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Articles de revues sur le sujet "LPS- binding proteins"
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Fenton, Matthew J., et Douglas T. Golenbock. « LPS-binding proteins and receptors ». Journal of Leukocyte Biology 64, no 1 (juillet 1998) : 25–32. http://dx.doi.org/10.1002/jlb.64.1.25.
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Hailman, E., H. S. Lichenstein, M. M. Wurfel, D. S. Miller, D. A. Johnson, M. Kelley, L. A. Busse, M. M. Zukowski et S. D. Wright. « Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. » Journal of Experimental Medicine 179, no 1 (1 janvier 1994) : 269–77. http://dx.doi.org/10.1084/jem.179.1.269.
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CD14 is a 55-kD protein found as a glycosylphosphatidylinositol (GPI)-anchored protein on the surface of monocytes, macrophages, and polymorphonuclear leukocytes, and as a soluble protein in the blood. Both forms of CD14 participate in the serum-dependent responses of cells to bacterial lipopolysaccharide (LPS). While CD14 has been described as a receptor for complexes of LPS with LPS-binding protein (LBP), there has been no direct evidence showing whether a ternary complex of LPS, LBP, and CD14 is formed, or whether CD14 binds LPS directly. Using nondenaturing polyacrylamide gel electrophoresis (native PAGE), we show that recombinant soluble CD14 (rsCD14) binds LPS in the absence of LBP or other proteins. Binding of LPS to CD14 is stable and of low stoichiometry (one or two molecules of LPS per rsCD14). Recombinant LBP (rLBP) does not form detectable ternary complexes with rsCD14 and LPS, but it does accelerate the binding of LPS to rsCD14. rLBP facilitates the interaction of LPS with rsCD14 at substoichiometric concentrations, suggesting that LBP functions catalytically, as a lipid transfer protein. Complexes of LPS and rsCD14 formed in the absence of LBP or other serum proteins strongly stimulate integrin function on PMN and expression of E-selectin on endothelial cells, demonstrating that LBP is not necessary for CD14-dependent stimulation of cells. These results suggest that CD14 acts as a soluble and cell surface receptor for LPS, and that LBP may function primarily to accelerate the binding of LPS to CD14.
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Qureshi, N., P. Y. Perera, J. Shen, J. J. Gao, A. Dhar, S. N. Vogel et D. C. Morrison. « NOVEL LPS-BINDING PROTEINS IN MURINE MACROPHAGES. » Shock 17, Supplement (juin 2002) : 28. http://dx.doi.org/10.1097/00024382-200206001-00082.
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Lei, M. G., et D. C. Morrison. « Specific endotoxic lipopolysaccharide-binding proteins on murine splenocytes. II. Membrane localization and binding characteristics. » Journal of Immunology 141, no 3 (1 août 1988) : 1006–11. http://dx.doi.org/10.4049/jimmunol.141.3.1006.
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Abstract We have characterized the binding of LPS to an 80-kDa LPS-binding protein detected by an LPS photoaffinity probe to be present on murine splenocytes. Specific binding of LPS to the 80-kDa protein is directly proportional to LPS concentration at low concentrations of LPS and is saturable at high concentrations of LPS. Binding is inhibited by both homologous and heterologous underivatized LPS as well as by polysaccharide-free lipid A, indicating a specificity for the biologically active component of LPS. Analysis of the kinetics of binding indicate a time-dependent increase over the first 15 min, but increases are not detected after this time. Binding of LPS to the 80-kDa LPS-binding protein is reduced but still readily detectable at 4 degrees C in the presence of azide. The presence of the 80-kDa LPS-binding protein in an isolated cytoplasmic membrane fraction of murine splenocytes as well as its release from intact splenocytes by octylglucoside suggest that this LPS-binding protein is membrane localized. The results are consistent with, but do not establish unequivocally, the identity of the 80-kDa LPS-binding protein as a specific membrane receptor for lipid A.
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Schumann, R. R., N. Lamping et A. Hoess. « Interchangeable endotoxin-binding domains in proteins with opposite lipopolysaccharide-dependent activities. » Journal of Immunology 159, no 11 (1 décembre 1997) : 5599–605. http://dx.doi.org/10.4049/jimmunol.159.11.5599.
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Abstract Host defense against microorganisms involves proteins that bind specifically to bacterial endotoxins (LPS), causing different cellular effects. Although LPS-binding protein (LBP) can enhance LPS activities, while bactericidal/permeability-increasing protein (BPI) and Limulus anti-LPS factor (LALF) neutralize LPS, it has been proposed that their LPS-binding domains possess a similar structure. Here, we provide evidence that the LBP/LPS-binding domain is, as in the LALF structure, solvent exposed and therefore available for LPS binding. Our investigations into the activity of LPS-binding domains of different LPS-binding proteins, in the context of LBP, provide the first functional analysis of these domains in a whole protein. We constructed domain exchange hybrid proteins by substituting 12 amino acids of the LBP/LPS-binding domain with those of BPI and LALF and expressed them in Chinese hamster ovary cells. Although discrete point mutations within the LPS-binding domain of LBP disrupted its specific functions, the hybrid proteins were still able to bind LPS and, in addition, retained the wild-type LBP activity of enhancing LPS priming for FMLP-induced oxygen radical production by neutrophils and transferring LPS aggregates to CD14. Although BPI and LALF display opposite activities to LBP, and LALF does not share any sequence homology with LBP, our data provide strong evidence that LBP, BPI, and LALF possess a solvent-exposed, interchangeable LPS binding motif that is functionally independent of LPS transport or neutralization.
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Hoess, A., Ch Ried, C. Wahl, R. Liddington, H. Wagner et L. Schneider-Mergener. « 110 THE LPS-BINDING DOMAIN OF ENDOTOXIN NEUTRALISING PROTEINS ». Shock 3, no 5 (mai 1995) : 34. http://dx.doi.org/10.1097/00024382-199505000-00111.
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Elass-Rochard, Elisabeth, Dominique Legrand, Valerie Salmon, Anca Roseanu, Mihaela Trif, Peter S. Tobias, Joel Mazurier et Genevieve Spik. « Lactoferrin Inhibits the Endotoxin Interaction with CD14 by Competition with the Lipopolysaccharide-Binding Protein ». Infection and Immunity 66, no 2 (1 février 1998) : 486–91. http://dx.doi.org/10.1128/iai.66.2.486-491.1998.
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ABSTRACT Human lactoferrin (hLf), a glycoprotein released from neutrophil granules during inflammation, and the lipopolysaccharide (LPS)-binding protein (LBP), an acute-phase serum protein, are known to bind to the lipid A of LPS. The LPS-binding sites are located in the N-terminal regions of both proteins, at amino acid residues 28 to 34 of hLf and 91 to 108 of LBP. Both of these proteins modulate endotoxin activities, but they possess biologically antagonistic properties. In this study, we have investigated the competition between hLf and recombinant human LBP (rhLBP) for the binding of Escherichia coli 055:B5 LPS to the differentiated monocytic THP-1 cell line. Our studies revealed that hLf prevented the rhLBP-mediated binding of LPS to the CD14 receptor on cells. Maximal inhibition of LPS-cell interactions by hLf was raised when both hLf and rhLBP were simultaneously added to LPS or when hLf and LPS were mixed with cells 30 min prior to the incubation with rhLBP. However, when hLf was added 30 min after the interaction of rhLBP with LPS, the binding of the rhLPS-LBP complex to CD14 could not be reversed. These observations indicate that hLf competes with rhLBP for the LPS binding and therefore interferes with the interaction of LPS with CD14. Furthermore, experiments involving competitive binding of the rhLBP-LPS complex to cells with two recombinant mutated hLfs show that in addition to residues 28 to 34, another basic cluster which contains residues 1 to 5 of hLf competes for the binding to LPS. Basic sequences homologous to residues 28 to 34 of hLf were evidenced on LPS-binding proteins such as LBP, bactericidal/permeability-increasing protein, and Limulus anti-LPS factor.
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El-Samalouti, Volker T., Jens Schletter, Helmut Brade, Lore Brade, Shoichi Kusumoto, Ernst T. Rietschel, Hans-Dieter Flad et Artur J. Ulmer. « Detection of Lipopolysaccharide(LPS)-Binding Membrane Proteins by Immuno-Coprecipitation with LPS and Anti-LPS Antibodies ». European Journal of Biochemistry 250, no 2 (décembre 1997) : 418–24. http://dx.doi.org/10.1111/j.1432-1033.1997.0418a.x.
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Amura, Claudia R., Takayuki Kamei, Noriko Ito, Michael J. Soares et David C. Morrison. « Differential Regulation of Lipopolysaccharide (LPS) Activation Pathways in Mouse Macrophages by LPS-Binding Proteins ». Journal of Immunology 161, no 5 (1 septembre 1998) : 2552–60. http://dx.doi.org/10.4049/jimmunol.161.5.2552.
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Abstract LPS binding to its receptor(s) on macrophages induces the synthesis of inflammatory mediators involved in septic shock. While the signaling mechanism(s) remains to be fully defined, the human LPS-binding protein (LBP) is known to regulate responses to LPS by facilitating its binding to CD14 on human monocytes. The structurally related bactericidal permeability increasing protein (BPI) differs from LBP by inhibiting LPS-induced human monocyte activation. We have demonstrated that, unlike the human monocyte response to LPS, both LBP and BPI inhibited LPS-stimulated TNF-α production in mouse peritoneal macrophages. In contrast, LPS-dependent nitric oxide release was not affected by LBP. LPS induces the phosphorylation of a number of proteins in a dose and time-dependent manner, however, the pattern of LPS-induced phosporylation was not reduced by either LBP or BPI under conditions that result in selective TNF-α inhibition. Further, activation of the transcription factor NF-κB in response to LPS was also not modified by either LBP or BPI. Finally, no differences were detected in TNF-α or inducible nitric oxide synthase mRNA accumulations induced by LPS in the presence or absence of either protein, whereas a slight decreased mRNA stability was observed in the group with LPS treatment. These results would suggest that many of the early signaling events contribute to LPS-induced macrophage signaling at a point preceding the divergence of pathways that differentially regulate TNF-α and NO production.
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Lamping, N., A. Hö, B. Yu, T. C. Park, S. D. Wright, C. Kirschning, D. Pfeil, F. Herrmann et R. R. Schumann. « IDENTIFICATION OF THE LIPOPOLYSACCHARIDE (LPS) BINDING SITE OF LPS BINDING PROTEIN (LBP) BY SITE-DIRECTED MUTAGENESIS, EVIDENCE FOR A SIMILAR LPS RECOGNITION MECHANISM IN DIFFERENT LPS BINDING PROTEINS ». Shock 7, Supplement (mars 1997) : 21–22. http://dx.doi.org/10.1097/00024382-199703001-00087.
Texte intégralThèses sur le sujet "LPS- binding proteins"
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SESTITO, STEFANIA ENZA. « LPS-binding proteins : interaction studies with natural and synthetic ligands ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/67756.
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L’obiettivo di questa tesi è elucidare alcuni aspetti dell’interazione tra proteine che legano il lipopolisaccaride (LPS) batterico e il loro ligando naturale o ligandi di sintesi.
LptC (Lipopolysaccharide transport C) è una proteina batterica che appartiene al sistema di trasporto Lpt, un sistema di 7 proteine essenziali che trasportano l’LPS sulla membrana esterna dei batteri Gram negativi dopo la sua biosintesi. Sebbene molti elementi della biosintesi dell’LPS siano stati elucidati, il preciso meccanismo di trasporto è ancora poco chiaro. Poiché LptC può essere considerata come proteina modello del sistema Lpt, in quanto presenta lo stesso folding delle altre proteine ed è la prima ad essere localizzata nel periplasma, abbiamo sviluppato ed ottimizzato un saggio di binding in vitro per studiare la sua interazione con l’LPS. Abbiamo ottenuto, per la prima volta, dettagliate informazioni sui parametri termodinamici e cinetici dell’interazione LptC-LPS. Abbiamo infatti dimostrato che in vitro il binding LptC-LPS è irreversibile con una Kd dell’ordine del μM. Considerando le analogie strutturali tra LptC e la proteina eucariotica CD14, appartenente al sistema recettoriale del TLR4, in modo analogo è stata studiata l’interazione di LptC con la molecola sintetica IAXO-102, un noto ligando di CD14. È emerso che IAXO-102 condivide lo stesso sito di legame dell’LPS e che l’interazione con la proteina è irreversibile con un’affinità inferiore a quella LptC-LPS. IAXO-102 può dunque essere considerato un prototipo per lo sviluppo di una nuova generazione di antibiotici che ha come target la biogenesi dell’LPS.
L’LPS è in grado di interagire con molte altre proteine, tra le quali quelle del sistema dell’immunità innata (TLR4, CD14, MD-2). Il riconoscimento dell’LPS da parte di questi recettori induce una forte risposta infiammatoria che termina con la produzione di citochine pro-infiammatorie e fattori immunomodulatori. Questa reazione infiammatoria è utile all’organismo, ma quando si manifesta in modo eccessivamente potente e sregolato induce sepsi, processi infiammatori e sindromi autoimmuni per le quali non è ancora disponibile un trattamento farmacologico. Una possibile soluzione al problema consiste nella ricerca e nello sviluppo di composti in grado di modulare questa eccessiva attivazione. Nella seconda parte di questo lavoro, sono riportate le caratterizzazioni biologiche di alcuni composti di sintesi con caratteristiche chimiche differenti. Di tutti i composti è stata valutata la tossicità mediante saggio dell’MTT e l’attività modulatoria del pathway del TLR4 utilizzando cellule HEK stabilmente trasfettate con i geni del TLR4, CD14 ed MD-2. Ulteriori caratterizzazioni sono state effettuate sui composti più promettenti, effettuando saggi in vitro su cellule HEK trasfettate con il complesso umano o murino TLR4•MD-2 e saggi in vivo. Infine, abbiamo investigato la possibile correlazione tra le note proprietà anti-infiammatorie di alcuni composti naturali, come i composti fenolici presenti nell’olio di oliva, e il pathway del TLR4. L’obiettivo di questo lavoro è duplice: individuare un lead compound come possibile modulatore del TLR4, ma anche discriminare quali caratteristiche chimiche siano importanti per ottenere questo effetto. Inoltre, le informazioni ottenute potrebbero essere estremamente utili per guidare il rational design di altri modulatori del TLR4.The purpose of this work is the elucidation of some aspects of the interaction between lipopolysaccharide (LPS) binding proteins and their natural ligand or synthetic compounds. LptC (Lipopolysaccharide transport C) is a bacterial protein belonging to Lpt complex, a molecular machinery composed of 7 essential proteins involved in the transport of LPS to the outer membrane in Gram negative bacteria after its biogenesis. Although many elements of LPS biosynthesis have been clarified, the precise mechanism of transport is still not completely understood. Since LptC can be considered as a model protein of Lpt complex, sharing the same folding of other proteins and being the first one in the periplasm, we have developed and optimized an in vitro binding assay to study its interaction with LPS. We have obtained, for the first time, detailed information about the thermodynamic and kinetic parameters of LptC-LPS binding. We have shown that the in vitro LptC-LPS binding is irreversible with a Kd of the order of μM. Considering the structural similarities between LptC and the eukaryotic protein CD14, belonging to TLR4 receptor system, the binding between LptC and the synthetic molecule iaxo-102, a known ligand of CD14, has been investigated. It is evident that iaxo-102 shares the same binding site of LPS and that the binding is irreversible with an affinity lower than that LptC-LPS. So, iaxo-102 can be considered as a lead compound for the development a new generation of antibiotics targeting the biogenesis of LPS. LPS also binds to other proteins, such as those of innate immunity TLR4, CD14 and MD-2. The LPS recognition by these receptors induces the production of pro-inflammatory cytokines and immunomodulators that trigger the inflammatory and immune responses. These reactions are useful for the organism, but when TLR4 activation is too strong or not well regulated induces sepsis, inflammation and autoimmune syndromes, which still lack a pharmacological treatment. A possible solution to solve this problem consists in the research and development of compounds which modulate this excessive activation. In the second part of thesis work, the biological characterization of some synthetic compounds, with different chemical features, have been reported. All compounds have been screened for their toxicity using MTT assay, and their modulatory activity on TLR4 pathway by using HEK cells stably transfected with TLR4, CD14 and MD-2 genes. The best compounds have been further characterized by in vitro assays on HEK cells transfected with the human or murine complex TLR4·MD-2 and in vivo studies. Finally, the possible correlation between the known anti-inflammatory properties of some natural compounds, such as the phenolic compounds of olive oil, and TLR4 activity has been investigated. The aim of this study is double: to find a lead compound active on TLR4 pathway, but also to discriminate which chemical features are important to obtain this effect. In addition, the information obtained could be very useful to guide the rational design of other TLR4 modulators.
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CIARAMELLI, CARLOTTA. « Synthesis and characterization of new small-molecule ligands of LPS binding proteins ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2015. http://hdl.handle.net/10281/77016.
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Lo scopo del presente lavoro è la progettazione, la sintesi e la caratterizzazione di nuove small molecules, attive come ligandi di LPS (lipopolisaccaridi)-binding proteins. Gli LPS, o endotossine batteriche, sono macromolecole anfifiliche ubiquitarie sulla membrana esterna dei batteri Gram-negativi. Le proteine che legano gli LPS studiate nel corso di questo progetto di tesi di dottorato appartengono a due categorie: le proteine batteriche di trasporto Lpt e il sistema recettoriale TLR4, che comprende anche i co-recettori LBP, CD14, MD2.
Le proteine Lpt, e in particolare la proteina LptC, sono responsabili del meccanismo di esportazione del LPS alla superficie cellulare, che è uno step fondamentale della via biosintetica dell’LPS. Pertanto, la biogenesi dell’LPS rappresenta un target ideale per lo sviluppo di nuovi antibiotici contro i batteri Gram-negativi. Inoltre, le strutture delle proteine Lpt sono state risolte, ma il meccanismo di trasporto è ancora da elucidare.
Nel presente lavoro di tesi sono stati utilizzate diverse tecniche per studiare l'interazione tra LPS e LptC, con particolare attenzione agli studi di interazione via NMR. Inoltre, un nuovo LPS fluorescente è stato prodotto ed è stato utilizzato come tool per studi di interazione LPS-LptC con tecniche di fluorescenza. Sono state anche sviluppate alcune nuove molecole sintetiche. Questi glicolipidi sono stati progettati e sintetizzati per ottenere ligandi di LptC e, in prospettiva, potenziali antibiotici contro i batteri Gram-negativi.
Il Toll-like receptor 4 (TLR4), il recettore dell'immunità innata, riconosce l’LPS aiutato da altre proteine (LBP, CD14 e MD-2) ed è responsabile dell'induzione della risposta infiammatoria. Molecole sintetiche in grado di modulare l'attività dei recettori dell’immunità innata sono un potente mezzo per studiare il sistema recettoriale TLR4 e hanno grande interesse farmacologico come adiuvanti vaccinali (agonisti), agenti antisepsi e anti-infiammatori (antagonisti).
L’attività biologica di glicolipidi con una funzione amminica (IAXO-102) come antagonisti del TLR4 è stata chiaramente dimostrata dal nostro gruppo di ricerca. La sintesi di molecole derivate da IAXO-102, che mantengano l'attività biologica del precursore, è stato un obiettivo di questo lavoro. In particolare, sono state portate a termine le sintesi di sonde fluorescenti, utilizzate per studi di interazione, derivati zwitterionici e molecole dimeriche. Nei nostri laboratori sono stati ottenuti anche antagonisti anionici del TLR4 con una struttura chimica più simile a Lipide A. Lo scopo di questo lavoro è stato valutare, tramite esperimenti NMR, la loro capacità di legare co-recettore dell'immunità innata MD-2.
Il carattere anfifilico degli analoghi sintetici del lipide A sintetizzati finora è spesso associato ad una bassa solubilità in acqua e a scarsa biodisponibilità. Invece, i composti attivi sul TLR4 di origine naturale hanno una migliore solubilità e biodisponibilità. La modifica chimica di queste strutture è molto utile per modulare l'attività biologica e per migliorare la specificità nei confronti del target. Di conseguenza, in una fase successiva di questo lavoro di tesi, è stata intrapresa la sintesi di nuove molecole con strutture chimiche ispirate ai modulatori naturali del TLR4. Recentemente è stato dimostrato che alcuni composti fenolici estratti da olio di oliva hanno una buona attività come antagonisti del TLR4. Pertanto, la sintesi di alcuni analoghi di queste molecole è stata eseguita per ottenere nuovi potenziali antagonisti del TLR4, con una migliore solubilità in acqua e una ridotta tossicità.The purpose of this work is the design, synthesis and characterization of new small molecules, active as ligands of two different lipopolysaccharide (LPS)-binding proteins. LPS, or bacterial endotoxin, is an amphiphilic macromolecule ubiquitous on the outer membrane of Gram-negative bacteria. The LPS binding proteins studied during this thesis project belong to two classes: the bacterial proteins of the Lpt transport machinery and the mammalian TLR4 receptor system, including the co-receptors LBP, CD14, MD-2. Lpt proteins, and in particular the protein LptC, are responsible for the export mechanism of LPS to the cell surface of Gram negative bacteria, which is a fundamental step of the LPS biosynthetic pathway. Therefore, the LPS biogenesis represents an ideal target for development of novel antibiotics against Gram-negative bacteria. Moreover, the structures of Lpt proteins have been elucidated, but very little is known about the mechanism of LPS transport. In this thesis work different techniques were used to study the interaction between LPS and LptC, particularly NMR binding studies. Moreover, a new fluorescent LPS was produced and it was used as a tool to perform LPS-LptC interaction studies with fluorescence techniques. Some new synthetic molecules were also developed during this thesis. Glycolipidic small molecules were designed and synthesized in order to obtain LptC ligands and, in perspective, potential antibiotics against Gram-negative bacteria. Toll-like receptor 4 (TLR4), the innate immunity receptor, recognizes LPS, helped by other proteins (LBP, CD14 and MD-2), and it is responsible for the induction of inflammatory responses. Synthetic small molecules able to modulate innate immunity receptors activity are a powerful mean to study the TLR4 receptor system and have great pharmacological interest as vaccine adjuvants (agonists), antisepsis and anti-inflammatory agents (antagonists). Antagonist activity on TLR4 receptor system of amino glycolipids (IAXO-102) was clearly demonstrated by our research group. The synthesis of molecules derived from IAXO-102 which retain the biological activity of the precursor was a target of this work. In particular, the synthesis of fluorescent probes, used for binding studies, zwitterionic derivatives and dimeric molecules were performed. Anionic TLR4 antagonists with a chemical structure more similar to Lipid A were also obtained in our labs. The aim of this work was the evaluation via NMR binding experiments of their ability to bind the innate immunity co-receptor MD-2. The amphiphilic character of the synthetic lipid A analogues synthesized so far is often associated with low water solubility and poor bioavailability. In this respect, the natural TLR4-active compounds have better solubility and bioavailability. The chemical modification of these structures is very helpful to modulate their biological activity and to enhance target specificity. Consequently, in a later stage of this work, the synthesis of new small molecules with chemical structures inspired to natural TLR4 modulators was pursued. Very recently it was found that some phenolic compounds from olive oil extracts presented a good activity as TLR4 antagonists. The synthesis of some analogues of these molecules was performed to obtain new potential TLR4 antagonists with better water solubility and reduced toxicity.
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Eckert, Jana Kristin. « Funktionelle Analyse von Mutanten des LPS-bindenden Proteins (LBP) ». Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15955.
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LBP vermittelt im Wirtsorganismus die direkte Immunantwort auf bakterielle Liganden wie das Lipopolysaccharid (LPS) von Gram-negativen oder Lipopeptide von Gram-positiven Bakterien. In dieser Arbeit wurde die Funktionsweise von LBP weiter aufgeklärt. Im ersten Teil der Arbeit wurde eine natürlich vorkommende Mutation des LBP (c998t), die an Position 333 zu einem Austausch der Aminosäure Prolin zu Leucin führt, hinsichtlich ihrer Auswirkungen auf Struktur und Funktionalität des Proteins untersucht. Westernblot-Analysen des rekombinant hergestellten Proteins und humaner Seren von Mutationsträgern weisen auf einen Zerfall des mutierten Proteins hin. Es kommt zu einer Beeinträchtigung der Bindung bakterieller Liganden und einer deutlichen Reduktion der LBP-vermittelten Zytokinausschüttung von Immunzellen. Der hier untersuchte Polymorphismus hat eine Allelfrequenz von 0,072 in einer gesunden europäischen Population. Genotypanalysen von Patientengruppen zeigten, dass es durch die Mutation zu einer deutlich erhöhten Mortalität bei Patienten mit septischen Komplikationen und einer durch Gram-negative Erreger verursachten Pneumonie kommt. Unsere Ergebnisse zur eingeschränkten Funktion des LBP-c998t bieten eine erste Erklärung dafür, wie diese Mutation vermutlich die Fähigkeit, Krankheiten zu bewältigen, beeinträchtigt. Innerhalb dieser Arbeit ging es um die Analyse der Bindung von bakteriellen Liganden an LBP. Dabei wurde eine potentiell gemeinsame Bindungsstelle für Liganden untersucht, die von Gram-positiven und Gram-negativen Bakterien stammen und später von den Toll-like Rezeptoren (TLRs) 2 und -4 erkannt werden. Dazu wurden Bindungsversuche zwischen Lipopeptiden und LPS mit einer zweiten LBP-Variante (LBP-E94/95) durchgeführt. Beim LPS führt dies zu einem Bindungsverlust. Auch für die Lipopeptide war durch die Mutationen die Interaktion mit LBP beeinträchtigt, was die These einer gemeinsamen Bindungsstelle von TLR2- und TLR4-Liganden an das Protein weiter unterstützt.LBP enhances the innate immune reaction against bacterial ligands like LPS from gram negative or lipopeptides from gram positive bacteria in the host. Here we investigated the function of LBP using two recombinant mutants of the protein. The first part of this work examines a natural occurring mutation of LBP (c998t) leading to an amino acid exchange of proline to leucine at position 333 with regard to the impact on structure and function of the protein. Western blot analyses of the recombinant protein and sera obtained from individuals differing in the LBP genotype indicate the disaggregation of the mutated protein. Thereby binding of bacterial ligands to LBP is diminished and the LBP mediated cytokine secretion of immune cells is reduced. The gene polymorphism leading to the occurrence of the mutation is present with an allelic frequence of 0.072. A recent study has shown that this LBP-SNP led to a higher mortality in patients with septic complications and gram negative pneumonia. The results presented here, showing the negative impact on the function of LBP due to the mutation, may therefore be a first explanation on how this mutation affects the ability of people to deal with disease. Within this work binding of ligands to LBP was also explored. It was investigated whether ligands which are later recognized by Toll-like receptors (TLRs) 2 and – 4 share a common binding site on LBP. Assays with immobilized lipopeptides and LPS were performed with a second mutated LBP (LBP-E94/95). LPS binding to LBP is diminished completely. Here we showed that binding of lipopeptide to LBP is affected likewise, furthermore supporting the hypothesis of a common binding site for TLR2- and TLR4- ligands.
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Szpryngiel, Scarlett. « Structure and lipid interactions of membrane-associated glycosyltransferases : Cationic patches and anionic lipids regulate biomembrane binding of both GT-A and GT-B enzymes ». Doctoral thesis, Stockholms universitet, Institutionen för biokemi och biofysik, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-131084.
Texte intégralRésumé :
This thesis concerns work on structure and membrane interactions of enzymes involved in lipid synthesis, biomembrane and cell wall regulation and cell defense processes. These proteins, known as glycosyltransferases (GTs), are involved in the transfer of sugar moieties from nucleotide sugars to lipids or chitin polymers. Glycosyltransferases from three types of organisms have been investigated; one is responsible for vital lipid synthesis in Arabidopsis thaliana (atDGD2) and adjusts the lipid content in biomembranes if the plant experiences stressful growth conditions. This enzyme shares many structural features with another GT found in gram-negative bacteria (WaaG). WaaG is however continuously active and involved in synthesis of the protective lipopolysaccharide layer in the cell walls of Escherichia coli. The third type of enzymes investigated here are chitin synthases (ChS) coupled to filamentous growth in the oomycete Saprolegnia monoica. I have investigated two ChS-derived MIT domains that may be involved in membrane interactions within the endosomal pathway. From analysis of the three-dimensional structure and the amino-acid sequence, some important regions of these very large proteins were selected for in vitro studies. By the use of an array of biophysical methods (e.g. Nuclear Magnetic Resonance, Fluorescence and Circular Dichroism spectroscopy) and directed sequence analyses it was possible to shed light on some important details regarding the structure and membrane-interacting properties of the GTs. The importance of basic amino-acid residues and hydrophobic anchoring segments, both generally and for the abovementioned proteins specifically, is discussed. Also, the topology and amino-acid sequence of GT-B enzymes of the GT4 family are analyzed with emphasis on their biomembrane association modes. The results presented herein regarding the structural and lipid-interacting properties of GTs aid in the general understanding of glycosyltransferase activity. Since GTs are involved in a high number of biochemical processes in vivo it is of outmost importance to understand the underlying processes responsible for their activity, structure and interaction events. The results are likely to be useful for many applications and future experimental design within life sciences and biomedicine.At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.
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Hallatschek, Werner. « Die Regulation des humanen Lipopolysaccharid bindenden Proteins (hLBP) ». Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2005. http://dx.doi.org/10.18452/15202.
Texte intégralRésumé :
Das Lipopolysaccharid Bindende Protein (LBP) ist ein überwiegend in der Leber synthetisiertes Akutphaseprotein. Es bindet den Zellwandbestandteil Lipopolysaccharid (LPS) Gram-negativer Bakterien und transportiert es zu zellulären Rezeptoren, wodurch das angeborene Immunsystem aktiviert wird. In dieser Arbeit wird die Regulation der LBP-Expression in Interleukin (IL)-1, IL-6 und Dexamethason (Dex) stimulierten humanen Hepatomzelllinien HuH-7 und HepG2 untersucht. Der wichtigste Stimulator ist dabei IL-6, dessen Wirkung über die Transkriptionsfaktoren (TF) Stat-3, C/EBP-beta und AP-1 vermittelt wird. Für alle 3 TF konnten aktive Bindungsstellen auf dem LBP-Promotor nachgewiesen werden. Für IL-1-Effekte die u. a. über den TF NF-kappaB vermittelt werden, konnten ebenfalls aktive Bindungsstellen nachgewiesen werden. Die Wirkung von Dex wird über Glucocorticoid Responsive Elements (GREs) vermittelt. Auf dem LBP-Promotor befinden, sich wie gezeigt werden konnte, mehrere aktive GREs, wobei einige verstärkend und einige hemmend wirken. Eine zu beobachtende Synergiewirkung von Dex und IL-6 wird durch die Aufregulation des IL-6-Rezeptors durch Dex verursacht. Die LBP-Expression kann durch TGF (Transforming Growth Factor)-beta gehemmt werden. Der TGF-beta-Signalweg über Smads ist in den Hepatomzellen aktiv, vermittelt aber nicht den TGF-beta-Hemmeffekt, sondern eine geringe stimulierende Wirkung, die bei alleiniger TGF-beta-Inkubation auftritt. Die inhibierende Wirkung von TGF-beta wird durch Gfi-1- und AP-1-Bindungsstellen vermittelt. Die Gfi-1-Bindungsstelle nimmt dabei, wie hier erstmals gezeigt werden konnte, eine herausragende Stellung ein. Die Aufklärung der LBP-Regulation und dabei besonders die Hemmung der LBP-Expression kann mittelfristig dazu beitragen, den klinischen Verlauf von inflammatorischen und infektiösen Erkrankungen zu beeinflussen und bietet daher Potenzial für neue Therapieansätze.Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein with the ability to bind and transfer LPS of Gram-negative bacteria. This soluble pattern recognition molecule represents an important defense principle of the host. Regulation of the hepatic acute phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host. Here were analyze the cooperation of Interleukin (IL)-1, IL-6, and Dexamethasone (Dex) at LBP expression in the hepatoma cell lines HuH-7 and Hep G2. The major inducer of LBP expression is IL-6. Within the LBP promoter numerously highly consensus binding sites such as AP-1, C/EBP-beta? and STAT3 are present, that confer transcriptional activity as shown by truncation and mutation experiments. Additionally, activate NF-kappaB sites activated by IL-1 were detected at the LBP promoter. By mutation experiments of the promoter furthermore were found differentially active glucocorticoid response elements (GREs). The promoter contains GREs enhancing the activity as well as inhibitory ones. The enhancing effect towards LBP expression by Dex was mediated by IL-6. Dex stimulated the expression of the IL-6 receptor and therefore upregulated the IL-6 pathway. Transforming Growth Factor (TGF)-beta is able to inhibit LBP expression in stimulated cells. An AP-1 binding site was identified mediating inhibitory TGF-beta effects towards LBP promoter activity. Furthermore it was shown that a growth factor independence (Gfi)-1 binding site localized near the AP-1 site is essential for mediating the TGF-beta inhibitory effect. The relevancy of the Gfi-1 site fore mediating TGF-beta effects indicates a novel mechanism for understanding inhibitory TGF-beta effects at the transcriptional level. In summary the complex regulation of LBP were elucidate which may help to eventually develop novel intervention strategies for acute phase, sepsis, and septic shock.
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ZAFFARONI, LENNY. « Production of recombinant human MD-2 and development of protein-ligand binding assays for the characterization of new TLR4 modulators ». Doctoral thesis, Università degli Studi di Milano-Bicocca, 2018. http://hdl.handle.net/10281/207343.
Texte intégralRésumé :
Il toll-like receptor 4 (TLR4) rappresenta un mediatore centrale dell’immunità innata ed adattativa in mammiferi. L’attivazione di TLR4 in risposta al lipopolisaccaride (LPS) batterico induce un rapido innesco di processi pro-infiammatori essenziali per una risposta immunitaria ottimale. L’attivazione di TLR4 mediata da LPS è un meccanismo che coinvolge la partecipazione di diverse proteine e culmina con la formazione del complesso attivato (TLR4/MD-2/LPS)2. MD-2 è il co-rettore di TLR4, e svolge un importante ruolo nell’interazione con LPS e la susseguente dimerizzazione del TLR4. MD-2 è la componente che interagisce con il ligando (LPS) nel complesso recettoriale TLR4/MD-2. Il legame di LPS al complesso TLR4/MD-2 induce la dimerizzazione del TLR4; mentre gli antagonisti del TLR4 sono in grado di legare il complesso TLR4/MD-2 ma non inducono la dimerizzazione del TLR4. L’attivazione non regolata del TLR4 è correlata ad un’ampia serie di problematiche prive di un trattamento farmacologico specifico. Esse includono disordini autoimmuni, infiammazione cronica, allergie, asma, infezioni e malattie del sistema nervoso centrale, cancro, e setticemia. L’inibizione del TLR4 tramite l’uso di piccole molecole sintetiche o naturali può quindi rappresentare una via per lo sviluppo di nuove terapie contro questa vasta gamma di problematiche. Questa tesi è parte di un studio originale di relazione struttura-attività (SAR) svolto su glicolipidi monosaccaridici sintetici nel contesto della modulazione del TLR4. In particolare, essa si focalizza sulla caratterizzazione del legame in vitro di nuovi glicolipidi monosaccaridici sintetici con il recettore MD-2 purificato. Per gli studi di interazione la proteina MD-2 umana (hMD-2) purificata e funzionale è stata espressa in cellule di lievito. Due diversi sistemi di espressione per la produzione di hMD-2 ricombinante sono stati testati: mammifero (HEK293T) e cellule di lievito (Pichia pastoris). La purificazione di hMD-2 da lievito è stata ottimizzata ottenendo una concentrazione finale di hMD-2 purificato di 30 μM. Per confrontare l’attività biologica di hMD-2 espresso nei diversi microorganismi è stato sviluppato un ELISA. hMD-2 da cellule di mammifero ha ottenuto l’attività biologica più elevata, seguito da hMD-2 espresso in P. pastoris. hMD-2 da E. coli ha ottenuto l’attività biologica più bassa dei tre. Date le rese più elevate di purificazione in lievito, hMD-2 espresso in P. Pastoris è stato utilizzato nei quattro diversi tipi di esperimenti di legame per studiare l’affinità di molecole naturali e sintetiche. I test di legame comprendono due ELISA con hMD-2 immobilizzato, un saggio fluorescente di spiazzamento, e misure di risonanza plasmonica di superficie (SPR). I due test ELISA sono basati su: i) spiazzamento dose-dipendente di un anticorpo da hMD-2 immobilizzato. L’anticorpo lega hMD-2 in una regione in prossimità del sito di legame del ligando; ii) spiazzamento di LPS biotinilato da hMD-2 immobilizzato. L’esperimento di florescenza è basato sullo spiazzamento di bis-ANS da hMD-2. Mentre la tecnica SPR è stata utilizzata per studiare la diretta interazione tra le molecole e hMD-2 immobilizzato. L’affinità per hMD-2 delle molecole analizzate (che risulta essere nell’intervallo del basso μM) è in linea con i risultati di attività biologica e la produzione di citochine in vitro in modelli cellulari. I risultati ottenuti da questi studi in vitro sul recettore hMD-2 purificato evidenziano l’interazione delle molecole con la tasca idrofobica di hMD-2, presentando differenze nei valori di affinità. Questi dati generati permettono uno studio sistemico dei modulatori del TLR4, creando buone prospettive per lo sviluppo di una nuova generazione di farmaci hits e leads che agiscano direttamente sul recettore TLR4.Toll-like receptor 4 (TLR4) represents a central mediator of innate and adaptive immune responses in mammals. TLR4 activation in response to bacterial lipopolysaccharides (LPS) results in the rapid triggering of pro-inflammatory processes essential for optimal host immune responses. TLR4 activation mediated by LPS is a complex event which involves several proteins (lipid binding protein (LBP), cluster of differentiation 14 (CD14), and myeloid differentiation 2 (MD-2)) and it ends with the formation of the activated (TLR4/MD-2/LPS)2 complex. The TLR4 co-receptor MD-2 plays an important role in the interaction with LPS and subsequent TLR4 dimerization. MD-2 alone binds to LPS, whereas TLR4 alone does not. MD-2 is the ligand-binding component of the TLR4/MD-2 receptor complex. LPS binding to TLR4/MD-2 induces TLR4 dimerization; whereas TLR4 antagonists binding to TLR4/MD-2 does not induce TLR4 dimerization. Deregulated TLR4 activation is related to an impressively broad spectrum of disorders still lacking specific pharmacological treatment. These include autoimmune disorders, chronic inflammations, allergies, asthma, infectious and central nervous system diseases, cancer, and sepsis. The TLR4 inhibition by small molecules of synthetic and natural origin provides access to new TLR-based therapeutics targeting this large array of diseases. This thesis is part of an original structure-activity relationship (SAR) study on synthetic monosaccharide glycolipids in the context of TLR4 modulation. Thesis work focuses on the in vitro binding characterization of new synthetic monosaccharide glycolipids with the purified receptor MD-2. Pure and functional human MD-2 (hMD-2) protein for binding studies has been obtained by expression in yeast cells. Two different expression systems for the production of recombinant hMD-2 were tested: mammalian (HEK293T) and yeast cells (Pichia pastoris). Recovery of hMD-2 from the medium of yeast cells was optimized, achieving a concentration of recombinant hMD-2 of 30 μM. An ELISA was developed in order to compare the biological activity of the hMD-2 expressed in different hosts. hMD-2 from mammalian cells obtained the highest biological activity, followed by the hMD-2 expressed by P. pastoris. hMD-2 expressed by E. coli presented the lowest biological activity of the three. Due to the higher yield of recovery achieved, hMD-2 expressed in P. pastoris was used in four different types of binding experiments to assess its affinity for natural and synthetic molecules. The binding tests comprise two plate based ELISA with immobilized hMD-2, a fluorescence displacement assay and surface plasmon resonance (SPR) measurements. The two ELISA tests were based on: i) dose-dependent displacement of a monoclonal antibody from immobilized hMD-2. The antibody binds to hMD-2 in a region proximal to ligand binding site; ii) displacement of biotin-LPS from immobilized hMD-2. The fluorescence experiment was based on the displacement of the bis-ANS from hMD-2, whereas the SPR technique was used to study the direct interactions between small ligands and immobilized hMD-2. The obtained binding affinities for hMD-2 of the tested molecules (which turned out to be in the low μM range) mirror their biological activity in modulating TLR4 signaling and cytokine production in vitro in cell models. The results obtained from these in vitro cell-free studies indicate that the tested molecules bind to the hMD-2 pocket, with differences in the affinity values. These data allow a systematic study on SAR for TLR4 modulators, opening the way for the development of a new generation of drug hits and leads targeting directly TLR4 signaling.
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Ghanim, Mustafa. « Les aspects génétiques des démences frontotemporales ». Paris 6, 2010. http://www.theses.fr/2010PA066039.
Texte intégral
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Agostini, Federico 1985. « Predictions of RNA-binding ability and aggregation propensity of proteins ». Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/318159.
Texte intégralRésumé :
RNA-binding proteins (RBPs) control the fate of a multitude of coding and non-coding transcripts. Formation of ribonucleoprotein (RNP) complexes fine-tunes regulation of post-transcriptional events and influences gene expression. Recently, it has been observed that non-canonical proteins with RNA-binding ability are enriched in structurally disordered and low-complexity regions that are generally involved in functional and dysfunctional associations. Therefore, it is possible that interactions with RNA protect unstructured protein domains from aberrant associations or aggregation. Nevertheless, the mechanisms that prevent protein aggregation and the role of RNA in such processes are not well understood. In this work, I will describe algorithms that I have developed to predict protein solubility and to estimate the ability of proteins and transcripts to interact. I will illustrate applications of computational methods and show how they can be integrated with high throughput approaches. The overarching goal of my work is to provide experimentalists with tools that facilitate the investigation of regulatory mechanisms controlling protein homeostasis.Las proteínas de unión de ARN son responsables de controlar el destino de una multitud de transcriptos codificantes y no codificantes. De hecho, la formación de complejos de ribonucleoproteínas (RNP) afina la regulación de una serie de eventos post-transcripcionales e influye en la expresión génica. Recientemente, se ha observado que las proteínas con capacidad no canónica de unión al ARN se enriquecen en las regiones estructuralmente desordenadas y de baja complejidad, que son las que participan generalmente en asociaciones funcionales y disfuncionales. Por lo tanto, es posible que interactuar con el ARN pudiera ser una manera de proteger las proteínas no estructuradas de asociaciones aberrantes o de agregación. Sin embargo, los mecanismos que impiden la agregación de proteínas y la función del ARN en tales procesos no están bien descritas. En este trabajo, se describen los me ́todos que he desarrollado para predecir la solubilidad de proteínas y para estimar la capacidad de transcriptos y proteínas de interactuar. De otra parte, voy a ilustrar sus aplicaciones y explicar como los métodos de bajo rendimiento han evolucionado a un mayor rendimiento. El objetivo final es proporcionar instrumentos a los investigadores experimentales que se pueden utilizar para facilitar la investigación de los mecanismos reguladores que controlan la homeostasis molecular.
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Ding, Peihui, et 丁佩惠. « Expression profile, molecular regulation and immuno-inflammatory function of LPS-binding protein in human oral keratinocytes ». Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B49617795.
Texte intégralRésumé :
Lipopolysaccharide (LPS)-binding protein (LBP) functions as a crucial molecule in innate immune responses to bacterial challenge. Our recent study shows the expression of LBP in human gingiva and its significant association with periodontal condition. Porphyromonas gingivalis is a keystone periodontopathogen with its LPS as a major virulence factor strongly involved in periodontal pathogenesis. Recent study has discovered that P. gingivalis LPS displays a significant lipid A structural heterogeneity. The present study investigated i) the expression profile of LBP in human oral keratinocytes (HOKs) stimulated by P. gingivalis LPS with penta-acylated (LPS1690) and tetra- (LPS1435/1449) lipid A structures as well as E. coli LPS; ii) the involvement of toll-like receptors (TLRs) and downstream signaling mechanisms in LBP expression; and iii) the effects of LBP and its crosstalk with the two isoforms of P. gingivalis LPS on the expression of cytokines and human β-defensins (hBD-2) in HOKs.
The expression of LBP mRNA and peptide was significantly up-regulated by P. gingivalis LPS1690 and E. coli LPS, while not by P. gingivalis LPS1435/1449. P. gingivalis LPS1690-induced LBP expression was through both TLR2 and TLR4, and the relevant down-stream signaling mechanisms were then further investigated. Western blot results showed that P. gingivalis LPS1690 activated the phosphorylation of IκBα, p65, p38 MAPK and SAPK/JNK, whereas E. coli LPS phosphorylated IκBα, p38 MAPK and SAPK/JNK. A nuclear translocation of NF-κB transcription factor was confirmed upon stimulation by both forms of LPS. Further blocking assay revealed that P. gingivalis LPS1690 induction of LBP was through NF-κB and p38 MPAK pathways, while E. coli LPS induction of LBP was mediated by NF-κB, p38 MPAK and JNK pathways. The effects of LBP and its crosstalk with P. gingivalis LPS1690 or LPS1435/1449 on the expression of cytokines and hBD-2 were further investigated. Interestingly, recombinant human LBP (rhLBP) per se could significantly up-regulate the expression of IL-6, IL-8 and TNF-α, while down-regulate hBD-2 expression. P. gingivalis LPS1690 or LPS1435/1449 modulated to different extents the rhLBP-induced cytokine expression. Notably, P. gingivalis LPS1690 significantly down-regulated rhLBP-induced IL-8 expression; whereas, P. gingivalis LPS1435/1449 down-regulated IL-8 expression more intensively (around 80% vs. 40% reduction). The key mediators of TLRs and their adaptors like CD180 and MD-1 were significantly down-regulated by rhLBP (fold changes: -2.44 and -9.62, respectively). Both CD180 and MD-1 mRNAs were up-regulated by P. gingivalis LPS1435/1449 (7.11 and 4.05 folds, respectively); while these two genes were reversely modulated by P. gingivalis LPS1690 (20.86 and -6.93 folds, respectively).
The present study demonstrates that P. gingivalis LPS with a lipid A structural heterogeneity differentially modulates LBP expression in HOKs. P. gingivalis LPS1690 promotes LBP expression in HOKs through TLR2 and TLR4 as well as NF-κB and p38 MAPK pathways in a way different from E. coli LPS. rhLBP per se significantly up-regulates the expression of IL-6, IL-8 and TNF-α, while down-regulates hBD-2 expression. P. gingivalis LPS with different lipid A structures down-regulates to different extents the rhLBP-induced expression of cytokines in HOKs, likely through fine-tuning of the CD180-MD1 complex and the relevant TLRs.published_or_final_version
Dentistry
Doctoral
Doctor of Philosophy
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Gonzalez, Daniel. « Les "phosphate binding protein" : entre import du phosphate et inhibition de la transcription virale ». Thesis, Aix-Marseille, 2014. http://www.theses.fr/2014AIXM4019.
Texte intégralRésumé :
Les « phosphate binding protein » (PBP) constituent une famille de protéines présentes de manière ubiquitaire chez les bactéries et plus marginalement chez les Eucaryotes. Impliquées dans l'import du phosphate extracellulaire chez les bactéries, les PBPs présentent un site de fixation du phosphate très bien caractérisé avec, notamment, une liaison hydrogène particulière nommée «low barrier hydrogen bond» (LBHB). Cette LBHB est impliquée dans la discrimination entre le phosphate et des anions proches chez les PBPs. Bien que cette discrimination semble nécessiter une haute conservation du site de fixation du phosphate, dans la nature différentes configurations sont observées. Au cours de ce travail, nous nous sommes intéressés à la PBP d'un organisme pathogène, C.perfringens qui présente un site de fixation alternatif. Avec, entre autre, une perte de la LBHB, cette PBP présente la plus faible capacité de discrimination testée à ce jour. Cette faible capacité de discrimination pourrait être liée au biotope de la bactérie ou bien à un phénomène d'adaptation fonctionnelle. D'autre part, certaines PBPs présentent des propriétés d'inhibition du VIH via l'étape de la transcription virale. Cependant, ces protéines sont particulièrement difficiles à produire en système hétérologue limitant l'étude fonctionnelle. Afin de lever ce verrou technique, nous avons développé une nouvelle méthodologie basée sur la phylogénie en vue de solubiliser notre modèle d'étude (HPBP). Nous avons obtenu un variant soluble de HPBP qui conserve ses activités antivirales permettant de débloquer les études fonctionnellesThe "phosphate binding protein" constitutes a family of proteins ubiquitously found in Prokaryotes but also more sparsely distributed in Eukaryotes. Involved in phosphate import, PBPs exhibits a well-characterized phosphate binding site with a peculiar hydrogen bond called "low barrier hydrogen bond" (LBHB). This LBHB is involved in the unique discrimination properties of PBPs, capable of discriminating phosphate from other similar anions such as arsenate of sulfate. Albeit this high discriminating property needs a high conservation of the phosphate binding pocket, different configurations are observed in nature. Herein, we have been interested in a PBP from a human pathogen, Clostridium perfringens, which presents an alternative phosphate binding site. Exhibiting a loss of the LBHB, C.perfringens PBP is the least discriminating PBP isolated so far. This weak discrimination property might be related to the environment of C.perfringens or to a functional adaptation of the PBP. On the other hand, PBPs issued from eukaryotic tissues exhibit HIV inhibition properties via a step not yet targeted in current therapies, i.e. the transcription. However, these proteins are difficult to obtain from human tissues and their expression in heterologous system remains impossible. We have developed a new methodology based on phylogeny in order to solubilise our study model, HPBP. Thus, we have obtained a soluble variant of HPBP which conserves the HIV-inhibiting properties. This unique tool both allow to unlock functional studies and lead to a better understanding on how PBPs are capable of inhibiting HIV
Livres sur le sujet "LPS- binding proteins"
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Wiklund, Olov, et Jan Borén. Pathogenesis of atherosclerosis : lipid metabolism. Oxford University Press, 2017. http://dx.doi.org/10.1093/med/9780198755777.003.0011.
Texte intégralRésumé :
Lipids are carried in plasma as microparticles, lipoproteins, composed of a core of hydrophobic lipids and a surface of amphipathic lipids. In addition, the particles carry proteins (i.e. apolipoproteins). The proteins have key functions in the metabolism as receptor ligands, enzymes or activators. Lipoproteins are classified based on density into: chylomicrons, VLDL, IDL, LDL, and HDL. Retention of apoB-containing lipoproteins (LDL, IDL, and VLDL) in the arterial intima is the initiating event in the development of atherosclerosis. Retention is mediated by binding of apoB to structural proteoglycans in the intima. Increased plasma concentration of apoB-containing lipoproteins is the main risk factor for atherosclerotic cardiovascular disease (CVD) and the causative role of LDL has been demonstrated in several studies. Lp(a) is a subclass of LDL and elevated Lp(a) is an independent risk-factor, primarily genetically mediated. Genetic data support that high Lp(a) causes atherosclerosis. Elevated triglycerides in plasma are associated with increased risk for CVD. Whether triglycerides directly induce atherogenesis is still unclear, but current data strongly support that remnant particles from triglyceride-rich lipoproteins are causal. HDL are lipoproteins that have been considered to be important for reversed cholesterol transport. Low HDL is a strong risk-factor for CVD. However, the causative role of HDL is debated and intervention studies to raise HDL have not been successful. Reduction of LDL is the main target for prevention and treatment, using drugs that inhibit the enzyme HMG-CoA reductase, i.e. statins. Other drugs for LDL reduction and to modify other lipoproteins may further reduce risk, and new therapeutic targets are explored.
Chapitres de livres sur le sujet "LPS- binding proteins"
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Hirata, M., M. Yoshida, K. Inada et T. Kirikae. « Investigation of Endotoxin Binding Cationic Proteins from Granulocytes ; Agglutination of Erythrocytes Sensitized with Re-LPS ». Dans Endotoxin, 287–99. Boston, MA : Springer US, 1990. http://dx.doi.org/10.1007/978-1-4757-5140-6_25.
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Lefèvre, G. « Le h-FABP (Heart Fatty Acid Binding Protein) ». Dans Les biomarqueurs en médecine d’urgence, 121–26. Paris : Springer Paris, 2012. http://dx.doi.org/10.1007/978-2-8178-0297-8_16.
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Taylor, A. H., M. Nedelman, D. Sherris, E. Brunt, J. Ghrayeb, D. Knight et G. A. Heavner. « Structure-function analysis of LPS neutralizing synthetic peptides derived from human LPS binding protein ». Dans Peptides 1994, 652–53. Dordrecht : Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-1468-4_298.
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Onur Yaman, Suzan, et Adnan Ayhanci. « Lipid Peroxidation ». Dans Lipid Peroxidation [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.95802.
Texte intégralRésumé :
Lipid peroxidation (LPO) is initiated by the attack of free radicals (eg OH ·, O2- and H2O2) on cellular or organelle membranes phospholipids or polyunsaturated fatty acids (PUFA), and with the formation of various types of aldehydes, ketones, alkanes, carboxylic acids and polymerization products. It is an autoxidation process that results. These products are highly reactive with other cellular components and serve as biological markers of LPO. Malondialdehyde (MDA), a toxic aldehyde end product of LPO, causes structural changes that mediate its oxidation, such as fragmentation, modification, and aggregation, especially in DNA and protein. The excessive binding of these reactive aldehydes to cellular proteins alters membrane permeability and electrolyte balance. Degradation of proteins leads to progressive degradation of the biological system mediated by oxidative stress. The chain reaction (CR) of LPO is initiated by the attack of free radicals on the PUFA of the cell membrane to form a carbon centered radical (R*). The O2 · - radical attacks the other lipid molecule to form lipid hydroperoxide (ROOH), thereby spreading the CR and forming the lipid peroxyl radical (ROO). These lipid hydroperoxides severely inhibit membrane functionality by allowing ions such as increased hardness and calcium to leak through the membrane. Damage to the lipid membrane and macromolecule oxidation can result in activation of necrotic or apoptotic tissue death pathways if severe enough.
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Janero, David R., Anisha Korde et Alexandros Makriyannis. « Ligand-Assisted Protein Structure (LAPS) : An Experimental Paradigm for Characterizing Cannabinoid-Receptor Ligand-Binding Domains ». Dans Methods in Enzymology, 217–35. Elsevier, 2017. http://dx.doi.org/10.1016/bs.mie.2017.06.022.
Texte intégralActes de conférences sur le sujet "LPS- binding proteins"
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Tokunaga, F., T. Miyata, T. Nakamura, T. Morita et S. Iwanaga. « LIPOPOLYSACCHARIDE-SENSITIVE SERINE-PROTEASE ZYMOGEN (FACTOR C) OF LIMULUS HEMOCYTES : IDENTIFICATION AND ALIGNMENT OF PROTEOLYTIC FRAGMENTS PRODUCED DURING THE ACTIVATION SHOW THAT IT IS A NOVEL TYPE OF SERINE-PROTEASE ». Dans XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644609.
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Limulus clotting factor, factor C, is a lipopolysaccharide (LPS)-sensitive serine-protease zymogen present in the hemocytes. It is a two-chain glycoprotein (M.W. = 123,000) composed of a heavy chain (M.W. = 80,000) and a light chain (M.W. = 43,000) T. Nakamura et al. (1986) Eur. J. Biochem. 154, 511-521 .On further studies of this zymogen, a single-chain factor C (M.W. = 123,000) was identified by Western blotting technique. The heavy chain had an NH2-terminal sequence of Ser-Gly-Val-Asp-, which was consistent with the NH2-terminal sequence of the single-chain factor C, indicating that the heavy chain is located in the NH2-terminal part of the zymogen. The light chain had an NH22-terminal sequence of Ser-Ser-Gln-Pro-. Incubation of the two-chain zymogen with LPS resulted in the cleavage of a Phe-Ile bond between residues 72 and 73 of the light chain. Concomitant with this cleavage, the A (72.amino acids) and B chains derived from the light chain was formed. The complete amino acid sequence of the A chain was determined by automated Edman degradation. The A chain contained a typical segment which is similar structuraly to those a family of repeats in human β2 -glycoprotein I, complement factors B, Clr, Cls, H, C4b-binding protein, 02, coagulation factor XIII b subunit, haptoglobin a chain, and interleukin 2 receptor. The NH2-terminal sequence of the B chain was Ile-Trp-Asn-Gly-. This chain contained the serine-active site sequence of -ASP-Ala-Cys-Ser-Gly-Asp-SER-Gly-Gly-Pro-.These results indicate that limulus factor C exists in the hemocytes in a single-chain zymogen form and is converted to an active serine-protease by hydrolysis of a specific Phe-Ile peptide bond. The correlation of limulus factor C and mammalian complement proteins was also suggested.
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Steiner, Robert F., et Lynn Norris. « Fluorescence Dynamics Of Calcium-Binding Proteins ». Dans OE LASE'87 and EO Imaging Symp (January 1987, Los Angeles), sous la direction de E. R. Menzel. SPIE, 1987. http://dx.doi.org/10.1117/12.966939.
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Ye, Xiaobing, et Shu F. Liu. « LPS Differentially Regulates Specificity Protein (Sp1) DNA Binding Activity In Heart, Lungs And Liver ». Dans American Thoracic Society 2010 International Conference, May 14-19, 2010 • New Orleans. American Thoracic Society, 2010. http://dx.doi.org/10.1164/ajrccm-conference.2010.181.1_meetingabstracts.a4951.
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Lan, Jianqing, et Robert F. Steiner. « Studies of the interactions between calcium-binding proteins and phosphofructokinase using fluorescent probes ». Dans OE/LASE '90, 14-19 Jan., Los Angeles, CA, sous la direction de Joseph R. Lakowicz. SPIE, 1990. http://dx.doi.org/10.1117/12.17749.
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Mao, Sun-Zhong, Xiaobing Ye et Shu F. Liu. « LPS Down-Regulates Specificity Protein 1 DNA Binding Activity Through Nf-&kB Signaling Pathway In Endotoxemic Mice ». Dans American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a5772.
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Bein, Kiflai, et George Leikauf. « Macrophages Mediate LPS-Induced Increased CCAAT/enhancer Binding Protein (C/EBP), Beta (C/EBP-Beta) Transcripts In Lung Epithelial Cells ». Dans American Thoracic Society 2011 International Conference, May 13-18, 2011 • Denver Colorado. American Thoracic Society, 2011. http://dx.doi.org/10.1164/ajrccm-conference.2011.183.1_meetingabstracts.a5099.
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Pannekok, H., A. J. Van Zonneveid, C. J. M. de vries, M. E. MacDonald, H. Veerman et F. Blasi. « FUNCTIONAL PROPERTIES OF DELETION-MUTANTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR ». Dans XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643724.
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Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.