Studying dynamics without explicit dynamics: a structure-based study of the export mechanism by AcrB

RND family proteins are transmembrane proteins identified as large spectrum drug transporters. A prototypical case in this superfamily, responsible for antibiotic resistance in selected gram negative bacteria, is AcrB. AcrB forms a trimer, which uses the proton motive force to efflux drugs, implementing a functional rotation mechanism. Unfortunately, the size of the system (1049 amino-acid per monomer and membrane) has prevented a systematic dynamical exploration, so that the mild understanding of this coupled transport jeopardizes our ability to counter it. To further our understanding, we present a novel strategy based on two key ingredients which are to study dynamics by exploiting information embodied in the numerous crystal structures of AcrB obtained to date, and to systematically consider subdomains, their dynamics, and their interactions. Along the way, we identify the subdomains responsible for dynamic events, refine the states (A,B,E) of the functional rotation mechanism, and analyze the evolution of intra-monomer and inter-monomer interfaces along the functional cycle. Our analysis paves the way to targeted simulations exploiting the most relevant degrees of freedom at certain steps, and also to a targeting of specific interfaces to block the drug efflux. More generally, our work shows that complex dynamics can be unveiled from static snapshots, and our strategy may be used on a variety of molecular machines of large size

The Hedgehog Receptor Patched Is Involved in Cholesterol Transport

International audienceBACKGROUND: Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened. METHODOLOGY/PRINCIPAL FINDINGS: Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol. CONCLUSION/SIGNIFICANCE: Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation

Heterologous Expression and Purification Systems for Structural Proteomics of Mammalian Membrane Proteins

Membrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are implicated in numerous diseases, such as cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer's disease. However, studies on these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at very low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the different heterologous expression systems, and the purification strategies, used to produce large amounts of pure mammalian MPs for structural proteomics

Heterologous expression of membrane proteins for structural analysis.

International audienceMembrane proteins (MPs) are responsible for the interface between the exterior and the interior of the cell. These proteins are involved in numerous diseases, like cancer, cystic fibrosis, epilepsy, hyperinsulinism, heart failure, hypertension and Alzheimer disease. However, studies of these disorders are hampered by a lack of structural information about the proteins involved. Structural analysis requires large quantities of pure and active proteins. The majority of medically and pharmaceutically relevant MPs are present in tissues at low concentration, which makes heterologous expression in large-scale production-adapted cells a prerequisite for structural studies. Obtaining mammalian MP structural data depends on the development of methods that allow the production of large quantities of MPs. This review focuses on the heterologous expression systems now available to produce large amounts of MPs for structural proteomics, and describes the strategies that allowed the determination of the structure of the first heterologously expressed mammalian MPs

Expression et purification du récepteur humain de la voie Hedgehog, Smoothened, dans une conformation native et stable

La voie de signalisation Hedgehog constitue l une des plus importantes voies dans le développement embryonnaire et la prolifération des cellules souches chez l adulte. Cette voie implique deux protéines membranaires, Patched et Smoothened, dont les dysfonctionnements sont associés à de très nombreux cancers. Durant ma thèse, j ai mis au point l expression hétérologue du récepteur humain Smoothened (hSmo) et sa purification pour une caractérisation structurale et fonctionnelle. L expression de hSmo a été réalisée dans la levure Saccharomyces cerevisiae. Utilisant la technique SPR, j ai démontré que hSmo exprimé à la membrane plasmique de la levure est dans sa conformation native capable de fixer son antagoniste. J ai ensuite mis au point la purification de hSmo et testé de nouvelles classes de surfactants. J ai ainsi trouvé les meilleures conditions qui stabilisent le récepteur hSmo en solution après purification. La caractérisation d une mutation ponctuelle au niveau de la 3ème boucle intracellulaire combinée à l utilisation des nouveaux surfactants ont permis d améliorer la stabilité de hSmo en solution. Les conditions que j ai mises au point permettront l étude structurale de hSmo et les essais de cristallisation. D autre part, les stratégies développées en SPR permettront la recherche des partenaires protéiques cytoplasmiques de ce récepteur, encore inconnus à ce jour, afin de mieux comprendre la signalisation en aval de Smoothened. Les données structurales ainsi que la découverte des partenaires protéiques cytoplasmiques de hSmo permettront l élaboration de nouvelles thérapies anticancéreuses.The Hedgehog pathway is one of the most important pathways in embryogenesis and in proliferation of adult stem cells. This pathway involves two transmembrane receptors, Patched and Smoothened whose dysfunctions have been linked to many human diseases including cancers. This study reports expression and purification of the human GPCR Smoothened, for structure-function relationship characterization. Therefore I developed the heterologous expression of Human Smoothened (hSmo) in the yeast S. cerevisiae. Using SPR technology, I showed that hSmo, expressed at the plasma membrane of yeast, is in its native conformation able to bind its antagonist, cyclopamine (CPN). Then, I developed the purification of hSmo by affinity chromatography and tested new surfactants. Results show that the new surfactants stabilize hSmo in solution after purification and are preserve antagonist-binding ability of Smo suggesting that purified hSmo maintains its native conformation in solution. In addition, characterization of a single mutation of Smoothened (hSmoG435R) combined to one of the surfactants, revealed an enhanced stability of the receptor. These established conditions will be useful for crystallization assays. SPR strategies developed in this study will also be used for the research of hSmo s cytoplasmic partners. Together, structural and functional data will contribute to the better understanding of Smo signaling and to the development of new cancer therapies.NICE-BU Sciences (060882101) / SudocSudocFranceF

Targeting the Multidrug Transporter Ptch1 Potentiates Chemotherapy Efficiency

One of the crucial challenges in the clinical management of cancer is resistance to chemotherapeutics. Multidrug resistance (MDR) has been intensively studied, and one of the most prominent mechanisms underlying MDR is overexpression of adenosine triphosphate (ATP)-binding cassette (ABC) transporters. Despite research efforts to develop compounds that inhibit the efflux activity of ABC transporters and thereby increase classical chemotherapy efficacy, to date, the Food and Drug Administration (FDA) has not approved the use of any ABC transporter inhibitors due to toxicity issues. Hedgehog signaling is aberrantly activated in many cancers, and has been shown to be involved in chemotherapy resistance. Recent studies showed that the Hedgehog receptor Ptch1, which is over-expressed in many recurrent and metastatic cancers, is a multidrug transporter and it contributes to the efflux of chemotherapeutic agents such as doxorubicin, and to chemotherapy resistance. Remarkably, Ptch1 uses the proton motive force to efflux drugs, in contrast to ABC transporters, which use ATP hydrolysis. Indeed, the “reversed pH gradient” that characterizes cancer cells, allows Ptch1 to function as an efflux pump specifically in cancer cells. This makes Ptch1 a particularly attractive therapeutic target for cancers expressing Ptch1, such as lung, breast, prostate, ovary, colon, brain, adrenocortical carcinoma, and melanoma. Screening of chemical libraries have identified several molecules that are able to enhance the cytotoxic effect of different chemotherapeutic agents by inhibiting Ptch1 drug efflux activity in different cancer cell lines that endogenously over-express Ptch1. In vivo proof of concept has been performed in mice where combining one of these compounds with doxorubicin prevented the development of xenografted adrenocortical carcinoma tumors more efficiently than doxorubicin alone, and without obvious undesirable side effects. Therefore, the use of a Ptch1 drug efflux inhibitor in combination with classical or targeted therapy could be a promising therapeutic option for Ptch1-expressing cancers

Expression et purification de protéines membranaires mammifères impliquées dans des pathologies

À l interface entre la cellule et le milieu extérieur, les protéines membranaires ont un rôle indispensable au fonctionnement cellulaire. Elles jouent un grand nombre de fonctions et peuvent être impliquées dans des pathologies graves. La connaissance de ces protéines membranaire peut permettre de mieux comprendre de nombreux phénomènes biologiques et pathologiques et leur localisation, accessible, en fait de bonne cibles thérapeutique. La connaissance de la structure des protéines apporte une quantité d information très importante sur leur fonctionnement. À ce jour si plusieurs dizaines de milliers de structures de protéines solubles ont pu être résolues, moins de deux cents structures de protéines membranaires sont connues. Cette différence provient des difficultés à produire des protéines membranaires pures et stables en quantités nécessaires à la détermination des structures tridimensionnelles. Les conditions d expression des protéines membranaires est très variables en fonction de la protéine. Notre équipe de recherche s intéresse au développement de stratégies pour l expression hétérologue et la purification de protéines membranaires impliquées dans des pathologies humaines dans le but d en étudier la structure et les relations structurefonction et par une approche protéine. Dans cette thèse sont présentés les résultats obtenus sur l expression et la purification des récepteurs humains de la voie de signalisation Hedgehog, Patched et Smoothened, dont le disfonctionnement est mis en cause dans de nombreux cancers mais également dans des maladies neuro-dégénératives. Ces protéines ont pu être exprimées dans différents systèmes hétérologues : les levures Saccharomyces cerevisiae et Pichia pastoris et dans les Cellules S2 de drosophile. Nous avons pu mettre en évidence que Smoothened était exprimé sous sa forme native dans les levures et qu il était possible de la purifier et la produire dans l optique d une étude structurale à partir de Pichia pastoris (de Rivoyre et al, FEBS letters, 2005). Patched et Smoothened ont également pu être exprimé stablement et fonctionnellement par des clones de cellules S2. Ce système qui permet la purification de ces protéines s est également avéré très intéressant pour une étude comparative du fonctionnement des protéines humaines et des protéines de drosophile (de Rivoyre et al, JBC, 2006).Membrane-bound proteins play a significant role in cell function due to their position in between the cell and the external medium. These proteines are for this reason, involved in a number of human diseases. Knowing membrane-bound proteins will allow us to better understand several biological and pathophysiological functions. Furthermore, their localisations make them interesting therapeutic targets. Knowing the structure of proteins gives a tremendous amount of information on their functions. To date, even if several thousands structures of soluble proteins have been solved; only less than two hundred structures of membrane-bound proteins are known. This important difference is partly due to the fact that membrane-bound proteins are difficult to obtain pure in a stable conformation in order to determine their three-dimensional structures. Recombinant expression of membrane-bound proteins has a high variability depending on the nature of the protein studied. Our research team is therefore interested in the development of recombinant expression and purification strategies of membrane-bound proteins involved in numerous human diseases, in order to study their functions but also to establish structurefunction relationships. My thesis work has focused on the expression and the purification of human receptors of the Hedgehog pathway, Patched and Smoothened. Alteration of these two proteins is known to be involved in numerous cancers but also in some neurological diseases. These two proteins have been expressed in two different systems : yeast cells Saccharomyces cerevisiae and Pichia pastoris and also in drosophila cells S2. We have been able to show that the protein Smoothened is expressed in Pichia pastoris in its native conformation in yeast cells an dit is therefore possible to purify it in order to perform structural studies. Patched and Smoothened have also been stably and functionaly expressed in S2 cells. This system is also interesting to perform comparative studies between drosophila and human proteins.NICE-BU Sciences (060882101) / SudocSudocFranceF