Dissertations / Theses on the topic 'Membrane protein band 3'
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1
Boulter, Jonathan Michael. "Structural and functional studies of the erythrocyte anion exchanger, band 3." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.297079.
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Young, Mark. "Studies of the transmembrane domain of the human erythrocyte anion exchanger (band 3)." Thesis, University of Bristol, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340324.
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Malik, Saira. "Protein-protein and protein-lipid interactions of band 3 in native and model membranes." Thesis, University of Oxford, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302943.
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Parker, Mark D. "Expression and anion transport studies on the human erythrocyte anion exchange protein (AE1, band 3) in the yeast Saccharomyces cerevisiae." Thesis, University of Bristol, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.310589.
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Stauffer, Kathrin. "On the structure and function of membrane-integrated segments of the human erythrocyte band 3 protein /." [S.l : s.n.], 1986. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Taylor, Andrew Mark. "Biophysical studies on the human erythrocyte anion transporter, band 3." Thesis, University of Oxford, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.360571.
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Clague, M. J. "A rotational dynamic study of Erythrocyte membrane proteins : Cytoskeletal restraints on band 3 and its aggregation by positively charged species." Thesis, University of Essex, 1988. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381251.
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Che, Alexis Pun Kit. "Studies of band 3 rotational mobility in normal and ovalocytic human red blood cell membranes by transient dichroism." Thesis, University of Essex, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.241212.
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Bruce, Lesley J. "A study of human erythrocyte band 3 variants." Thesis, University of Bristol, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.240590.
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Wang, Lin. "Complementation and membrane assembly studies of human erythrocyte anion exchanger (AE1, band 3)." Thesis, University of Bristol, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.388102.
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Denbow, Cynthia J. "Membrane Domain of Plant 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase: Targeting, Topology, and Function." Diss., Virginia Tech, 1997. http://hdl.handle.net/10919/30472.
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The rate limiting step in isoprenoid biosynthesis is catalyzed by 3-hydroxy-3-methylglutaryl CoA reductase (HMGR, EC 1.1.1.34). In plants, HMGR is encoded by small gene families whose members are differentially expressed. In tomato, hmg2 was previously isolated and sequenced. We report the isolation and sequence analysis of a clone (pCD4) encompassing exon I of tomato hmg1 which encodes the putative membrane domain. Sequence comparisons of plant HMGR proteins reveal two hydrophobic stretches within the amino terminus which are highly conserved among species. Using in vitro transcription and translation systems, the membrane domain structure of two tomato HMGR isoforms, HMG1 and HMG2, were analyzed. Results from these experiments reveal that tomato HMGRs are targeted to microsomal membranes in a cotranslational fashion that does not involve cleavage of an N-terminal targeting peptide. Membrane topography of HMGR was revealed by protease protection studies, indicating that both tomato HMGRs span the membrane two times such that both the C- and N-termini are located within the cytosol. HMG2 but not HMG1 was glycosylated in the in vitro system. Deletion of the hmg1 5' untranslated regions and sequences encoding the first six highly charged amino acids resulted in inefficient translation in vitro. However, targeting to microsomes was unchanged. HMG1 membrane domain was tagged with a FLAG epitope to facilitate in vivo studies. Agrobacterium-mediated transformation was used to introduce the tagged hmg1 gene into two Nicotiana tabacum cell lines, BY-2 and KY-14. The slow growth kinetics of KY-14 prevented effective recovery of transformed lines, however, Northern analyses of BY-2 showed that the hmg1 transgene was expressed. Comparisons of BY-2 and KY-14 revealed differences in defense responses to elicitor treatment. BY-2 cells showed minimal defense capabilities, whereas KY-14 cells were rapidly induced as indicated by increased HMGR enzyme activity and browning of the cells. HMGR enzyme activity was decreased in both KY-14 and BY-2 cells following sterol treatment, but the reduction was more pronounced in KY-14 cells. Thus transgenic BY-2 cells may be useful in future in vivo immunolocalization studies, but analyses of HMGR transcriptional regulation and regulated degradation will require use of the more responsive KY-14 cells..Ph. D.
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Schischmanoff, Olivier. "Etude de l'interaction ankyrine-protéine bande 3 dans les sphérocytoses héréditaires." Paris 5, 1993. http://www.theses.fr/1993PA05P180.
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Wu, Hao. "Structural and functional characterization of scaffold protein par-3 /." View abstract or full-text, 2008. http://library.ust.hk/cgi/db/thesis.pl?BICH%202008%20WU.
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Adams, Charlotte Lynne 1971. "Tandem MS/MS analysis of band 3 protein from young and old erythrocytes." Diss., The University of Arizona, 1997. http://hdl.handle.net/10150/282574.
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Every day, billions of senescent human erythrocytes are removed from the circulation. The mechanism of recognition involves the formation of a neo-antigen on senescent cells, which binds autologous immunoglobulin and targets the senescent cell for phagocytosis. This neo-antigen is derived from an existing integral membrane protein, band 3. The molecular mechanisms underlying the formation of this neo-antigen during the aging process are poorly understood, but oxidative damage is suggested to be a critical event. Several post-translational modifications have been associated with aging that may contribute to altered antigenicity of the band 3 molecule, either directly by forming a covalent modification that contributes to the neo-antigen epitope or indirectly by altering the conformation of the protein, exposing hidden epitopes. Tandem mass spectral analysis was performed on tryptic digests of the band 3 protein from young and old erythrocytes. Six oxidations of methionyl residues were detected, one of which lies adjacent to a region of band 3 proposed to form an epitope of the neo-antigen and one of which lies between proposed antigenic regions. Studies of vitamin E deficiency and supplementation strongly support oxidation as a pivotal event in alteration of band 3 with aging, and the oxidized methionines identified in this study may represent the critical sites of damage. A possible deamidation was also identified in an antigenic region of band 3. Deamidation is suggested to serve as a molecular timeclock for proteins, and conversion of a glutamine to a glutamic acid may alter the antigenicity at this critical region of band 3. This work represents the first application of tandem MS/MS methodologies to a large integral membrane protein. Forty-four of the 75 band 3 tryptic peptides were characterized, covering 61% of the band 3 polypeptide. Many of the tryptic peptides did not meet criteria for proper peptide analysis. Of the remaining peptides, 95.3% of the band 3 molecule was characterized, with 55% sequenced by MS/MS. Since only partial sequence information is expected for this type of analysis, these percentages represent a tremendously successful application of the technique.
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Nilsson, Johan. "Membrane protein topology : prediction, experimental mapping and genome-wide analysis /." Stockholm, 2004. http://diss.kib.ki.se/2004/91-7349-963-3/.
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McPhee, Helen Kennedy. "A study of the membrane binding properties of the matrix protein from human respiratory syncytial virus." Thesis, Durham University, 2009. http://etheses.dur.ac.uk/3/.
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This thesis describes the structure of the matrix (M) protein from human respiratory syncytial virus (RSV), and its interactions with model membranes composed of a range of lipids, which have been characterized using a variety of techniques. The M protein was expressed in E. coli with a histidine tag and purified by immobilised metal affinity chromatography (IMAP). The protein was found to have acquired a mutation at position 254 from methionine to arginine, but this did not appear to affect its behaviour and both the native and mutated form of the protein were used in several experiments. Efforts to produce 2D crystals of the M protein by incubation with lipid and detergent were unsuccessful, but in several detergent-free incubations, particularly where the lipid DPPE was included, extended helical structures were formed that bore a striking resemblance to the structure of filamentous RSV virions with the viral envelope removed. These helical assemblies appeared to grow from liposomes, and demonstrate the inherent ability of RSV M to self assemble into arrays of this nature. Samples of purified protein were provided to Dr Victoria Money who was able to crystallize M and solve its structure to a resolution of 1.6 Å, revealing two domains linked by a region with little secondary structure that is potentially flexible, and a large positively charged area on the surface extending across both domains. The overall structure is somewhat similar to the crystal structure of the Ebola virus matrix protein VP40. Circular dichroism (CD) studies of M showed that it contains significantly less secondary structure in solution than in the crystal structure. Cross-linking experiments suggested that RSV M preferentially forms dimers, tetramers and hexamers in solution, in common with several other viral matrix proteins. Experiments performed on a Langmuir trough revealed that the M protein partitions into phospholipid monolayers, regardless of whether the lipid head group is phosphocholine or phosphoethanolamine. It interacts differently with monolayers containing sphingomyelin, which in combination with cholesterol forms phase separated liquid-ordered (Lo) microdomains. RSV M was found to bind extrinsically to the sphingomyelin-containing Lo domains. This did not occur when the protein was in contact with monolayers composed of only phospholipids and cholesterol that are also known to form Lo domains, showing that RSV M has a specific interaction with sphingomyelin. The evidence for this hypothesis obtained from Langmuir pressure-area isotherms was supported by direct visualization of the monolayer at the surface using Brewster angle microscopy (BAM), and examination of monolayers deposited on a modified silicon surface by atomic force microscopy (AFM). The findings described herein suggest that specific interactions with sphingomyelin may provide a mechanism for localization of the matrix protein, and therefore targeting of the site of viral assembly, to particular regions of the host cell membrane. The structure of the protein, with its two domains linked by a potentially flexible region, would allow for changes in protein conformation whilst bound to the membrane: it is possible that such changes could play a key role in the function of RSV M.
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Christians, Arne [Verfasser], and Stefan [Akademischer Betreuer] Wiemann. "Funktionelle Charakterisierung des putativen Tumorsuppressors "Epithelial Membrane Protein 3" / Arne Christians ; Betreuer: Stefan Wiemann." Heidelberg : Universitätsbibliothek Heidelberg, 2014. http://d-nb.info/1180300955/34.
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Jones, Graham K. "Expression and anion transport studies on the human erythrocyte anion exchange protein (AE1, band 3)." Thesis, University of Bristol, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.245676.
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Ramakrishnan, Sarathiraja. "Encapsulation of omega-3 fatty acids by premix membrane emulsification." Doctoral thesis, Universitat Rovira i Virgili, 2013. http://hdl.handle.net/10803/145770.
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In food and pharma industry, fish oils are highly demanded due to the many associated health benefits of omega-3 fatty acids. However, the delivery of fish oil through food is a major challenge as this oil is susceptible to oxidation and to produce off-flavors. To prevent these undesired effects, microencapsulation techniques are used. Typically oil encapsulation is carried out by two steps (i) emulsion preparation (ii) drying. The main objective is to study the effect of the emulsification method, emulsion formulation, membrane type and microcapsule formulation on relevant physic-chemical parameters of fish oil microcapsules.The approach of the project is to combine, for the first time, the advantages of using a low energy emulsification technique (membrane emulsification) and spray drying to obtain food-grade fish oil microcapsules. The results show a clear improvement in the oil encapsulation efficiency (OEE) when decreasing the droplet size of the emulsion and increasing the amount of wall material. The combination of a polysaccharide with a protein has been found to improve protection against oxidation during storage of the microcapsules, while the addition of denatured proteins as a part of the microcapsule wall material enhances OEE but does not improve the mechanical strength of the microcapsules.El aceite de pescado es altamente valorado en la industria alimentaria por su demostrada actividad en la prevención y tratamiento de numerosas patologías, asociada a su contenido en ácidos grasos omega-3. La incorporación de aceite de pescado en alimentos presenta algunas dificultades relacionadas con su rápida oxidación y su característico aroma y sabor. La encapsulación del aceite de pescado retrasa la oxidación y permite enmascarar sus propiedades sensoriales. Tradicionalmente, la encapsulación se lleva a cabo combinando una etapa de emulsificación seguida de secado por atomización. El objetivo principal del trabajo es estudiar el efecto del método de emulsificación y la formulación de la emulsión y las microcápsulas en los parámetros físico-químicos más relevantes de las microcápsulas. En este proyecto se combina por primera vez la emulsificación por membranas con el secado por atomización para obtener microcápsulas de aceite de pescado aplicables a la industria alimentaria. Los resultados muestran una clara mejora en la eficiencia de encapsulación del aceite cuando se reduce el tamaño de gota de la emulsión y se incrementa la cantidad de material de pared de las microcápsulas. La combinación de un polisacárido con una proteína para la formación de la pared mejora la estabilidad oxidativa de las microcápsulas durante el almacenamiento. Por otra parte la adición de proteínas desnaturalizadas para reforzar las paredes de las microcápsulas ha resultado en una mejora de la eficiencia de encapsulación de aceite pero no ha mejora su resistencia mecánica
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Kilelee, Erin M. "Modeling the interaction of the platelet microbicidal protein tPMP-1 with the cell membrane." View electronic thesis (PDF), 2009. http://dl.uncw.edu/etd/2009-3/r3/kileleee/erinkilelee.pdf.
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Darbyson, Angie L. "ORP-3 Rescues ER Membrane Expansions Caused by the VAPB-P56S Mutation in Familial ALS." Thèse, Université d'Ottawa / University of Ottawa, 2013. http://hdl.handle.net/10393/29054.
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A mutation in ER membrane protein VAPB is responsible for causing a familial form of ALS (ALS8). The VAPB-P56S mutation causes protein aggregation and a nuclear envelope defect, where retrograde transport is disrupted. Over-expression of a FFAT peptide from OSBP1 reduces the size of VAPB-P56S aggregates and restores retrograde transport. A screen was performed on FFAT-motif containing ORPs to determine if any could rescue the mutant phenotype. ORP3 successfully reduced aggregate size and restored transport to the nuclear envelope. ER membrane protein Sac1, a PI4P phosphatase cycles between the ER and Golgi and becomes trapped in expanded ERGIC compartments with VAPB-P56S. Loss of Sac1 in the ER leads to an increase in intracellular PI4P. ORP3 may increase Sac1 phosphatase activity by acting as a lipid sensor. We propose that VAPB, Sac1 and ORP3 are interacting partners that together modulate levels of PI4P. Disruptions in the gradient of PI4P may result in the vesicle trafficking defects observed in VAPB-P56S cells.
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Wenham, Matt. "The role of Adaptor Protein 3 in cytotoxic T lymphocytes." Thesis, University of Oxford, 2009. http://ora.ox.ac.uk/objects/uuid:1a5c1fd6-c8cc-454f-81a5-b9a5a18c1540.
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Cytotoxic T lymphocytes (CTL) kill virally infected and tumourigenic cells via the regulated secretion of specialised secretory lysosomes. These secretory lysosomes contain cytolytic effector molecules, such as perforin and granzymes, which are able to induce apoptosis in target cells. Secretion occurs at the contact point between the CTL and its target, in a highly structured region termed the immunological synapse (IS). Upon formation of the IS, CTL undergo polarisation of their microtubule cytoskeleton and movement of the microtubule organising centre (MTOC) to the IS. Secretory lysosomes are then able to polarise along microtubules, fuse with the plasma membrane and deliver their effector molecules to the IS. The Adaptor Protein 3 complex (AP-3) sorts transmembrane proteins to lysosomes and deficiency in AP-3 results in missorting of proteins from the lysosomal to plasma membrane. CTL from AP-3 deficient patients, who suffer from Hermansky-Pudlak Syndrome Type 2 (HPS2), show reduced killing of target cells. This thesis describes two new patients with HPS2, both with homozygous mutations in the AP3B1 gene, which codes for the β3A subunit of the AP-3 complex. CTL from the new HPS2 patients show reduced cytotoxicity, which is shown here to be due to impaired secretory lysosome polarisation towards the IS. This impairment is common to HPS2 CTL, but varies between patients. In order to determine differences between HPS2 and wild type CTL, the localisation of a range of lysosomal, cytolytic, transmembrane, inhibitory and activation marker proteins is examined. This shows that in HPS2 CTL, LAMP1, CD63 and CD9 are potential AP-3 cargos. In addition, a possible effect on the key lytic effector perforin is identified. Preliminary experiments to allow proteomic comparison of HPS2 and wild type CTL are also presented. Further investigation of these results will help to shed light on the mechanisms involved in secretory lysosome polarisation in CTL.
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Lefnaier, Wafa. "Potential Role for the Sarcolemmal Membrane Associated Protein Isoform 3 (SLMAP3) in Cardiac Remodeling Post Myocardial Infarction." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/35713.
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ABSTRACT
The Sarcolemmal Membrane Associated Protein 3 (SLMAP3) is a tail-anchored membrane protein, which is ubiquitously expressed in tissues including myocardium. It is a component of subcellular membranes and the centrosome, and it appears to serve distinct roles in cell growth and membrane biology. In addition, mutations in SLMAP have been linked to Brugada syndrome, which leads to cardiac dysfunction and death. Here, we have examined the effects of different levels of SLMAP3 on postnatal heart function, pre and post myocardial infarction (MI).Transgenic (TG) mice with a cardiac specific expression of SLMAP3 isoform were generated and assessed with echocardiography to measure function, immunohistochemistry for histology, TUNEL assay for apoptosis, Masson’s trichrome staining for fibrosis, and Western blots for protein expression.
Baseline echocardiography of 8 weeks old TG mice showed a normal cardiac function that was expressed in ejection fraction percent (%EF=66%±7.42), which was similar to those of wild type mice (%EF=67%±9.36), p<0.05, n=20-25 (in each group). MI was induced by permanent ligation of left anterior descending (LAD) artery in 9 week old WT & TG mice, while sham was the control. No death was recorded in SLMAP3 TG mice up to one year post MI, whereas 70% of WT mice had deceased, p<0.01, n=17-18 (in each group). Cardiac function was assessed by echocardiography (at 4 week post MI) showed a partially restored ejection fraction percent (%EF~49.2%±17.02) in SLMAP3 TG mice post MI compared to (%EF~36.4%±15.25) in WT mice post MI, p<0.05, n=15-16 (in each group). Furthermore, infarct size (IS) as well as collagen area (CA) post MI were significantly attenuated (IS~43%±8.82, CA~35%±5.15) in SLMAP3 TG myocardium in comparison to WT (IS~53%±9.30, CA~47%±7.36), p<0.05, n=20- 22 (each group). Moreover, expression of the heart failure biomarker galectin3 was markedly
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attenuated (1.8±0.20) in SLMAP3 TG hearts post MI compared to (3.2±0.35) in WT, p<0.01, n=4-5 (in each group). The apoptotic index in SLMAP3 TG myocardium assessed by TUNEL was markedly decreased (77±11.48) in comparison to WT (112±15.32), p<0.05, (n=20-22 in each group). Further, expression of proapoptotic proteins (Caspase3 and Bax) was significantly attenuated in SLMAP3 TG (p<0.05, n=4-5 in each group) while the expression of the prosurvival proteins (Bcl2 and caveolin3) was significantly upregulated (p<0.05, n=4-5 (in each group) in post MI. These data indicate that increased SLMAP3 levels serve to protect myocardium post MI through mechanisms which promote cell survival and limit cardiac fibrosis. Strategies to increase SLMAP3 level in myocardium may provide new therapeutic options in the treatment of heart failure.
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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.
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Fujita, Koji. "Clostridium perfringens enterotoxin binds to the second extracellular loop of claudin-3, a tight junction integral membrane protein." Kyoto University, 2001. http://hdl.handle.net/2433/150516.
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Voss, Matthias. "Signal peptide peptidase-like 3 (SPPL3) is a type II membrane protein-selective sheddase that regulates cellular N-glycosylation." Diss., Ludwig-Maximilians-Universität München, 2014. http://nbn-resolving.de/urn:nbn:de:bvb:19-181243.
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Intramembrane proteolysis - hydrolysis of membrane proteins within or close to their membrane-spanning regions - is a crucial cellular process that is conserved throughout all kingdoms of life. It is executed by distinct classes of polytopic membrane proteins, the intramembrane-cleaving proteases, that provide a hydrophilic, proteinaceous environment accommodating membrane protein substrates as well as water molecules within the hydrophobic membrane interior and catalyse peptide bond hydrolysis. In particular, intramembrane-cleaving aspartyl proteases have received attention as the presenilins, the catalytic subunits of the γ-secretase complex, were identified as key players in Alzheimer's disease pathophysiology. In addition to presenilins, mammalian genomes harbour presenilin homologues which include signal peptide peptidase (SPP) and SPP-like (SPPL) proteases. Among these, the Golgi-resident protease SPPL3 stands out as it is highly conserved among metazoa and SPPL3 orthologues are also found in plants. However, due to the lack of known substrates, SPPL3 has thus far hardly been characterised. Hence, the purpose of this study was to identify its substrates and elucidate its physiological function(s).
In the first part of this study, the foamy virus envelope glycoprotein (FVenv) was identified as the first substrate of SPPL3. This allowed to study SPPL3's proteolytic activity in detail, with a focus on its substrate selectivity and sensitivity towards previously characterised inhibitors of intramembrane-cleaving aspartyl proteases. Importantly, this study revealed in addition that two other intramembrane-cleaving proteases, SPPL2a and SPPL2b, also endoproteolyse FVenv. SPPL2b in particular had been studied in detail before and therefore SPPL3- and SPPL2b- mediated endoproteolysis of FVenv were examined in parallel to directly compare these phylogenetically related intramembrane-cleaving proteases. This uncovered an unexpected idiosyncrasy of SPPL3 that clearly sets SPPL3 apart from other intramembrane-cleaving aspartyl proteases: SPPL3 endoproteolysed full-length FVenv and did not require the substrate's prior tailoring by another proteolytic activity - an otherwise common phenomenon among intramembrane-cleaving aspartyl proteases.
In the second part, the physiological function of SPPL3 was investigated. Alterations in the cellular levels of proteolytically active SPPL3 turned out to impact the composition of N-glycans attached to endogenous cellular glycoproteins. SPPL3 over-expression was accompanied by a decrease in glycoprotein molecular weight, i.e. a hypoglycosylation phenotype, while loss of SPPL3 expression in cell culture models but also in vivo resulted in a hyperglycosylation phenotype. This led to the identification of Golgi glycan-modifying enzymes such as GnT-V and β3GnT1 as novel physiological substrates of SPPL3. Loss or reduction of SPPL3 expression, for instance, led to a marked intracellular accumulation of these enzymes, explaining the more extensive N-glycan elaboration and the hyperglycosylation phenotype observed under these conditions. At the same time secretion of these enzymes was reduced under these conditions. Together with additional observations such as the mapping of the SPPL3 cleavage site to the membrane-spanning region of GnT-V, this study demonstrates that SPPL3-mediated intramembrane proteolysis of such glycan-modifying enzymes liberates their active site-harbouring ectodomains. Acting in this manner, SPPL3 controls the intracellular pool of active glycan-modifying enzymes.
Importantly, the finding that SPPL3 proteolytically cleaves full-length glycan-modifying enzymes and sheds their ectodomains is well in line with the observations made for FVenv and suggested that SPPL3 acts functionally equivalent to classical sheddases or rhomboid proteases but much unlike all other characterised mammalian intramembrane-cleaving aspartyl proteases. To examine whether these observations hold also true on a global cellular scale, a proteomic approach was undertaken in the third part of the study to define the SPPL3 degradome of HEK293 cells in conditions of SPPL3 over-expression. On the one hand, this led to the identification of numerous novel, mostly Golgi-resident candidate SPPL3 substrates and, considering the physiological implications, suggests that SPPL3 is very intricately linked to Golgi function. On the other hand, this approach supports the initial hypothesis that SPPL3 acts as a cellular type II membrane protein-selective sheddase.
Taken together, this study provides the first in-depth characterisation of the intramembrane protease SPPL3 and reveals the cellular function of SPPL3. SPPL3 displays considerable and marked differences to other intramembrane-cleaving aspartyl proteases and emerges as a fundamental cellular sheddase that exhibits strong selectivity for type II-oriented, Golgi-resident membrane proteins. Products of SPPL3-mediated endoproteolysis of these Golgi factors are secreted and/or may be subject to intracellular degradation which compromises their catalytic activity. Thus, SPPL3 indirectly controls protein glycosylation in the Golgi apparatus.Intramembranproteolyse, die hydrolytische Spaltung von Membranproteinen innerhalb ihrer Transmembrandomänen, ist ein wichtiger zellulärer Prozess, der in allen Reichen der Lebewesen konserviert ist. Die Intramembranproteolyse wird von unterschiedlichen Klassen von polytopen Membranproteinen, den Intramembranproteasen, ausgeführt, die eine hydrophile Proteinumgebung innerhalb des hydrophoben Bereichs der Membran bilden. In dieser Umgebung finden sowohl Membranproteinsubstrate als auch Wassermoleküle Platz und Hydrolyse der Peptidbindung wird durch die Intramembranproteasen katalysiert. Vor allem die Aspartylintramembranproteasen haben sehr viel Aufmerksamkeit auf sich gezogen, da die Preseniline, die katalytischen Untereinheiten des γ-Sekretasekomplexes, als Schlüsselenzyme in der Pathophysiologie der Alzheimer-Erkrankung identifiziert wurden. Neben den Presenilinen finden sich in Säugetiergenomen auch Presenilinhomologe, welche sowohl die Signalpeptidpeptidase (SPP) als auch SPP-ähnliche (SPP-like, SPPL) Proteasen umfassen. Aus diesen sticht die im Golgi lokalisierte Protease SPPL3 heraus, da sie in vielzelligen Tieren hochkonserviert ist und sich SPPL3-Orthologe auch in Pflanzen finden. Gleichzeitig ist SPPL3 jedoch, vor allem aufgrund der Tatsache, dass keine Substrate beschrieben wurden, bisher kaum charakterisiert. Daher war es das Ziel dieser Arbeit Substrate von SPPL3 zu identifizieren und seine physiologische Funktion(-en) aufzuklären. Im ersten Teil dieser Arbeit wurde ein erstes SPPL3-Substrat, das foamy-virale Hüllglykoprotein (FVenv), identifiziert. Dies erlaubte die proteolytische Aktivität von SPPL3 und insbesondere seine Substratselektivität sowie seine Sensitivität gegenüber Inhibitoren von Aspartylintramem- branproteasen im Detail zu untersuchen. Es konnte ebenfalls festgestellt werden, dass zwei weitere Intramembranproteasen, SPPL2a und SPPL2b, FVenv ebenfalls endoproteolytisch spalten. Vor allem SPPL2b war bereits zuvor im Detail untersucht worden und daher konnte die SPPL3- beziehungsweise SPPL2b-vermittelte Endoproteolyse von FVenv verglichen werden. Dies offenbarte eine unerwartete Eigenheit von SPPL3, welche SPPL3 deutlich von anderen Intramembranproteasen, vor allem den anderen Aspartylintramembraneproteasen, unterschiedet: SPPL3 spaltete das FVenv-Holoprotein und bedarf keinem vorherigen Zuschneiden des Substrates durch eine andere proteolytische Aktivität - ein sonst sehr verbreitetes Phänomen bei Intramembranproteasen. Im zweiten Teil sollte die physiologischen Funktion von SPPL3 untersucht werden. Dabei zeigte sich, dass Änderungen in der zellulären SPPL3-Aktivität große Auswirkungen auf die Zusammensetzung der N-Glykane auf endogenen zellulären Glykoproteinen haben. SPPL3- Überexpression ging mit einer Reduktion des Molekulargewichts untersuchter Glykoproteine, einem so genannten Hypoglykosylierungsphänotyp, einher, während der Verlust der SPPL3- Expression im Zellkulturmodell aber auch in vivo in einem Hyperglykosylierungsphänotyp resultierte. Dies führte zur Identifizierung von glykanmodifizierenden Enzymen im Golgi wie beispielsweise GnT-V und β3GnT1 als physiologische SPPL3-Substrate. Verlust oder Reduktion der SPPL3-Expression führte zu einer starken intrazellulären Akkumulation dieser Substrate, was die unter diesen Bedingungen beobachtete umfassendere N-Glykan- ausarbeitung sowie den Hyperglykosylierungsphänotyp erklärte. Gleichzeitig wurde die Sekretion dieser Enzyme unter diesen Bedingungen stark beeinträchtigt. Zusammen mit weiteren Beobachtungen wie der Bestimmung der SPPL3-Schnittstelle im Bereich der Transmembrandomäne von GnT-V zeigte diese Studie, dass die von SPPL3 vermittelte Intramembranproteolyse solcher glykan-modifizierenden Enzyme deren Ektodomainen freisetzt und SPPL3 folglich auf diese Weise das intrazelluläre Reservoir aktiver glykan-modifizierender Enzyme kontrolliert. Hervorzuheben ist dabei, dass diese Erkenntnis in guter Übereinstimmung mit den für FVenv gemachten Beobachtungen steht. Dies legt wiederum nahe, dass SPPL3 in funktioneller Hinsicht mit einer klassischen Sheddase oder einer Rhomboidprotease gleichgestellt ist, sich jedoch von allen anderen charakterisierten Aspartylintramembranproteasen in Säugetieren unterscheidet. Um herauszufinden, ob diese Beobachtungen auch auf globaler zellulärer Ebene Bestätigung finden, wurde eine Proteomanalyse im dritten Teil dieser Arbeit durchgeführt, um das SPPL3-Degradom in HEK293-Zellen unter Überexpressionsbedingungen zu definieren. Dies führte einerseits zur Identifizierung vieler weiterer neuer, zumeist im Golgi lokalisierter SPPL3-Substratkandidaten und legte andererseits unter physiologischen Gesichtspunkten nahe, dass SPPL3 sehr mit der Funktion des Golgi-Apparats verwoben ist. Andererseits unterstützten diese Ergebnisse sehr deutlich die formulierte Hypothese, dass SPPL3 als zelluläre Typ-II-Membranprotein-selektive Sheddase agiert. Zusammengefasst liefert diese Arbeit die erste ausführliche Charakterisierung der Intramem- branprotease SPPL3. SPPL3 zeigt erhebliche Unterschiede zu anderen Aspartylintramembran- proteasen und erweist sich damit als bedeutende zelluläre Sheddase mit einer auffälligen Selektivität für Typ-II-orientierte, im Golgi lokalisierte Membranproteine. Produkte der Endoproteolyse dieser Golgi-Proteine werden sekretiert und/oder werden möglicherweise intrazellulär degradiert, was wiederum ihre eigentliche intrazelluläre Funktion beeinträchtigt. Folglich kontrolliert SPPL3 indirekt die Proteinglykosylierung im Golgi-Apparat.
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Beuming, Thijs. "Structure/function studies of membrane proteins : from molecular modeling and ligand binding to protein-protein interactions of the dopamine transporter /." Access full-text from WCMC:, 2007. http://proquest.umi.com/pqdweb?did=1432803991&sid=3&Fmt=2&clientId=8424&RQT=309&VName=PQD.
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Kimura(Takemoto), Sayaka. "Molecular cloning and characterization of CLICK-3/CaMKIγ, a novel membrane-anchored neuronal Ca[2+]/calmodulin-dependent protein kinase (CaMK)." Kyoto University, 2003. http://hdl.handle.net/2433/148486.
Full textAbstract:
Kyoto University (京都大学)0048
新制・課程博士
博士(医学)
甲第10449号
医博第2648号
新制||医||844(附属図書館)
UT51-2003-T275
京都大学大学院医学研究科脳統御医科学系専攻
(主査)教授 月田 承一郎, 教授 中西 重忠, 教授 武藤 誠, 教授 成宮 周
学位規則第4条第1項該当
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Adhikari, Anirban. "Regulation of guanine nucelotide exchange in inhibitory G protein alpha subunit by activator of G protein signaling 3 and novel regulatory peptides." Embargoed access until after 12/19/2006, 2005. http://www4.utsouthwestern.edu/library/ETD/etdDetails.cfm?etdID=114.
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Braun, Anja Catharina [Verfasser], and Monilola [Akademischer Betreuer] Olayioye. "Regulation of endocytic membrane trafficking by the GTPase-activating protein Deleted in Liver Cancer 3 (DLC3) / Anja Catharina Braun. Betreuer: Monilola Olayioye." Stuttgart : Universitätsbibliothek der Universität Stuttgart, 2015. http://d-nb.info/1071089188/34.
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Voss, Matthias [Verfasser], and Christian [Akademischer Betreuer] Haass. "Signal peptide peptidase-like 3 (SPPL3) is a type II membrane protein-selective sheddase that regulates cellular N-glycosylation / Matthias Voss. Betreuer: Christian Haass." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2014. http://d-nb.info/1069743631/34.
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Ainsworth, Julia. "Comparison of p53 and MAGI-3 regulation mediated by the E6 protein from high-risk human papillomavirus types 18 and 33." Thesis, McGill University, 2007. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=112368.
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The HPV E6-p53 interaction is well-understood, but not for all high-risk HPV types. In addition, HPV E6 p53-independent functions are gaining recognition for their importance in cellular transformation but require clarification. Thus, the aim of this study was two-fold: (1) to gain insight into the p53-E6 interaction for high-risk HPV-33 and, (2) to explore how high-risk HPV E6 proteins targets cellular MAGI-3 for degradation.In vivo and in vitro results indicated that E6 from HPV types 18 and 33 interacted similarly with p53 although, variants of the HPV-33 E6 prototype demonstrated interesting disparities. Of note was HPV-33 E6 variant 2, which degraded p53 more efficiently than prototype HPV-33 E6 and HPV-18 E6. The E6 protein from HPV types 18 and 33 also potently degraded MAGI-3 via a different pathway than that used for p53. Specifically, proteasome inhibition did not interfere with MAGI-3 degradation and MAGI-3 was not ubiquitinated in the presence of the E6 protein.
Therefore, the results described herein enhance our understanding of high-risk HPV type 33 E6 and the E6-MAGI-3 interaction.
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Zamzami, Omar M. "Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) peptides as inducers of regulatory cells to treat autoimmune haemolytic anaemia." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources. Online version available for University member only until May, 2014, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=59565.
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Siebertz, Hanna Margarete [Verfasser], Stefan [Akademischer Betreuer] Gründer, and Gerhard [Akademischer Betreuer] Müller-Newen. "Approach to the structural basis of silencing of acid-sensing ion channel 3 by the integral membrane protein stomatin / Hanna Margarete Siebertz ; Stefan Gründer, Gerhard Müller-Newen." Aachen : Universitätsbibliothek der RWTH Aachen, 2019. http://d-nb.info/1195237464/34.
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Alves, Kelly Cristina Nascimento. "Contribuições ao estudo da não-idealidade de soluções proteicas." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/3/3137/tde-29072013-161832/.
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O estudo de soluções proteicas visando à modelagem e à simulação de processos de recuperação e purificação de bioprodutos passa necessariamente pelo estudo da não idealidade, em sentido termodinâmico, destas soluções. Para sistemas em que a concentração de proteína seja baixa, situação comumente presente nestes processos, a principal maneira de avaliar experimentalmente a não idealidade é por meio da determinação da pressão osmótica gerada pela proteína. Deste modo, os objetivos deste trabalho foram: estudar a influência de co-solventes na pressão osmótica de soluções proteicas, verificar a integridade das estruturas secundária e terciária das proteínas nessas soluções, e modelar termodinamicamente os dados de pressão osmótica obtidos. A pressão osmótica foi determinada diretamente por osmometria de membrana, usando soluções de referência com mesma concentração de co-solvente e pH, mas isentas de proteínas. Obtiveram-se dados de pressão osmótica, em função da concentração proteica, de cinco diferentes proteínas (albumina de soro bovino, imunoglobulina G humana, ovalbumina, -lactoglobulina e lisozima) em soluções contendo co-solventes como o polietileno glicol (de diversos tamanhos de cadeia) e sais (sulfato de amônio, sulfato de sódio e cloreto de sódio). Cada conjunto de dados foi obtido em pH e concentração de co-solvente constantes. Observou-se que a presença de co-solventes altera a pressão osmótica, mas este efeito é dependente da proteína, do co-solvente e sua concentração, e do pH da solução. Medidas de fluorescência e de dicroísmo circular das mesmas proteínas permitiram confirmar que elas mantêm sua integridade estrutural nesses meios, o que justifica o uso de equações volumétricas de estado com parâmetros constantes. Os dados de pressão osmótica em função da concentração proteica foram correlacionados por meio de uma equação volumétrica de estado, que combina um termo de esferas rígidas adesivas e um termo de perturbação de ordem zero (aproximação aleatória). O modelo proposto, embora simples, foi suficiente para correlacionar adequadamente o comportamento experimental.The study of protein solutions aiming at the modeling and simulation of downstream processes entails the study of non-ideality (in thermodynamic sense) of these solutions. For systems wherein the protein concentration is low a situation often encountered in these processes the most important technique to experimentally evaluate this non-ideality is the determination of the osmotic pressure generated by the protein in solution. Thus, the objectives of this work were: to study the influence of co-solvents on the osmotic pressure of protein solutions, to verify the absence of change in the secondary and tertiary structure of proteins in these solutions, and to thermodynamically model the obtained osmotic pressure data. The osmotic pressure was directly measured through membrane osmometry, using protein-free reference solutions with the same pH and co-solvent concentration. The data of osmotic pressure were obtained as a function of protein concentration for five different proteins (bovine serum albumin, human immunoglobulin G, ovalbumin, - lactoglobulin and lysozyme) in solution with co-solvents such as polyethylene glycol (of various chain sizes) and salts (ammonium sulfate, sodium sulfate and sodium chloride). Each data set was obtained at constant pH and co-solvent concentration. It was observed that the presence of co-solvents do shift the osmotic pressure, but this effect is dependent on the protein, the co-solvent and its concentration, and the solution pH. Measurements of fluorescence and circular dichroism of these proteins confirmed that they maintain their structure unchanged in the media, which corroborates the use of volumetric equations of state with constant parameters. The osmotic pressure data as a function of protein concentration were correlated using a osmotic equation of state comprising a repulsive term of adhesive hard spheres and a zero-order perturbation term (random approximation). The proposed model, though simple, was sufficient to properly correlate the experimental behavior.
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Hallstrom, Kelly N. "The Epithelial Transmembrane Protein PERP Is Required for Inflammatory Responses to S. typhimurium Infection: A Dissertation." eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/807.
Full textAbstract:
Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea.
Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity.
In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation.
Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.
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Hallstrom, Kelly N. "The Epithelial Transmembrane Protein PERP Is Required for Inflammatory Responses to S. typhimurium Infection: A Dissertation." eScholarship@UMMS, 2010. http://escholarship.umassmed.edu/gsbs_diss/807.
Full textAbstract:
Salmonella enterica subtype Typhimurium (S. Typhimurium) is one of many non-typhoidal Salmonella enterica strains responsible for over one million cases of salmonellosis in the United States each year. These Salmonella strains are also a leading cause of diarrheal disease in developing countries. Nontyphoidal salmonellosis induces gastrointestinal distress that is characterized histopathologically by an influx of polymorphonuclear leukocytes (PMNs), the non-specific effects of which lead to tissue damage and contribute to diarrhea.
Prior studies from our lab have demonstrated that the type III secreted bacterial effector SipA is a key regulator of PMN influx during S. Typhimurium infection and that its activity requires processing by caspase-3. Although we established caspase-3 activity is required for the activation of inflammatory pathways during S. Typhimurium infection, the mechanisms by which caspase-3 is activated remain incompletely understood. Most challenging is the fact that SipA is responsible for activating caspase-3, which begs the question of how SipA can activate an enzyme it requires for its own activity.
In the present study, we describe our findings that the eukaryotic tetraspanning membrane protein PERP is required for the S. Typhimuriuminduced influx of PMNs. We further show that S. Typhimurium infection induces PERP accumulation at the apical surface of polarized colonic epithelial cells, and that this accumulation requires SipA. Strikingly, PERP accumulation occurs in the absence of caspase-3 processing of SipA, which is the first time we have shown SipA mediates a cellular event without first requiring caspase-3 processing. Previous work demonstrates that PERP mediates the activation of caspase-3, and we find that PERP is required for Salmonella-induced caspase-3 activation.
Our combined data support a model in which SipA triggers caspase-3 activation via its cellular modulation of PERP. Since SipA can set this pathway in motion without being cleaved by caspase-3, we propose that PERP-mediated caspase-3 activation is required for the activation of SipA, and thus is a key step in the inflammatory response to S. Typhimurium infection. Our findings further our understanding of how SipA induces inflammation during S. Typhimurium infection, and also provide additional insight into how type III secreted effectors manipulate host cells.
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Di, Guilmi Anne-Marie. "Étude de l'interaction de l'adénovirus humain de sérotype 3 avec les cellules HeLa." Université Joseph Fourier (Grenoble ; 1971-2015), 1994. http://www.theses.fr/1994GRE10058.
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Les travaux decrits dans ce rapport concernent l'interaction de l'adenovirus humain de serotype 3 avec les cellules hela. Dans un premier temps, nous avons defini les caracteristiques de la liaison du virus ad3 avec les cellules hela a 37c et a 4c. Pour ces deux conditions de temperature, nous avons montre que le virus ad3 a la propriete de se lier avec deux affinites distinctes sur ses sites recepteurs. Nous avons demontre que les virus ad2 et ad3 (appartenant respectivement aux sous-groupes c et b) ne partagent pas les memes sites de liaison a la surface des cellules hela. La fibre est la proteine virale dont la fonction est de se lier au recepteur. Nous avons produit cette proteine dans le systeme de baculovirus. La fibre ad3 recombinante presente une morphologie et une structure correctes. L'activite fonctionnelle de la fibre a ete mise en evidence par l'efficacite de l'inhibition de la fixation du virus ad3, a la surface des cellules hela. Dans le but d'identifier le recepteur cellulaire de l'ad3 nous avons applique la technique du vopba en utilisant la fibre ad3 recombinante et le virus ad3. A partir des cellules hela, le virus et la fibre ad3 ont detecte une proteine membranaire de 130kda avec laquelle ils interagissent. Cette reconnaissance presente un caractere specifique. Des travaux similaires effectues sur des cellules a549, permissives a l'infection par le virus ad3, n'ont cependant pas permis de detecter cette proteine 130
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Hierso, Régine. "Implication du stress oxydant dans la physiopathologie de la drépanocytose : crises vaso-occlusives, taux d'anticorps anti-bande 3 et oxydation du globule rouge." Thesis, Antilles, 2015. http://www.theses.fr/2015ANTI0045/document.
Full textAbstract:
A partir du défaut premier de la drépanocytose, la polymérisation de l’hémoglobine S (HbS), se développe toute une série de processus anormaux qui contribuent au développement d’une réponse inflammatoire et d’un stress oxydant dus à une hypoxie-reperfusion traumatisante et à l’auto-oxydation de l’HbS. L’exacerbation du stress oxydatif semble participer de manière active aux mécanismes physiopathologiques de la maladie et jouer un rôle dans la survenue des crises vaso-occlusives (CVO). Les travaux menés dans le cadre de cette thèse avaient pour objectif de mieux documenter les effets délétères du stress oxydant sur le globule rouge et son impact dans le développement des CVO. Nous avons, en premier lieu, évalué in vitro l’impact du stress oxydant sur la rhéologie sanguine des patients drépanocytaires SS et SC à l’aide d’un agent à fort potentiel oxydant, le t-butyl hydroperoxide (TBHP). Nous avons montré que les globules rouges des patients drépanocytaires (GR SS) produisent en présence du TBHP davantage de radicaux libres que les GR provenant de sujets contrôles (GR AA) et que ces GR SS présentent un système de défense anti-oxydant diminué. L’induction d’un stress oxydant accentue les altérations rhéologiques déjà présentes chez les patients drépanocytaires (i.e, déformabilité diminuée, diminution de l’indice d’agrégation, augmentation du seuil de désagrégation) tandis qu’il induit chez les sujets contrôles un profil hémorhéologique altéré proche de celui déjà préexistant chez les patients drépanocytaires. Ces résultats suggèrent que le stress oxydant, en participant aux anomalies hémorhéologiques associées à la drépanocytose, pourrait être l’un des facteurs favorisant la survenue des complications drépanocytaires. Nous nous sommes de plus attachés à documenter directement l’impact du stress oxydant dans le développement des CVO en analysant des prélèvements sanguins provenant de patients drépanocytaires SS en crise et à l’état de base. Il s’agissait : 1) de tester l’hypothèse selon laquelle la protéine bande 3, protéine de la membrane du GR, est une cible majeure des espèces réactives de l’oxygène qui provoquent au niveau de cette protéine l’apparition d’épitopes de senescence reconnus par des auto anticorps anti-bande 3 ; 2) d’évaluer l’évolution de marqueurs moléculaires et cellulaires pro- et anti-oxydants ; 3) d’étudier l’évolution des paramètres hémorhéologiques ; 4) d’explorer l’activité du système nerveux autonome, considéré comme un marqueur potentiel de sévérité. Nous avons mis en évidence au cours des CVO : 1) une exacerbation du stress oxydant ; 2) une diminution du taux d’anticorps anti-bande 3 et une augmentation de la concentration plasmatique de microparticules érythrocytaires, suggérant que ces deux processus sont liés au phénomène de clusterisation de la protéine bande 3 déclenché par le stress oxydant ; 3) une exacerbation des anomalies hémorhéologiques se traduisant par une réduction de la déformabilité érythrocytaire, une augmentation de l’agrégation érythrocytaire et du seuil de désagrégation ; 4) une altération du système nerveux autonome marqué par un retrait de l’activité parasympathique, ce déséquilibre étant accentué au cours des CVO. Les travaux de cette thèse se traduisent par à une meilleure compréhension de la physiopathologie extrêmement complexe de la drépanocytose en précisant l’impact du stress oxydant dans le déclenchement des CVO, première cause d’hospitalisation des sujets drépanocytaires. Les données obtenues, qui mettent en évidence des marqueurs pertinents de ce stress oxydant au cours de la CVO du sujet drépanocytaire, pourront favoriser la mise en œuvre de nouvelles pistes thérapeutiques anti-oxydantes et une amélioration in fine de la prise en charge des patientsBesides the primary defect of sickle cell disease, hemoglobin S (HbS) polymerization, other abnormal processes may contribute to the development of an inflammatory response and to an oxidative stress caused by traumatic hypoxia-reperfusion and autoxidation of HbS. The exacerbation of oxidative stress seems to participate actively in the pathophysiology of the disease and play a role in vaso-occlusive crisis (VOC). The aim of this thesis was to document the deleterious effects of oxidative stress on the red blood cell and its impact in the development of VOC. First, we have evaluated the impact of tert-butyl hydroperoxide-induced oxidative stress on blood rheology of SS and SC sickle cell patients. We have shown that sickle red blood cells (SS RBC) produce more free radicals in the presence of tert-butyl hydroperoxide (TBHP) than control subject red blood cells (AA RBC). Furthermore, SS RBC have a decreased anti-oxidant defense system. Induction of oxidative stress increases the rheological alterations already present in sickle cell patients (ie, decreased deformability, reduced aggregation, increased disaggregation threshold). In control subjects, oxidative stress induces an altered hemorheological profile close to that already present in sickle cell patients. These results suggest that oxidative stress by modulating the hemorheological abnormalities associated with sickle cell disease, could be one of the factors promoting the occurrence of sickle cell complications. Then, we have studied the impact of oxidative stress in the development of VOC, analyzing blood samples from SS patients in crisis and at steady state. 1) We have tested the hypothesis that the protein band 3 of RBC, is a major target of reactive oxygen species, which cause the appearance of senescence epitopes of this protein recognized by auto anti-band 3 antibodies; 2) We have evaluated pro- and anti-oxidants molecular and cellular markers; 3) We have studied the evolution of hemorheological parameters; 4) We have explored the activity of the autonomic nervous system, seen as a potential marker of severity. Our results show during VOC: 1) an exacerbation of the oxidative stress; 2) a decrease in anti-band 3 antibodies levels and an increase in the plasma concentration of erythrocyte microparticles, suggesting that these two processes are linked to the clustering phenomenon of band 3 protein triggered by oxidative stress; 3) an exacerbation of hemorheological abnormalities resulting in a reduction of SS RBC deformability, increased aggregation and disaggregation threshold; 4) an impairment of the autonomic nervous system marked by a withdrawal of parasympathetic activity and this imbalance is accentuated during VOC. This work allows a better understanding of the complex pathophysiology of sickle cell disease, highlighting the impact of oxidative stress in the development of VOC, the leading cause of hospitalization of sickle cell subjects. The data obtained, which reveal relevant markers of oxidative stress during VOC, could promote the implementation of new antioxidant therapeutic approaches and help improving sickle cell patients care
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Raphael, Mariana Lopes Teixeira. "Estudo da imunogenicidade da proteína de classe 3 (PorB) purificada da membrana externa de Neisseria miningitidis: imunização intranasal/intramuscular em camundongos adultos e neonatos utilizando Bordetella pertussis como adjuvante." Universidade de São Paulo, 2008. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-13012009-172350/.
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As proteínas de classe 3 são candidatas na preparação de uma vacina contra a doença meningocócica. O objetivo deste estudo é determinar a imunogenicidade da proteína de classe 3 purificada da cepa de Neisseria meningitidis do sorogrupo B juntamente com a capacidade adjuvante de whole cells de Bordetella pertussis. Foram imunizados camundongos BALB/c neonatos em um intervalo de 3 a 12 dias entre 1 e 4 doses da proteína de classe 3 mais adjuvante, pela via intranasal e no 21º dia pela via intramuscular com a proteína de classe 3 emulsificada com hidróxido de alumínio. Os resultados demonstraram que após 2 doses pela via intranasal e 1 dose pela via intramuscular houve rápido estímulo das células imunes nos camundongos adultos BALB/c e neonatos BALB/c e outbred. Todos os soros foram analisados por ELISA e immunoblot. O adjuvante B. pertussis administrado pelas vias intranasal ou intramuscular, aumentou a resposta imune comparada com os controles. Anticorpos bactericidas e de alta afinidade foram produzidos.Proteins of class 3 sound candidates in the preparation of vaccine against meningococcal illness. The aim of this study was to determine the immunogenicity of class 3 proteins purified of Neisseria meningitidis of the serogroup B along with whole cells of Bordetella pertussis as adjuvant. BALB/c and outbred neonate mice between 3 and 12 days old were immunized with 1 to 4 doses of the purified class 3 proteins with or without adjuvant given by the intranasal route, and on the 21st day the animals received an intramuscular dose of the class 3 proteins with or without aluminum hydroxide. The results demonstrated that after 2 doses by the intranasal route and 1 dose intramuscular there was a rapid stimulation of the immune cells in BALB/c adult mice as well as BALB/c and outbred neonates mice. All sera were analyzed by ELISA and immunoblot. The adjuvant B. pertussis used in the present investigation and given via the intranasal or intramuscular route increased the immune response compared with the controls. High affinity and bactericidal antibodies were produced.
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Correia, Patrícia Maria Dias. "Identification and characterization of potential therapeutic targets for spinal cord repair." Master's thesis, Universidade de Aveiro, 2017. http://hdl.handle.net/10773/22055.
Full textAbstract:
Mestrado em Biomedicina MolecularTraumatic spinal cord injury (SCI) is a devastating event that leads to loss of neurological functions below the vertebral level of the lesion. As adult neurons from central nervous system (CNS) fail to regenerate when injured, the consequences of SCI are partially or totally irreversible. The lack of regeneration ability of CNS neurons has been studied for years but still no effective treatment was found for this pathology; only steroids are validated and recognized as a pharmacologic treatment attempt, but just limit the lesion extent. This work focused on finding putative candidate genes involved in regeneration that could be targeted for therapy. A bioinformatics analysis based on studies with rodent SCI models, where a regenerative treatment attempt was applied and functional recovery was observed, was performed and some common regulated genes were found in the analysed studies. KIF4A and MPP3 genes were highlighted for further experimental studies in a regenerative model: a rodent model of peripheral nervous system (PNS) injury, with crush or transection of the sciatic nerve. Our results demonstrated that KIF4A and MPP3 are expressed and regulated in the lesioned sciatic nerve and in the corresponding dorsal root ganglia (DRG). Moreover, these genes also showed protein distribution in spinal cord tissue sections, in sciatic nerve and in DRG cuts, revealing that they are neuronal specific. These results represent important remarks to instigate further studies regarding the role of these genes in regenerative processes of lesioned neuronal tissues and the possibility of becoming important therapeutic targets in spinal cord injuries or related pathologies affecting the spinal cord integrity
A lesão traumática da medula espinal é um evento devastador que leva à perda de funções neurológicas abaixo do nível vertebral da lesão. Devido à falta de capacidade regenerativa dos neurónios adultos do sistema nervoso central, quando lesionados, as consequências das lesões são parcial ou totalmente irreversíveis. A falta de capacidade de regeneração dos neurónios do SNC tem sido estudada há anos, mas ainda não foi encontrado um tratamento efetivo para esta patologia; apenas os esteroides são validados e reconhecidos como um tratamento farmacológico, mas só limitam a extensão da lesão. Este trabalho centrou-se na procura de genes hipoteticamente envolvidos em regeneração do sistema nervoso, que possam ser candidatos a alvos de terapia para lesões na medula. Foi realizada uma análise bioinformática baseada em estudos com modelos de roedores com lesão da medula espinal, onde uma tentativa de tratamento regenerativo foi aplicada e observou-se recuperação funcional, e foram levantados os genes regulados comuns aos três estudos. Os genes KIF4A e MPP3 foram destacados para estudos experimentais adicionais num modelo regenerativo: um modelo de roedor, de lesão do sistema nervoso periférico, com esmagamento ou corte do nervo ciático. Os resultados demonstraram que os genes KIF4A e MPP3 são expressos e regulados no nervo ciático lesionado e nos gânglios da raiz dorsal correspondentes. Além disso, estes genes também mostraram distribuição proteica em secções de tecido de medula espinhal, de nervo ciático e em cortes de DRG, desvendando que possam ser específicos de tecido neuronal. Estes resultados representam observações importantes para instigar estudos adicionais sobre o papel destes genes nos processos regenerativos de tecidos neuronais lesionados e a possibilidade de se tornarem alvos terapêuticos importantes para lesões ou patologias relacionadas que afetem a integridade da medula espinal.
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Vallet, Lara Dominique. "The Role of Erythrocyte Membrane Proteins in Haemolytic Anaemias in South African Populations." Thesis, 2006. http://hdl.handle.net/10539/1794.
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Faculty of Science
School of Pathology(Molecular Medicine and Haematology).
9707563v
tridium@acenet.co.zaThe erythrocyte carries gases between the cells and the lungs, and has to distort to negotiate narrow splenic sinuses and capillaries. This distortion necessitates a high surface area to volume ratio that is maintained by the erythrocyte membrane skeleton, a network of proteins including spectrin and protein 4.1. The skeleton anchors the lipid bilayer through attachment to integral membrane proteins, notably the anion exchange protein, band 3. Abnormalities of the erythrocyte membrane proteins cause loss of cell elasticity and ultimately the erythrocytes become prematurely trapped in the spleen where they are phagocytosed, resulting in haemolytic anaemia. Three mutations causing band 3-deficient hereditary spherocytosis (HS), a haemolytic anaemia characterised by spherical erythrocytes, were located using restriction enzyme analysis and DNA sequencing. Proband A (Black) is heterozygous for band 3 Pinhal (R490H) and has mild clinical symptoms. Proband B and his mother (Caucasian) are heterozygous for band 3 Bicetre (R490C) and have severe anaemia requiring transfusions and splenectomy, respectively. These results confirm codon 490 as a hotspot for mutations and indicate the effect of different amino acid substitutions in the same position on clinical severity. Proband C (Black) is homozygous for a novel mutation (E508K) for which her parents are heterozygous. The proband is severely affected and transfusion- dependent whereas her father has moderate anaemia and her mother is asymptomatic. It is speculated that a secondary factor modulates their clinical symptoms. All of these mutations occur in a CpG dinucleotide, a common source of DNA mutations, and are located within the highly conserved exon 13, which encodes the third to fifth α-helices and the second extracellular loop of the transmembrane region of band 3. The mutations are likely to alter the conformation of band 3, impairing its insertion into the erythrocyte membrane. No causative mutations were located in another 12 band 3-deficient HS kindred using restriction enzymes and single strand conformation polymorphism analysis. Ten protein 4.1-deficient patients with hereditary elliptocytosis, a haemolytic anaemia characterised by elliptical erythrocytes, were also studied. Immunoblot analyses ruled out abnormally sized protein 4.1 and three known DNA mutations were excluded using restriction enzyme analysis. Further studies are required to elucidate the cause of the haemolytic anaemia in these kindred. This study advanced our knowledge of the molecular basis of HS in South African kindred and highlighted the susceptibility of CpG dinucleotides to mutations.
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Vogelaar, Nancy Swick. "Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor." Thesis, 1989. https://thesis.library.caltech.edu/7948/1/Vogelaar%201989.pdf.
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Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.
Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.
The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.
Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.
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Tang, Meng-Ju, and 湯孟儒. "Role of Epithelial Membrane Protein 3 inHuman Hepatocellular Carcinoma Cells." Thesis, 2013. http://ndltd.ncl.edu.tw/handle/87941803841473005612.
Full textAbstract:
碩士中山醫學大學
生化暨生物科技研究所
101
The epithelial membrane proteins-3 (EMP-3) comprise a subfamily of small hydrophobic membrane proteins. The putative four-transmembrane domain structure as well as the genomic structure is highly conserved among family members. EMP3 were mapped to chromosomes 19q13.3. EMP3 is expressed in many tissues, and functions in cell growth, differentiation, invasion and apoptosis have been reported. However, its involvement in human hepatocellular carcinoma (HCC) progression remains unclear. In our study, we found that EMP3 protein expression is higher in poor differentiated HA22T/VGH and SK-Hep-1 cells than in moderate and well differentiated Huh-7 and HepG2 cells, and HCC cancer tissue correlated significantly with tumor differentiation (P < 0.02). To verify the biological function of EMP3 in HCC cells, we found that Lentivirus-mediated EMP3 interference inhibited growth as assessed by the MTT assay. Likewise, inhibition of EMP3 expression induced cell-cycle arrest in SK-Hep-1 and Huh-7 cells by up-regulating the expression of p21Cip1 and p27Kip1, down-regulating the expression of Cyclin D1、Cyclin E and Skp2 protein expressions. Knockdown of EMP3 decreased migration and invasion in SK-Hep-1 and Huh-7 cells, and down-regulation of MMP9 and uPA. We examined the effect of down-regulation of EMP3 on the activation of ERK1/2 and Akt signaling. Western blot analysis showed that the expression of phosphorylated Akt was significantly decreased in EMP3 knockdown cells. Moreover, overexpression of Akt abolished EMP3-mediated cell migration and invasion. Conversely, inhibition of Akt phosphorylation remarkably reduced the migration and invasion of HCC cells with decreased expression of EMP3. We also found that EMP3 interacted with p85 (regulatory subunit of phosphoinositide 3-kinase [PI3K]) by immunoprecipitation and immunofluorescence assay. Conversely, inhibition of endogenous EMP3 by shRNA significantly reduced the interaction of p85 in SK-Hep-1 cells. In agreement with these findings, Moreover, EMP3 expression positively correlates with p85 (R=0.3851, P <0.01)、p-Akt (R=0.3789, P< 0.01)、MMP9 (R=0.3697, P < 0.02) and uPA (R=0.4986, P < 0.001) in tumor tissues from patients with HCC. From literature showed that the constitutive overexpression of EMP3 in HEK-293 cells led to cell blebbing and cell death, therefore, we found that P2X7R protein expression is higher in poor differentiated HA22T/VGH and SK-Hep-1 cells than in moderate and well differentiated Huh-7 and HepG2 cells. In addition, immunoprecipitation and immunofluorescence analysis revealed that EMP3 associated with P2X7R. Conversely, EMP3 depletion reduced the P2X7 expression. Collectively, our results show that overexpression of EMP3 promotes human HCC progression by enhancing cell proliferation and invasiveness. Therefore, EMP3 is a master regulator of human HCC growth and might be a useful molecular target for HCC prognosis and treatment.
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Etzkorn, Manuel. "Protein precipitates, aggregation kinetics and membrane protein receptors characterized by solid-state NMR." Doctoral thesis, 2008. http://hdl.handle.net/11858/00-1735-0000-0006-B647-3.
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Brizida, Carolina Almeida Lavado. "Interaction between a Chlamydia trachomatis inclusion membrane protein (Inc) and host cell 14-3-3 proteins." Master's thesis, 2020. http://hdl.handle.net/10362/113711.
Full textAbstract:
Chlamydia trachomatis is an obligate intracellular bacterium that causes genital and ocular infections
in humans. Its multiplication in host cells occurs exclusively inside a membrane-bound compartment,
known as inclusion. To enter, multiply and exit host cells, C. trachomatis transports proteins through a
type III secretion system into the host cell. These proteins include Incs, which insert into the inclusion
membrane, modifying it, and manipulating the host cell in various ways. In this work, the interaction of
an Inc protein (CT006) from C. trachomatis with 14-3-3 proteins of the host cell was studied. There are
seven isoforms of 14-3-3 proteins known in mammals that in general have the capacity to bind to several
signaling proteins, being able to regulate several cellular processes. It was observed that all 14-4-3
isoforms (β/ε/η/γ/σ/τ/ζ) are recruited to the periphery of the inclusion membrane, probably in an Inc
CT006-independent-manner, but only 14-3-3β/η/γ/σ interact with Inc CT006. By analyzing the primary
structure of Inc CT006 it was realized that although the protein has potential 14-3-3 binding motifs, none
seems to be exclusively necessary for the interaction. However, any one of the motifs when present
and active can promote the interaction. 14-3-3 proteins are also thought to interact with Inc CT006
through their conserved binding groove. In summary, this work supports the idea that 14-3-3 proteins
have a relevant role in the intracellular multiplication of C. trachomatis. Future studies with C.
trachomatis with the gene encoding Inc CT006 inactivated or overexpressed, and/or with host cell with
reduced levels of 14-3-3 proteins, might clarify how the interaction of Inc CT006 with 14-3-3 proteins
contribute to the intracellular multiplication of C. trachomatis.
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Aoh, Quyen Le. "Regulation of epidermal growth factor receptor trafficking by secretory carrier membrane protein 3." 2009. http://proquest.umi.com/pqdweb?did=1801419091&sid=2&Fmt=2&clientId=3507&RQT=309&VName=PQD.
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James, Christina. "Analysis of the Interactome and Membrane Insertion of VAPB, a Tail- Anchored Protein at the Inner Nuclear Membrane." Doctoral thesis, 2020. http://hdl.handle.net/21.11130/00-1735-0000-0005-13F3-3.
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Chang, Shu-Hui, and 張淑慧. "Molecular mechanism of EBV latent membrane protein-1 induced inhibition of tissue inhibitor of metalloproteinase-3." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/86392507708066462674.
Full textAbstract:
碩士高雄醫學大學
公共衛生學研究所碩士班
94
Epstein-Barr virus is a prototype gamma herpes virus which is widely spreaded infection in the adults. EBV has been implicated in the pathogenesis of several human malignancies such as Burkitt’s lymphoma, Hodgkin’s disease and nasopharyngeal carcinoma (NPC). Latent membrane protein 1 (LMP-1) is the oncoprotein of EBV associated NPC. Some evidences showed that LMP-1 enhanced the MMP-9 expression in the EBV-infected human epithelial cell lines C33A. In addition, numerous studies indicated that many NPC patients have lymph-node metastasis. In the physical condition, MMPs are regulated by natural inhibitor called tissue inhibitor of metalloproteinases (TIMPs). Until now, there are four kinds of TIMPs: TIMP-1, TIMP-2, TIMP-3 and TIMP-4. The biological function of TIMP-3 is different from the others. First, it could inhibit ADAM-17 (a disintergrin and metaloproteinases) , ADAMT-4 and ADAMT-5 (ADAM with thrombospondin domain). TIMP-3 could inhibit TNF-α release from the cells and regulate cellular inflammation. Second, TIMP-3 could promote cancer cells to undergo apoptosis. Third, TIMP-3 could inhibit the metastasis and angiogenesis of cancer cells. Fourth, TIMP-3 played a key role in the inhibition of tumor growth. Some evidences showed that the TIMP-3 promoter was hypermethylated in the tumor, and its expression was significantly down-regulated in tumors.The goal of our study is to elucidate the molecular mechanism by which LMP-1 regulates TIMP-3 in the NPC cell line TW04. Our results showed that TIMP-3 was decreased in LMP-1-expressing TW04 cells. Promoter activity assays indicated that the -44 / +28 region of TIMP-3 promoter was regulated by LMP-1. By using different kinds of protein kinase inhibitors, it was showed that the TIMP-3 mRNA level was restored after treatment with p38 kinase inhibitor SB203580. We found that the LMP-1 induced TIMP-3 inhibition could increase the migration and invasion of TW04 cells. While co-expression with TIMP-3 attenuated LMP-1-evoked the migration and invasion. According to our results, we suggest that LMP-1 might increase the cell migration and invasion through p38 mediated down-regulation of TIMP-3.
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Wu, Shih-Jung, and 鄔式絨. "Outer membrane protein, OprI/OprF, modulates the susceptibility of Pseudomonas aeruginosa to cationic peptide, (R3S1)3." Thesis, 2008. http://ndltd.ncl.edu.tw/handle/40169852136839280541.
Full textAbstract:
碩士國立臺灣大學
微生物學研究所
96
Short cationic antimicrobial peptides are widely present in living organisms for innate defense against invading microorganisms. They are generally amphipathic, small and cationic with at least two positive residues. We demonstrated a synthetic cationic peptide, (R3S1)3, possessing antimicrobial activity, especially against Pseudomonas aeruginosa. Our results showed that the outer membrane proteins of P. aeruginosa, OprI and OprF, interact with the peptide. The antimicrobial activity of (R3S1)3 were repressed by excess amounts of rOprI, rOprF, anti-OprI or anti-OprF. The bacterial membrane became permeable, the chromatin condensed in cytosol and blebs formed on outer membrane of bacteria after (R3S1)3 treatment analyzed by TEM. In addition, the (R3S1)3 translocated across the cytoplasmic membrane, localized in the cytosol of P. aeruginosa and bound to intracellular target, like nucleic acids, analyzed by immunohistochemistry. The (R3S1)3 exerted not only antimicrobial activity but also penetrated eukaryotic cell and nuclear membranes. These action mechanism of antimicrobial peptide (R3S1)3 against P. aeruginosa through the bacterial outer membrane proteins, OprI/OprF, provide important clues for development of new antimicrobial agents.