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Structural and mechanistic studies of liver ABC transporters

English title Structural and mechanistic studies of liver ABC transporters
Applicant Locher Kaspar
Number 189111
Funding scheme Project funding (Div. I-III)
Research institution Institut für Molekularbiologie und Biophysik Deptartement für Biologie ETH Zurich
Institution of higher education ETH Zurich - ETHZ
Main discipline Biophysics
Start/End 01.10.2019 - 30.09.2023
Approved amount 1'348'838.00
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All Disciplines (2)

Discipline
Biophysics
Biochemistry

Keywords (16)

Membrane protein; Cryo-EM; Function; Electron microscopy; Bile salt; Canalicular; Nanodisc; Membrane; Liver; ATP-binding cassette; Transporter; BSEP; Structure; ABC transporter; Mechanism; Phospholipid

Lay Summary (German)

Lead
Strukturelle und mechanistische Untersuchung von Transportproteinen der Leber
Lay summary

Eine der wesentlichen Funktionen der Leber ist die Erzeugung von Galle. Dieser Prozess beginnt mit Hepatozyten (Leberzellen), die die Bestandteile der Galle in Canaliculi absondern, winzige Kanäle, die die Primärgalle sammeln und zu den Gallengängen führen. Eine Schlüsselrolle in diesem Sekretionsschritt kommt in die Kanalmembran eingebetteten Transportproteinen zu. Zwei davon, genannt BSEP und ABCB4, sind in der Biomedizin von besonderer Bedeutung, da ihre Funktionsstörung zu einer Reihe von Gesundheitsproblemen führen kann, einschließlich der vererbten progressiven Cholestase oder Gallensteinen. Darüber hinaus können Arzneimittel die Funktion von BSEP oder ABCB4 beeinträchtigen und so zu einer durch Arzneimittel verursachten Leberschädigung führen.
Dieses Forschungsprojekt zielt darauf ab, die strukturellen und mechanistischen Eigenschaften von menschlichem BSEP und ABCB4 zu untersuchen. Es sollen insbesondere hochaufgelöste Strukturen von humanem BSEP und ABCB4 in unterschiedlichen Funktionszuständen mithilfe der Einzelpartikel-Kryo-Elektronenmikroskopie bestimmt werden.Zudem sollen funktionelle Studien es erlauben, kinetische Reaktionsparameter zu bestimmen und mechanistische Hypothesen zu formulieren.
Die geplanten Studien werden das Verständnis einer Schlüsselfunktion der Leber durch mechanistische Einblicke in spezifische, durch ABC-Transporter katalysierte Reaktionen erweitern. Darüber hinaus können Erkenntnisse darüber, wie BSEP und ABCB4 durch niedermolekulare Verbindungen (einschließlich häufig verwendeter Arzneimittel) gehemmt werden, helfen, die Toxizität von zukünftigen Medikamenten besser abzuschätzen.

Direct link to Lay Summary Last update: 07.10.2019

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196862 Structural and mechanistic investigations of membrane-integral enzymes involved in eukaryotic protein N-glycosylation pathway 01.01.2021 Project funding (Div. I-III)
166672 Structural and mechanistic studies of components of bacterial protein N-glycosylation pathway and of vitamin B12 transport 01.04.2016 Project funding (Div. I-III)
170808 Acquisition of a Talos Arctica transmission electron microscope for single particle analysis and cryo-tomography 01.05.2017 R'EQUIP
125762 NCCR TransCure: From transport physiology to identification of therapeutic targets (phase I) 01.11.2010 National Centres of Competence in Research (NCCRs)

Abstract

One of the essential functions of the liver is the generation of bile. This process starts with hepatocytes secreting bile constituents into canaliculi, tiny canals that collect the primary bile and lead it to the bile ducts. A key role in the secretion from hepatocytes is attributed to three transport proteins embedded in the canalicular membrane: The bile salt export pump BSEP (ABCB11), the phospholipid “floppase” ABCB4, and the cholesterol transporter ABCG5/G8. The function of these transporters guarantees the correct concentration ratio of the bile constituents. BSEP and ABCB4 are particularly relevant in biomedicine because their dysfunction can lead to a number of health problems including inherited progressive cholestasis or gall stones. In addition, drugs that interfere with BSEP or ABCB4 can lead to drug-induced liver injury (DILI).This project aims at understanding the structural and mechanistic properties of human BSEP and ABCB4. They belong to the B-subfamily of ABC transporters and share structural features with the multidrug transporter P-glycoprotein (ABCB1), but exhibit clearly distinct, non-overlapping functions. Over the past years, researchers have investigated the physiological roles of these bile-generating ABC transporters in vivo and using cellular in vitro systems. However, an in-depth understanding of their physiological activity and their inhibition by small-molecule compounds requires structural and mechanistic insight at high resolution. Until recently, this would have constituted a daunting “mission impossible” due to the difficulties involved in generating well-ordered 3D crystals of such dynamic membrane proteins for X-ray crystallographic structure determination. However, the advent of single-particle cryo-EM has allowed such projects to be tackled with an excellent likelihood of success. The goals of this project are the following:• We will determine high-resolution cryo-EM structures of human BSEP and ABCB4 reconstituted in lipidic nanodiscs and trapped in distinct functional states, including apo states, nucleotide- and substrate-bound states, as well as inhibited states containing bound small-molecule compounds.• We will develop in vitro transport and ATPase assays using purified and reconstituted BSEP and ABCB4 to determine kinetic reaction parameters and formulate and explore mechanistic hypotheses. In combination with mutagenesis, structure-function studies will be pursued to probe transport and inhibition mechanisms and to trap distinct functional states for structural studies.• To explore the physiological context of BSEP and ABCB4, we will use cryo-electron tomography to visualize canalicular structures, initially in the HepG2 model cell line that forms canaliculi in cell culture. If successful, the method may then be applied to primary liver cell lines or expanded to study rodent or human liver biopsies.The combination of high-resolution structures and functional analyses will allow us to (i) generate “molecular movies” that capture essential conformational transitions in the transport cycles of BSEP and ABCB4, (ii) visualize specific interactions with substrates, nucleotides, and inhibitors, and (iii) rationalize disease- causing mutations. To achieve the required “side-chain resolution”, specific conformational antibody fragments will be generated. These include Fab fragments derived from IgG-type antibodies and nanobodies derived from cameloid heavy-chain-only antibodies.The planned studies will advance our understanding of a key function of the liver by providing mechanistic insight into specific, ABC transporter-catalyzed reactions. In addition, insight obtained into how BSEP and ABCB4 are inhibited by small-molecule compounds (including commonly used drugs) may have a translational value in rationalizing the associated drug-induced liver injury. This may provide early decision tools in drug development, aiming at minimizing or avoiding hepatotoxicity. The cryo-electron tomographic studies of canalicular structures will provide novel insight into the physiological context of BSEP and ABCB4 and may reveal differences in the ultrastructure of healthy vs. cholestatic liver samples.
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