O gene AGT1 do locus mal1g, do sistema de transporte de maltose de S. cerevisiae, codifica uma proteína de 67 kDa, que tem 75% de similaridade e 58% de identidade com o transportador de maltose do locus MAL1. Sua expressão é ativada por genes reguladores constitutivos do sistema MAL, e é reprimida pela glicose. A cepa AP68-7A carrega o gene AGT1, e possivelmente o gene transportador MAL31, e transporta trealose ao final da primeira fase de crescimento anaeróbico. SDS-PAGE comparando proteínas de membranas de células reprimidas pela glicose (taxa de transporte <0,5 U/mg), com aquelas de células induzidas por α-metilglicosídeo (taxa de transporte de ~35 U/mg), verificou-se 2 bandas (PMs 57 e 66 kDa) exclusivas em membranas de células induzidas. As 2 bandas foram isoladas por três métodos diferentes (cromatografia de troca iônica, lavagens da membrana com tampão de alta força iônica e cromatografia de afinidade) e testadas para ligação de 14C-trealose. A ligação foi enriquecida ~3 X após a cromatografia de troca iônica. O transporte de trealose na cepa AP77-4C (que não tem nenhum dos genes transportadores dos loci MAL) foi recuperado após sua transformação com plasmídeo YEp366-AGT1. De membranas plasmáticas destas células (transporte de trealose ~25 U/mg) foram isoladas por cromatografia de afinidade, 2 bandas cujos PMs em SDS-PAGE são idênticos aos das proteínas isoladas das membranas da cepa AP68-7A. Estes resultados permitem concluir que o transportador de trealose de leveduras é codificado pelo gene AGT1. / The AGT1 gene presente in the mal1g locus from S. cerevisiae maltose transport system encodes a 67 kDa protein which shares 75% similarity and 58% identity with the maltose transporter protein encoded in MAL6 locus. The expression of this gene is regulatory genes from MAL system and is repressed by glucose. The strain AP68-7A which harbors the AGT1 gene and probably the MAL31 transporter gene, expresses trehalose transport activity at the end of first anaerobic growth. The comparison from the SDS-PAGE of membrane proteins from glucose repressed cells (trehalose transport activity of <0.5 U/mg), and α-methylglucoside induced cells (trehalose transport activity of ~35 U/mg), revealed 2 bands (Mr 57 and 66 kDa) present only in the induced cells membranes. Those bands were isolated by 3 different methods (ionic exchange chromatography, high strength ionic washes and affinity chromatography, and tested for 14C-trehalose binding. Both bands bind trehalose and this activity was enriched about 3 times after the ionic exchange chromatography. The trehalose transport activity was recovered by strain AP77-4C, (which does not harbor any MAL transporter gene) after its transformation with a plasmid containing the AGT1 gene. From the membranes of these cells (trehalose transport activity of ~25 U/mg) 2 bands were isolated by affinity chromatography with similar Mrs to those isolated from AP68-7A strain. The results permit to conclude that the AGT1 gene encodes the yeast trehalose transporter.

L’enveloppe des bactéries à Gram négatif est composée de plusieurs polysaccharides dont certains, comme le peptidoglycane et les lipopolysaccharides, sont essentiels à leur survie et sont impliqués dans les interactions entre ces bactéries et leur environnement (e.g. résistance à des agents antimicrobiens et reconnaissance par le système immunitaire de l’hôte). La biosynthèse de ces polymères nécessite la translocation de leurs sous-unités à travers la membrane interne, ce qui requiert un lipide "porteur", l’undécaprényl phosphate (C55-P). Ce lipide est formé par la déphosphorylation de son précurseur, l’undécaprényl pyrophosphate (C55-PP), qui est lui-même généré par synthèse de novo ou par un recyclage. Chez Escherichia coli, les protéines membranaires BacA, YbjG, PgpB et LpxT présentent une activité C55-PP phosphatase et participe donc de façon redondante au recyclage du C55-P. Pour mieux comprendre le rôle physiologique spécifique de ces protéines dans la biogénèse de l’enveloppe, notre travail a porté sur l’étude des mécanismes d’action, de la fonction et de la régulation de deux de ces enzymes, PgpB et LpxT.L’activité de la protéine PgpB a été caractérisée in vivo et in vitro afin de mieux comprendre son rôle et sa capacité à participer à deux voies métaboliques essentielles, le recyclage du C55-P et la biosynthèse du phosphatidylglycérol. Nous avons mis en évidence des résidus d’acides aminés qui étaient essentiels à la catalyse des deux substrats naturels et d’autres qui n’avaient pas le même rôle dans l’hydrolyse des deux substrats, nous permettant de proposer des mécanismes différenciés. En outre, des données calorimétriques ont montré que PgpB pouvait être grandement stabilisée par la liaison d’un substrat. Hypothétiquement, le changement structural sous-jacent serait susceptible de libérer de l’énergie mobilisée pour le flip du produit à travers la membrane. La protéine LpxT est responsable d’une modification constitutive des lipopolysaccharides en transférant le phosphate libéré du C55-PP sur la partie lipide A. Dans certaines conditions de stress, l’activité de LpxT est spécifiquement inhibée par un petit peptide (PmrR) pour permettre à d’autres modifications de s’opérer. Nous avons montré que la modification catalysée par LpxT était déterminante pour permettre à E. coli de résister aux acides biliaires et de coloniser efficacement le tractus intestinal de l’hôte. LpxT constitue ainsi un point de contrôle clé chez E. coli pour résister à différent agents antimicrobiens qui ciblent les lipopolysaccharides mais qui possèdent des propriétés physico-chimiques opposées. En outre, nous avons montré que LpxT et PmrR formaient un complexe stable mais que de façon inattendue, cette liaison n’inhibait pas l’activité phosphotransférase de l’enzyme in vitro. Nous proposons que PmrR inhibe l’activité de LpxT in vivo en modifiant sa capacité à interagir avec ses partenaires de la voie de biosynthèse des lipopolysaccharides. La biogénèse des polymères de l’enveloppe est absolument nécessaire à la survie des bactéries et les modifications structurales de ces éléments sont autant de stratégies plus ou moins spécifiques qui participent au fitness et à un mode de vie particulier des bactéries. Les C55-PP phosphatases qui participent activement à ces processus constituent ainsi autant de cibles potentielles intéressantes pour la conception de nouveaux antibiotiques. / The bacterial cell envelope is composed of many polysaccharides such as the peptidoglycan and the lipopolysaccharides (LPS) that are required for survival and are involved in the interactions that the bacteria establish with their surroundings, including antimicrobial resistance and host immune system recognition. The biosynthesis of these polymers requires the translocation of the sugar sub-units across the plasma membrane, which implies an essential lipid carrier, the undecaprenyl phosphate lipid (C55-P). This lipid is generated by the dephosphorylation of its precursor, C55-PP, itself arising from either de novo synthesis or recycling. The Escherichia coli bacterial species possesses four membrane proteins (BacA, YbjG, PgpB and LpxT) exhibiting a C55-PP phosphatase activity, which all contribute redundantly to C55-P recycling. To highlight the specific physiological role of these enzymes in the cell wall biogenesis, our work was focused on the characterization of the mechanism of action, the function and the regulation of two of these phosphatases, PgpG and LpxT.The PgpB activity was characterized both in vivo and in vitro to decipher its role and ability to participate in two essential metabolic pathways, the C55-P recycling and the phosphatidylglycerol biosynthesis. We identified essential residues of PgpB involved in the hydrolysis of both natural substrates, C55-PP and PGP, and some others that function differently in the hydrolysis of these two lipids, allowing us to propose different reaction mechanisms for this enzyme. In addition, calorimetric data showed that substrate binding greatly stabilized the PgpB protein. Hypothetically, the underlying structural change would release energy allowing translocation of the lipid across the membrane. LpxT is responsible for a constitutive modification of the LPS by transferring the phosphate released from C55-PP to lipid A. Under certain environmental stresses, the LpxT activity is specifically inhibited by a small peptide (PmrR), thereby allowing other modifications of the lipid A structure to take place. We showed that the LpxT-dependent modification accounts for bile acids resistance in E. coli and is critically important for E. coli colonization in the host’s gut. LpxT constitutes a key critical control point in E. coli to resist different antimicrobial agents with opposite physical-chemical properties but which all target the LPS. In addition, we demonstrated that PmrR forms a stable complex with LpxT, but unexpectedly, this binding did not inhibit the phosphotransferase activity of the enzyme in vitro. We propose that PmrR inhibits LpxT activity in vivo by modulating its ability to interact with its protein partners that are involved in LPS biosynthesis. The biogenesis of cell wall polymers is essential for bacterial survival and structural changes in these components participate more or less specifically to the fitness and particular lifestyles of bacteria. The C55-PP phosphatases which participate actively in these processes therefore constitute interesting potential targets for new antibiotics design.

No description available.

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23225号 / 工博第4869号 / 新制||工||1760(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 跡見 晴幸, 教授 大塚 浩二 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Integral membrane proteins (IMPs) assume critical roles in cell biology and are key targets for drug discovery. Given their involvement in a wide range of diseases, the structural and functional characterization of IMPs are of significant importance. However, this remains notoriously challenging due to the difficulties of stably purifying membrane-bound, hydrophobic proteins. Compounding this, many diseases are caused by IMP mutations that further decrease their stability. One such example is cystic fibrosis (CF), which is caused by misfolding or dysfunction of the epithelial cell chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). Roughly 70% of CF patients world-wide harbor the ΔF508-CFTR mutation, which interrupts CFTR’s folding, maturation, trafficking and function. No existing treatment sufficiently addresses the consequences of ΔF508, and the substantial instability that results from this mutation limits our ability to study ΔF508-CFTR in search of better treatments. To that end, my colleagues at Sanofi generated homology models of full-length wild-type and ΔF508-CFTR +/- second-site suppressor mutations (SSSMs) V510D and R1070W, and performed molecular dynamics (MD) simulations for each model. Using information obtained from this analysis, I tested several hypotheses on the mechanism by which ΔF508 destabilizes full-length CFTR and how SSSMs suppress this effect. Leveraging studies of the purified NBD1 subdomain and of full-length CFTR in a cellular context, I confirmed the prediction of a key salt-bridge interaction between V510D and K564 important to second-site suppression. Furthermore, I identified a novel class of SSSMs that support a key prediction from these analyses: that helical unraveling of TM10, within CFTR’s second transmembrane domain, is an important contributor to ΔF508-induced instability. In addition, I developed a detergent-free CFTR purification method using styrene-maleic acid (SMA) copolymer to extract the channel directly from its cell membrane along with the surrounding lipid content. The resulting particles were stable, monodisperse discs containing a single molecule of highly-purified CFTR. With this material, I optimized grid preparation techniques and carried out cryo-EM structural analysis of WT-hCFTR which resulted in 2D particle class averages which were consistent with an ABC transporter shape characteristic of CFTR, and a preliminary 3D reconstruction. This result establishes a foundation for future characterization of ΔF508-CFTR in its native state. I have also applied this SMA-based purification method to the facilitated glucose transporter GLUT1 (SLC2A1). SLC2A1 mutations contribute to a rare and developmentally debilitating disease called GLUT1-deficiency syndrome. Using SMA, I successfully extracted GLUT1 in its native state. With the application of this method, I was able to purify endogenous GLUT1 from erythrocytes, in complex with several associated proteins as well as the surrounding lipids, in its monomeric, dimeric and tetrameric forms without the use of cross-linking or chimeric mutations. These results point to the potential for studying isolated IMPs without the use of destabilizing detergents and thereby offer a pathway to analysis of wild-type and mutant membrane protein structure, function and pharmacodynamics.

Colicin A is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of target bacteria. In the present thesis, we aimed at studying the closed channel state, the membrane insertion mechanism, the acidic pH induced molten globule state and the interaction of colicin A in living E. coli cells. For that, we used Electron Paramagnetic Resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) method to explore the structural details of colicin A. The EPR studies of the membrane-bound colicin A (reconstituted into proteoliposomes) suggest the transmembrane orientation of the hydrophobic hairpin in the closed channel state. The pH dependent membrane insertion studies indicate that the membrane binding efficiency is significantly enhanced at pH < 3. Moreover, in the presence of a membrane potential, the pH induced membrane-bound state is able to open channels in the liposomes. The membrane-bound conformation (induced by acidic pH) is similar to the conformation of reconstituted colicin A which support the umbrella model for the closed channel state of colicin A. The studies on pH dependent conformational changes suggest that colicin A forms a molten globule at pH 2. The molecular details of pH induced conformational changes were analyzed by molecular dynamic simulations. The results of the MD simulations agree with the EPR results. Conformational changes of colicin A upon interaction with living E. coli cells could also be followed. Comparison between colicin A in wild type (WT) cells and tolB knock-out mutants suggest that the observed conformational changes originate from colicin A which has been already translocated to the inner membrane.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第23405号 / 理博第4740号 / 新制||理||1679(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 高田 彰二, 教授 川口 真也, 准教授 立川 正志 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(エネルギー科学) / 甲第21193号 / エネ博第367号 / 新制||エネ||72(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 木下 正弘, 教授 森井 孝, 教授 片平 正人 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21379号 / 農博第2303号 / 新制||農||1071(附属図書館) / 学位論文||H30||N5152(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 栗原 達夫, 教授 植田 充美, 教授 小川 順 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM