles virus-infected cells. The products of these recombinants were found to be soluble. F protein expressed by vaccinia virus recombinants in primate and murine cells were observed to cause syncytia formation in the absence of measles virus hemagglutinin (H) co-expression. This H-independent fusion could not be inhibited by Z-D-Phe-L-Phe-Gly. When the adenovirus system was used, F protein could not facilitate fusion unless measles virus hemagglutinin was present as well.
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Postigo, Peláez Miguel Ángel. "Construction of a Fusion Gene : to anchor a truncated version of the inflammatory receptor NLRP3 to the cell membrane." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17573.
Full textAbstract:
Inflammasomes are a group of protein complex that regulate inflammation throughcomplex signal transduction, although their specific mechanisms and structures have notbeen fully described. As the protein that kickstarts assembly of a type of inflammasome,NLRP3 is a key regulator of inflammation and may play a relevant role in the developmentof inflammatory diseases. In this project it has been attempted to perform a Gene Fusionbetween a segment of NLRP3 and regions of Toll-Like Receptor 4 by means of overlapextensionPCR, a technique that employs hybrid primers to create an overlap between bothsequences that can be filled by a polymerase, causing them to merge. Results suggest GeneFusion was successful, however cloning and expression of the construct have not beenachieved so far. If expressed as a fusion protein, the added transmembrane domain willanchor two domains of NLRP3 to the membrane, allowing more precise study of thecomposition and functionality of the inflammasome. Removal of the terminal domain ofNLRP3 will help determine its implication and relevance in the assembly process of theprotein complex.
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Gaddy, Jennifer Angeline. "Acinetobacter baumannii Virulence Attributes: The Roles of Outer Membrane Protein A, Acinetobactin-mediated Iron Acquisition Functions, and Blue Light Sensing Protein A." Miami University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=miami1289178632.
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Klimyte, Edita M. "ELUCIDATING BINDING, FUSION AND ENTRY OF HUMAN METAPNEUMOVIRUS." UKnowledge, 2016. http://uknowledge.uky.edu/biochem_etds/28.
Full textAbstract:
Human metapneumovirus (HMPV) is a respiratory pathogen in the Paramyxoviridae family that infects nearly 100% of the world population. This enveloped RNA virus causes severe viral respiratory disease in infants, the elderly, and immunocompromised patients worldwide. Despite its prevalence and importance to human health, no therapies are available against this pathogen. Entry of paramyxoviruses into host cells generally requires the coordinated activity of the attachment glycoprotein, G, which interacts with a cell receptor, and the fusion glycoprotein, F, which promotes subsequent fusion of viral and cellular membranes. However, HMPV F is the primary viral protein mediating both binding and fusion for HMPV. Previous work that showed HMPV F mediates attachment to heparan sulfate proteoglycans (HSPGs), and some HMPV F fusion activity can be promoted by acidic pH. The work presented here provides significant advances in our understanding of the fusion and binding events during HMPV infection. We demonstrated that low pH promotes fusion in HMPV F proteins from diverse clades, challenging previously reported requirements and identifying a critical residue that enhances low pH promoted fusion. These results support our hypothesis that electrostatic interactions play a key role in HMPV F triggering and further elucidate the complexity of viral fusion proteins. Additionally, we characterized the key features of the binding interaction between HMPV and HSPGs using heparan sulfate mimetics, identifying an important sulfate modification, and demonstrated that these interactions occur at the apical surface of polarized airways tissues. We identified differences in particle binding related to the presence or absence of the HMPV G and SH glycoproteins. Lastly, we characterized paramyxovirus infection in cystic fibrosis bronchial epithelial cells, identifying a potential specific susceptibility to HMPV infection in these individuals. The work presented here contributes to our understanding of HMPV infection, from mechanisms of early events of entry to clinical scenarios.
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Hennerdal, Aron, Jenny Falk, Erik Lindahl, and Arne Elofsson. "Internal duplications in alpha-helical membrane protein topologies are common but the nonduplicated forms are rare." Stockholms universitet, Institutionen för biokemi och biofysik, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-51243.
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Many alpha-helical membrane proteins contain internal symmetries, indicating that they might have evolved through a gene duplication and fusion event Here, we have characterized internal duplications among membrane proteins of known structure and in three complete genomes We found that the majority of large transmembrane (TM) proteins contain an internal duplication The duplications found showed a large variability both in the number of TM-segments included and in their orientation Surprisingly, an approximately equal number of antiparallel duplications and parallel duplications were found However, of all 11 superfamilies with an internal duplication, only for one, the AcrB Multidrug Efflux Pump, the duplicated unit could be found in its nonduplicated form An evolutionary analysis of the AcrB homologs indicates that several independent fusions have occurred, including the fusion of the SecD and SecF proteins into the 12-TM-protein SecDF in Brucella and Staphylococcus aureus In one additional case, the Vitamin B-12 transporter-like ABC transporters, the protein had undergone an additional fusion to form protein with 20 TM-helices in several bacterial genomes Finally, homologs to all human membrane proteins were used to detect the presence of duplicated and nonduplicated proteins This confirmed that only in rare cases can homologs with different duplication status be found, although internal symmetry is frequent among these proteins One possible explanation is that it is frequent that duplication and fusion events happen simultaneously and that there is almost always a strong selective advantage for the fused formauthorCount :4
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Ding, Daniel N. "Membrane display of a fusion protein containing the ice-nucleation protein from Pseudomonas syringae and ScFv against c-myc oncoprotein in recombinant Escherichia coli." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape8/PQDD_0001/MQ42060.pdf.
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Bharadwaj, Prashant R. "Yeast as a Model for Studying Aβ Aggregation, Toxicity and Clearance." Thesis, Edith Cowan University, Research Online, Perth, Western Australia, 2011. https://ro.ecu.edu.au/theses/404.
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Alzheimer’s disease (AD) is a progressive neurodegenerative disorder of the central nervous system, characterised by acute memory loss and behavioural symptoms. The AD brain is characterized by the presence of senile amyloid plaques associated with degenerating neurites and inflammatory processes. The major protein component of these amyloid deposits is the amyloid beta (Aβ) protein. The Aβ protein is a 40 or 42 amino acid cleavage product of APP (Amyloid Precursor Protein) which is produced in low levels in the normal ageing brain. Although senile amyloid plaques is the major pathological hallmark of AD brains, accumulating evidence has been presented to show that increased levels of soluble forms of Aβ42 correlate with the clinical manifestations and severity of the disease. Increased accumulation (both intracellular and extracellular) and toxicity of Aβ42 peptide in the brain play pivotal roles in neurodegeneration and loss of memory functions in the AD brain. Therefore reducing the toxicity of Aβ42 and increasing its clearance from the brain has been considered to be main targets for AD therapeutics. The search for a disease modifying therapy for AD has been very difficult with the majority of agents failing in later stages of clinical trials. The incomplete understanding of drug-target mechanisms and the lack of high-throughput screening systems for identifying selective target based drugs have been some of the main issues expressed for the failure of AD drugs. Yeast offer a simple eukaryotic model for studying pathological mechanisms and compared to other models there is availability of various experimental tools applicable for high throughput analysis of protein-protein, gene-gene and gene-protein interactions and associated cellular functions. It can also offer a versatile model for initial screening in drug development for various human diseases, including AD. Yeast models have been utilised for studying AD related proteins including APP and its processing enzymes (secretases) and tau phosphorylation.
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Böhm, Daniela [Verfasser]. "Investigation of the mechanism of Epstein-Barr virus Latent Membrane Protein 1 mediated NF-kB activation / Daniela Böhm." Berlin : Freie Universität Berlin, 2010. http://d-nb.info/102450249X/34.
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Andreazza, Camilla. "Mechanism of atlastin-mediated membrane fusion and identification of atlastin functional partners involved in endoplasmic reticulum dynamics using Drosophila as a model." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423008.
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Abstract
The biogenesis and maintenance of the endoplasmic reticulum (ER) requires membrane fusion. ER homotypic fusion is driven by the large GTPase atlastin however the mechanisms governing atlastin-mediated membrane fusion are unknown.
Using a structure-function analysis we investigated the mechanistic basis of nucleotide-dependent membrane fusion by Drosophila atlastin. Domain analysis of atlastin shows that a conserved region of the C-terminal cytoplasmic tail is absolutely required for fusion activity. Atlastin lacking the C-terminal domain are incapable of driving membrane fusion. When functional Drosophila atlastin is expressed, the normally reticular ER is lost and variably sized puncta are now stained with ER markers. However, overexpressing ER localized truncated atlastin essentially does not disrupt ER morphology demonstrating that it is a less or non-functional protein in vivo. Deletion analysis has allowed to establish that a middle domain folded as a 3 helix bundle (3HB) is important for oligomerization. Mutations disrupting the structure of the helices within the 3HB inactivate atlastin by preventing tethering and the subsequent fusion of ER membranes. Furthermore, increasing the distance of atlastin complex formation from the membrane inhibits fusion, suggesting that this distance is crucial for atlastin to promote fusion.
Recently it has been shown that atlastin interacts with other ER tubule-forming proteins such as reticulon and REEP/Yop/DP1 families. We found that in Drosophila Dp1 and reticulon interact genetically with atlastin. The lethality associated with null atlastin mutations is largely suppressed by the simultaneous loss of reticulon function. Furthermore, the hyperfusion phenotype caused by atlastin overexpression in COS-7 cells is rescued by coexpression of Dp1 or reticulon. This result is supported by experiment in the fly eye. Expression of atlastin in the eye causes a small and rough eye phenotype. Overexpression of Dp1 in an eye simultaneously expressing atlastin resulted in the rescue of the atlastin-dependent phenotype. Instead, loss of reticulon exacerbates the rough eye phenotype caused by atlastin overexpression. These data show that atlastin exibits a strong, antagonistic interaction with both reticulon and Dp1 suggesting that these proteins are likely to exert opposing functions to regulate ER structure. We speculate that a balance between atlastin, reticulon, and Dp1 activities is important in maintaining and determining the morphology of the ER.Riassunto La biogenesi e il mantenimento del Reticolo Endoplasmatico (ER) richiedono fusione delle membrane. La fusione omotipica delle membrane dell'ER dipende dalla GTPasi atlastina, tuttvia i meccanismi che governano il processo di fusione mediato da questa proteina sono ancora sconosciuti. Attraverso un’analisi struttura-funzione, abbiamo esaminato i meccanismi alla base del processo di fusione nucleotide-dipendente di atlastina nell’organismo modello Drosophila melanogaster. L'analisi dei domini di atlastina mostra che una regione conservata nella coda citoplasmatica C-terminale è indispensabile per la sua attività fusogena. Atlastina priva del dominio C-terminale non permette la fusione delle membrane. L’espressione di atlastina wild-type di Drosophila causa alterazione del caratteristico fenotipo reticolare dell'ER e marcatori del Reticolo evidenziano dilatazioni delle membrane reticolari di varie dimensioni. Al contrario, l'espressione di atlastina tronca della regione C-terminale non altera la morfologia del ER dimostrando che questa forma troncata è poco o non funzionale in vivo. L'espressione di atlastina tronca ha permesso di identificare un dominio intermedio costituito da un fascio di tre eliche che sono importanti per l'oligomerizzazione di atlastina. Mutazioni che distruggono la struttura delle alfa-eliche di questo dominio inattivano atlastina prevenendo l'avvicinamento e la conseguente fusione delle membrane del Reticolo Endoplasmatico. Inoltre, l’aumento della distanza di formazione del complesso di atlastina dalle membrane destinate a fondersi inibisce la fusione, suggerendo che questa distanza è cruciale per atlastina per promuovere la fusione. Recentemente è stato visto che atlastina interagisce con altre proteine coinvolte nel determinare la morfologia dell'ER appartenenti alle famiglie reticulon e REEP/Yop/DP1. I nostri studi hanno dimostrato che Dp1 e reticulon interagiscono funzionalmente con atlastina. La letalità dovuta a mutazione nulla di atlastina è recuperata dalla simultanea perdita di funzione di reticulon. Inoltre, l fenotipo di iperfusione causato da sovraespressione di atlastina in cellule COS-7 è recuperato co-esprimendo Dp1 o reticulon. Questo risultato è supportato da esperimenti nell'occhio di Drosophila. Espressione di atlastina wild type nell'occhio di Drosophila causa un occhio piccolo e rovinato. La sovraespressione simultanea di Dp1 e atlastina nell'occhio risulta nel recupero del fenotipo causato dall'espressione di atlastina. Al contrario, l'assenza di reticulon aumenta il fenotipo di occhio piccolo e rovinato dovuto a espressione di atlastina. Questi risultati mostrano una forte interazione genetica antagonistica tra atlastina e reticulon o Dp1, suggerendo che questa proteine esercitano funzioni opposte per regolare la struttura dell'ER. Ipotizziamo che un bilanciamento tra le attività di atlastina, reticulon e Dp1 sia importante nel mantenimento e nel determinare la morfologia del Reticolo Endoplasmatico.
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Baljinnyam, Bolormaa. "Untersuchungen zur F-proteinvermittelten Fusion von Paramyxoviren." Doctoral thesis, [S.l.] : [s.n.], 2003. http://deposit.ddb.de/cgi-bin/dokserv?idn=968791549.
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Ojelabi, Ogooluwa A. "Small Molecule Modulation of GLUT1-Mediated Glucose Transport." eScholarship@UMMS, 2017. https://escholarship.umassmed.edu/gsbs_diss/950.
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The glucose transport protein, GLUT1, is highly expressed in rapidly proliferating cells, including cancer cells, while decreased GLUT1 levels are found in diseases such as GLUT1 deficiency syndrome and Alzheimer’s. There is increased interest in developing GLUT1 inhibitors as novel anticancer therapeutics, and the discovery of compounds that directly stimulate GLUT1 function. This work investigates how small molecules stimulate and/or inhibit GLUT1-mediated glucose transport, either directly or through the AMPK pathway.
Using sugar transport assays and docking analyses to explore Ligand–GLUT1 interactions and specificity of binding, we show that: 1) Ligands inhibit GLUT1 by competing with glucose for binding to the exofacial or endofacial sugar binding sites; 2) Subsaturating inhibitor concentrations stimulate sugar uptake; 3) Ligands inhibit GLUT1–, GLUT3– and GLUT4–mediated sugar uptake in HEK293 cells; and 4) Inclusion of a benzonitrile head group on endofacial GLUT1 inhibitors confers greater inhibitory potency.
Furthermore, we investigated AMPK-regulated GLUT1 trafficking in cultured blood-brain barrier endothelial cells, and show that inhibition of GLUT1 internalization is not responsible for increased cell surface levels of GLUT1 observed with AMPK activation in these cells.
This study provides a framework for screening candidate GLUT1 inhibitors for specificity, and for optimizing drug design and delivery. Our data on transport stimulation at low inhibitor concentrations support the idea that GLUT1 functions as a cooperative oligomer of allosteric alternating access subunits.
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Ishikawa, Raga. "Development of Engineered Extracellular Vesicle-Liposome Hybrid Using Baculovirus-Expression System." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263686.
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