Apolipoprotein A-I (apoA-I) and ATP-Binding Cassette A1 (ABCA1) transporter play important roles in nascent high density lipoprotein (nHDL) biogenesis – the first step in the reverse cholesterol transport pathway. Based on the crystal structure of a C-terminally truncated form of apoA-I (apoA-I(1-184)) determined in the laboratory, structurally designed and naturally occurring mutants of apoA-I were conformationally characterized in solution. The function of these mutants in nHDL formation was assessed in ABCA1-transfected HEK293 cells. An apoA-I mutant designed to destabilize the N-terminal helical bundle at the first hinge region, 38/40G, exhibited a locally reduced α-helical content, destabilized overall structure, and increased lipid binding ability in solution, indicating a destabilized N-terminal helical bundle. In the cellular system, 38/40G showed significantly enhanced nHDL forming ability, suggesting that a destabilized N-terminal bundle will facilitate nHDL formation. Other designed N-terminal mutants (Q41A, P66G, G65A, V67P, T68P, 65/67/68P) and the naturally occurring mutants (R153P, L178P, and insertion mutant apoA-INashua) all showed either unchanged or destabilized overall structure, unchanged lipid binding abilities in solution and unchanged nHDL formation and cholesterol efflux promotion from the cells. Mutants designed to progressively extend the C-terminus (1-184, 1-198, 1-209, 1-220, 1-231) yielded progressively increased nHDL formation and cholesterol efflux, suggesting that the C-terminus of apoA-I is critical for these two activities. Central Helix 5 triple glycine mutation (H5 3xG) designed to lock the monomer conformation of apoA-I resulted in reduced nHDL formation but unaffected cholesterol efflux, suggesting that hindering apoA-I monomer to dimer conversion could retard nHDL formation. Remarkably, studies of cholesterol efflux and nHDL particle formation indicated that the two processes might be two uncoupled events. Analysis of the nHDL particles revealed the presence of ganglioside (GM1) in the complexes. Cross-linking data demonstrated binding of apoA-I to ABCA1-expressing cells. The binding level of apoA-I mutants to ABCA1-expressing cells was positively correlated with nHDL forming ability of these mutants. ABCA1 was isolated from FreeStyle™ HEK293-F cells in suspension by detergent solubilization and was shown to have ATPase activity. A direct interaction between apoA-I and amphipol solubilized- ABCA1 in solution was detected for the first time. Furthermore, the successful purification of ABCA1 has laid the foundation of structure determination of this protein in the future.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21820号 / 農博第2333号 / 新制||農||1067(附属図書館) / 学位論文||H31||N5192(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 和光, 教授 三芳 秀人, 教授 三上 文三 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

In this study, we examined the effects of thyroid hormone on regulatory processes of cholesterol metabolism. Specifically, the pathways of cholesterol synthesis and cholesterol efflux were investigated. Hepatic HMG-CoA reductase (HMGR) is the rate-limiting enzyme in cholesterol synthesis. Hypothyroid rats exhibit decreased expression of this gene, which can be induced by subsequent treatment with thyroid hormone. The mechanism of this activation was previously unknown. Utilizing in vivo electroporation, we identified HMGR promoter elements necessary for the induction of HMGR by thyroid hormone. The -316/-321 element, the sterol response element, and nuclear factor-y site were all found to be necessary to induce HMGR promoter activity by thyroid hormone. We used electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) studies to demonstrate increased binding of upstream transcription factor-2 (USF-2) to the -316/-321 element in the HMGR promoter in response to thyroid hormone. Finally, co-electroporation of the wild-type HMGR plasmid with siRNA to USF-2, SREBP-2, or NF-Y nearly abolished the T3 induction as measured by promoter activity. Microarray and real-time PCR analysis demonstrated an induction of the apolipoproteins ApoA-I and ApoA-IV mRNA by T3. Serum levels of ApoA-I and ApoA-IV proteins were induced by T3. We collected serum from rats treated with or without T3 and used these sera in an in vitro macrophage efflux model. We found that T3 promoted cholesterol efflux via the ABCA1 cholesterol transporter and not via the ABCG1 transporter. We propose that the induction of serum ApoA-I and ApoA-IV by thyroid hormone promotes cholesterol efflux via the ABCA1 cholesterol transporter. Hepatic ABCG5 and ABCG8 are cholesterol transporters that promote biliary secretion of cholesterol. We utilized EMSAs to scan the shared ABCG5/G8 rat promoter for a thyroid hormone response element (TRE). We identified a TRß binding site at -392/-376 of the ABCG8 promoter. Collectively, these observations provide new insight into the cholesterol-lowering function of thyroid hormone.

La apoA-I cumple un rol muy importante en el transporte reverso del colesterol (TRC), es el componente mayoritario de las lipoproteínas de alta densidad (HDL) que desempeñan diversas funciones en las distintas etapas del TRC. Resultados previos de este laboratorio permiten postular la hipótesis de que la región central de la apoA-I, formada por el par de hélices tipo Y, estaría involucrada en la interacción con la membrana celular, que sería importante para el eflujo de lípidos y la movilización de depósitos intracelulares de colesterol (como el disponible a ser esterificado por ACAT) hacia la membrana plasmática. Como la conformación del dominio central es influenciada por el tamaño y composición lipídica (contenido de colesterol) de las HDL, también se postula que esto podría modular la capacidad de interacción con la membrana celular y el consecuente eflujo lipídico. El objetivo general de este trabajo fue someter a prueba esta hipótesis y aportar información relevante para entender los mecanismos implicados en las etapas iniciales del TRC, como en la interacción de las HDL con membranas celulares y el eflujo celular de lípidos. Como objetivos específicos, nos propusimos: 1) Reconstituir partículas discoidales HDL similares a las pre-β-HDL del plasma, de diferente composición y tamaño, mediante la técnica de diálisis con el detergente colato. Estas fueron comparadas en cuanto a su capacidad de unirse a la membrana celular, y de promover el eflujo de colesterol y fosfolípidos de dos líneas celulares diferentes: CHO-K1 (células de ovario de hámster chino) y RAW 264.7 (macrófagos murinos). 2) Estudiar en comparación con apoA-I salvaje, la funcionalidad y las respuestas celulares a dos mutantes de deleción de un residuo de lisina en las regiones de hélices tipo Y: una con la deleción en la región central de la hélice 4 (ΔK107) y la segunda con la deleción en la posición homóloga de la hélice 10 (ΔK226). La primera de estas mutantes es una variante natural cuyos portadores presentan un metabolismo alterado de las HDL e incrementado riesgo aterogénico, por lo que los resultados de estos estudios también podrían ayudar a la comprensión de los síntomas presentados por estos pacientes. Es de esperar que estas mutaciones desplacen en ~100º la orientación relativa entre las caras hidrofílica e hidrofóbica de la hélice anfipática a ambos lados de la mutación, lo que puede afectar tanto la interacción con lípidos como con los receptores celulares.

A large number of proteins interact with biological membranes, either integrated in the membrane (PepTSo2), embedded on a membrane surface (5-lipoxygenase) or encircling a cutout of lipid bilayer (apolipoprotein1 (apoA-I). They function as transporters, receptors or biocatalysts in cellular processes like inflammation or cholesterol transport which are touched upon here. Malfunction of specific membrane proteins are the cause for several diseases or disorders. Knowledge of protein structure supports understanding of its mechanism of function. Here, transmission electron microscopy (TEM) was used for structure determination. To obtain structure information to high resolution for membrane proteins, normally surrounded by lipids, demands specific methods and materials for stabilization. Stabilized in detergent the structure of the bacterial transporter PepTSo2 was shown to form a tetramer even bound to substrate. However, with a protein based stabilizer, Salipro, the structure of PepTSo2 could be determined to high resolution. High density lipoprotein (HDL) in blood plasma, involved in the removal of cholesterol from peripheral tissues, have a central role in cardiovascular function, metabolic syndrome and diabetes. The HDL-particle is composed of two copies of ApoA1 and around hundred lipid molecules. From TEM data, for the first time the clearly discoidal shape could be shown by 3-dimendional reconstructions. These were used for modelling the ApoA1 protein dimer by a "biased fitting" procedure. The results indicate how ApoA1 folds around a lipid bilayer in a disc-shaped structure. Modified HDL called nanodiscs were here used to show the Ca2+ dependent binding of 5-lipoxygenase on the nanodisc bilayer and thereby increased production of the inflammatory mediator leukotrieneA4. Dimerization of 5-lipoxygenase inactivates these functions. / <p>QC 20170323</p>

No description available.

Η HDL είναι ένα μείγμα λιποπρωτεϊνικών σωματιδίων υψηλής πυκνότητας, που ανάλογα με τη σύσταση τους σε λιπίδια μπορούν να είναι δισκοειδή ή σφαιρικά. Η κύρια αθηροπροστατευτική δράση της HDL, οφείλεται στο γεγονός ότι η συγκεκριμένη λιποπρωτεΐνη συλλέγει την περίσσεια χοληστερόλης από τους περιφερικούς ιστούς και τη μεταφέρει στο ήπαρ όπου καταβολίζεται. Επιπλέον, έχει αντιφλεγμονώδη και αντιοξειδωτική δράση. Η κύρια πρωτεΐνη της HDL είναι η απολιποπρωτεΐνη Α-Ι (apoA-I). Ωστόσο, πρόσφατα αποδείχθηκε ότι σε πειραματόζωα με έλλειψη στην apoA-I και κατά συνέπεια στην κλασσική HDL, η απολιποπρωτεΐνη Ε (apoE) αλληλεπιδρά με τον μεταφορέα λιπιδίων ABCA1 προάγοντας την de novo σύνθεση HDL σωματιδίων. Στην παρούσα μελέτη, στόχος ήταν η εύρεση της περιοχής της apoE που είναι υπεύθυνη για την λειτουργική αλληλεπίδραση με τον ABCA1 για το σχηματισμό HDL. Για το σκοπό αυτό, ανασυνδυασμένοι αδενοϊοί που εξέφραζαν καρβοξυ-τελικές συντετμημένες μορφές της apoE4 (AdGFP-E4[1-259], AdGFP-E4[1-229], AdGFP-E4[1-202], AdGFP-E4[1-185]), χορηγήθηκαν σε ποντίκια με έλλειψη στην ApoA-I σε δόση 8x108 pfu και πέντε μέρες μετά τη μόλυνση δείγματα πλάσματος αναλύθηκαν για το σχηματισμό HDL. Κλασματοποίηση των λιποπρωτεϊνών του πλάσματος με υπερφυγοκέντρηση σε διαβάθμιση πυκνότητας καθώς και FPLC χρωματογραφία αποκάλυψε ότι όλες οι συντετμημένες μορφές της apoE4 προάγουν το σχηματισμό HDL. Ανάλυση ηλεκτρονικής μικροσκοπίας με αρνητική χρώση των HDL κλασμάτων, επιβεβαίωσε ότι όλες οι συντετμημένες μορφές της apoE4 είναι ικανές να προάγουν το σχηματισμό σωματιδίων με διάμετρο στην περιοχή της HDL. Τα δεδομένα αυτά οδηγούν στο συμπέρασμα ότι η αμινοτελική περιοχή της apoE που εκτείνεται από τα αμινοξέα 1 έως 185 αρκεί για το σχηματισμό HDL σωματιδίων in vivo. Αυτά τα ευρήματα, ανοίγουν το δρόμο στην έρευνα για το σχεδιασμό βιολογικών φαρμάκων με βάση την apoE για τη θεραπεία της δυσλιπιδαιμίας, της αθηροσκλήρωσης και της στεφανιαίας νόσου. / HDL is a mixture of high density lipoprotein particles that depending on the lipid composition may be discoidal or spherical. The main atheroprotective property of HDL is reverse cholesterol transport, a process that unloads excess cholesterol from peripheral tissues and transports it to the liver for catabolism. HDL has also anti-inflammatory and antioxidant properties. The main protein of HDL is apolipoprotein A-I (apoA-I). However, recently it was shown that in the absence of apoA-I and consequently classical HDL, apolipoprotein E (apoE) interacts functionally with the lipid transporter ABCA1, promoting the de novo synthesis of HDL-like particles. The present study focused on the identification of the domain of apoE that is responsible for the functional interaction with ABCA1 and the formation of apoE-containing HDL. Recombinant attenuated adenoviruses expressing carboxy-terminal truncated forms of apoE4 (apoE4[1-259], apoE4[1-229], apoE4[1-202], and apoE4[1-185]) were administered to apoA-I-deficient mice at a low dose of 8x108 pfu and five days post-infection plasma samples were isolated and analyzed for HDL formation. Fractionation of plasma lipoproteins of the infected mice by density gradient ultracentrifugation and FPLC revealed that all forms were capable of promoting HDL formation. Negative staining electron microscopy analysis of the HDL density fractions confirmed that all C-terminal truncated forms of apoE4 promoted the formation of particles with diameters in the HDL region. Taken together, these data establish that the aminoterminal 1 to 185 region of apoE suffices for the formation of HDL particles in vivo. These findings may have important ramifications in the design of apoE-based biological drugs for the treatment of dyslipidemia, atherosclerosis and coronary heart disease.