Drug transport mechanism of P-glycoprotein monitored by single molecule fluorescence resonance energy transfer

In this work we monitor the catalytic mechanism of P-glycoprotein (Pgp) using single-molecule fluorescence resonance energy transfer (FRET). Pgp, a member of the ATP binding cassette family of transport proteins, is found in the plasma membrane of animal cells where it is involved in the ATP hydrolysis driven export of hydrophobic molecules. When expressed in the plasma membrane of cancer cells, the transport activity of Pgp can lead to the failure of chemotherapy by excluding the mostly hydrophobic drugs from the interior of the cell. Despite ongoing effort, the catalytic mechanism by which Pgp couples MgATP binding and hydrolysis to translocation of drug molecules across the lipid bilayer is poorly understood. Using site directed mutagenesis, we have introduced cysteine residues for fluorescence labeling into different regions of the nucleotide binding domains (NBDs) of Pgp. Double-labeled single Pgp molecules showed fluctuating FRET efficiencies during drug stimulated ATP hydrolysis suggesting that the NBDs undergo significant movements during catalysis. Duty cycle-optimized alternating laser excitation (DCO-ALEX) is applied to minimize FRET artifacts and to select the appropriate molecules. The data show that Pgp is a highly dynamic enzyme that appears to fluctuate between at least two major conformations during steady state turnover.Comment: 10 pages, 7 figure

A P-glikoprotein membrán mikrokörnyezete és működésének gátolhatósága modulátor és antitest együttes alkalmazásával = P-glycoprotein membrane microenviroment and inhibition of pumping by the combined application of modulators and specific antibody

A multidrog rezisztencia hátterében gyakran a P-glikoprotein (Pgp) pumpa működése áll, melynek gátlására jelentős erőfeszítések történnek világszerte. Az UIC2 antitest a fehérjét katalitikus ciklusa egy fázisában ismeri fel, és ott blokkolja. Az így elért gátlás azonban általában kisfokú, mert az antitest a sejtfelszíni Pgp-knek csak egy hányadához kötődik - vizsgálataink utóbbi jelenség megértésére és befolyásolására irányultak. Megállapítottuk, hogy egy korábbi munkáinkban leírt teszt segítségével azonosítható szubsztrát/modulátor (S/M) csoport bármelyik tagja jelenlétében az antitest az összes sejtfelszíni Pgp-hez kötődik és azt inaktív állapotba hozza. Ez a S/M-ok önmagukban hatástalan koncentrációja mellett valósul meg, és in vivo is megtörténik, mint azt xenotranszplantációs kísérletekben demonstráltuk. Megállapítottuk, hogy az UIC2 által mindig felismert Pgp-k hányada (pool I) lipid tutaj- (raft)- asszociált, klasztereket alkot és az UIC2 kötés nyomán fokozottan internalizálódik. Számos olyan fehérjét azonosítottunk, melyek a Pgp molekuláris környezetében vannak: ezek nagy része az aktomiozin-kapcsolt trafficking apparátus része. Az antitest által csak egyes S/M-ok jelenlétében felismert Pgp-k szoliter molekulák. Mindkét pool funkcionális Pgp molekulákat tartalmaz, a pool I valamilyen endogén szubsztrát által indukált ATPáz aktivitás során csapdázódik az UIC2 jelenlétében. A kétféle lipid környezet az eltérő oldékonyságú S-ok optimális transzportját szolgálhatja. | P-glycoprotein (Pgp), an ABC transporter often responsible for the multidrug resistance of cancer cells, is recognized and inhibited by the UIC2 mAb that freezes the transporter in a particular phase of its catalytic state. However, the degree of this inhibition is mitigated by the lack of its binding to all cell surface Pgps, a phenomenon poorly understood. We have demonstrated that Pgp is present in two distinct subpopulations on the cell surface: Both pools contain functional Pgp molecules, but only pool I is recognized in the absence of exogeneous substrates/modulators (S/M-s), while pool II Pgps bind UIC2 only upon addition of certain S/M-s, identified by a test introduced by us earlier. In the presence of any of this group of S/M-s administered at such a low concentration which is insufficient to cause Pgp inhibition when used alone, the mAb can completely inhibit the pump also in vivo, as demonstrated in xenotransplantation experiments. Pgp molecules of the two pools could be distinguished in terms of membrane microdomain localization, clustering propensity, cytoskeletal anchorage, intracellular molecular neighbours, and trafficking characteristics. Endocytosis of Pgp involves the actin microfilament system, and primarily targets the raft associated pool I, when an ATP- conformational state is stabilized. The presence of the pump in two different lipid environment may be optimal for the transport of S-s with different solubilities

Development of Immobilised Biopolymer Stationary Phases based on the Efflux Transporters

The body is continuously exposed to a variety of toxins and metabolic waste products but is able to rid itself of these by using various detoxification mechanisms such as enzymes and transmembrane transporters. A large number of the transporters which play an essential role in the detoxification mechanisms are found in the liver, kidney and intestines. The largest family of transporters is the Solute Carrier (SLC) Superfamily with 255 members in humans. While most SLC transporters are highly specialized, a number of these transporters are polyspecific, generalized transporters that play a major role in the elimination process. Accordingly, the aim of this research programme was to develop novel methodology to enable the study of the interaction of drugs and related compounds with these transporters, in a rapid and facile manner. In the initial studies the application of affinity chromatography was carried out with the use of α1-acid glycoprotein (AGP) column to achieve enantioselective separation of the drug ketamine and its metabolites. The enantioselective separation of ketamine and norketamine from plasma samples was achieved and the assay conducted was sensitive and reproducible. Building upon the experience gained in this work on affinity chromatography and LC-MS, these useful tools for the determination of chiral drugs in biological fluids were also used in the study of drug-protein interactions. With the nicotinic acetylcholine receptors it was possible to explore the use of immobilized liquid chromatographic stationary phases containing drug transporters in on-line high throughput screening (HTS). I Thus having demonstrated that a stationary phase containing immobilized membranes can be used to identify substrate/inhibitors of an expressed receptor/transporter the next and principal phase of the programme was to adapt the methodology to other target biopolymers such as P-glycoprotein which over the last decade has been focused upon for its role in drug resistance in cancer treatments. In particular liquid chromatographic columns containing the drug transporters, P-glycoprotein (Pgp) and human organic cation transporter 1 (hOCT1) were prepared, evaluated and exploited. Initial studies were conducted to confirm the functionality of a stably transfected cell line expressing Pgp (Pgp-(+), LCC6/MDR1 cell line) through a comparison with the non-transfected cell line (Pgp-(-), LCC6 cell line). Initially membranes from the Pgp(+) and Pgp(-) cell lines were then immobilized on immobilized artificial membranes. However, although the resulting column could actively bind the Pgp substrates, strong non-specific interactions with the IAM backbone led to large retention times and peak tailing. A more successful approach was to immobilize on the surface of open tubular capillaries. Such columns were used to sort compounds with or without affinity for Pgp, by comparing the differential retention time on the Pgp (+) and Pgp (-) columns. In this way the non-specific interactions with the constituents of the cellular membrane were compensated for. The results from the sorting by differential chromatography were compared with the behavior of the same compounds in Caco-2 monolayers cultured in 96-well transwell plates, the standard method for the determination of substrates for Pgp. A group of 14 compounds II previously characterized as substrates or non-substrates of Pgp were studied using the chromatographic and Caco-2 methods. In the parallel chromatographic screen, the value o

Advance crew procedures development techniques: Procedures generation program requirements document

The Procedures Generation Program (PGP) is described as an automated crew procedures generation and performance monitoring system. Computer software requirements to be implemented in PGP for the Advanced Crew Procedures Development Techniques are outlined

The Personal Genome Project-UK, an open access resource of human multi-omics data

Integrative analysis of multi-omics data is a powerful approach for gaining functional insights into biological and medical processes. Conducting these multifaceted analyses on human samples is often complicated by the fact that the raw sequencing output is rarely available under open access. The Personal Genome Project UK (PGP-UK) is one of few resources that recruits its participants under open consent and makes the resulting multi-omics data freely and openly available. As part of this resource, we describe the PGP-UK multi-omics reference panel consisting of ten genomic, methylomic and transcriptomic data. Specifically, we outline the data processing, quality control and validation procedures which were implemented to ensure data integrity and exclude sample mix-ups. In addition, we provide a REST API to facilitate the download of the entire PGP-UK dataset. The data are also available from two cloud-based environments, providing platforms for free integrated analysis. In conclusion, the genotype-validated PGP-UK multi-omics human reference panel described here provides a valuable new open access resource for integrated analyses in support of personal and medical genomics

Beneficial Bacteria Isolated from Grapevine Inner Tissues Shape Arabidopsis thaliana Roots

We investigated the potential plant growth-promoting traits of 377 culturable endophytic bacteria, isolated from Vitis vinifera cv. Glera, as good biofertilizer candidates in vineyard management. Endophyte ability in promoting plant growth was assessed in vitro by testing ammonia production, phosphate solubilization, indole-3-acetic acid (IAA) and IAA-like molecule biosynthesis, siderophore and lytic enzyme secretion. Many of the isolates were able to mobilize phosphate (33%), release ammonium (39%), secrete siderophores (38%) and a limited part of them synthetized IAA and IAA-like molecules (5%). Effects of each of the 377 grapevine beneficial bacteria on Arabidopsis thaliana root development were also analyzed to discern plant growth-promoting abilities (PGP) of the different strains, that often exhibit more than one PGP trait. A supervised model-based clustering analysis highlighted six different classes of PGP effects on root architecture. A. thaliana DR5::GUS plantlets, inoculated with IAA-producing endophytes, resulted in altered root growth and enhanced auxin response. Overall, the results indicate that the Glera PGP endospheric culturable microbiome could contribute, by structural root changes, to obtain water and nutrients increasing plant adaptation and survival. From the complete cultivable collection, twelve promising endophytes mainly belonging to the Bacillus but also to Micrococcus and Pantoea genera, were selected for further investigations in the grapevine host plants towards future application in sustainable management of vineyards