A humán ABCG2 multidrog transzporter működésének vizsgálata = Investigation of the mechanism of action of the human ABCG2 multidrug transporter

A humán ABCG2 egy plazma membrán glikoprotein, amely a szervezet méregtelenítésében játszik szerepet, ezen kívül ellenállóvá teheti a rákos sejteket bizonyos gyógyszerekkel szemben. Az ABCG2 az őssejtek egyik jellegzetes fehérjéje. Jelen kutatás során a következő főbb eredményeket értük el: 1. Változatos szubsztrát specificitással és transzport aktivitással bíró mutáns változatokat hoztunk létre a vad-típustól mindössze a 482-es aminosav pozícióban eltérő ABCG2 mutánsok előállításával, amelyek hasznosíthatók lehetnek például terápiás sejtek védelmében. 2. Igazoltuk, hogy az ABCG2-Q141K polimorfizmus csökkent aktivitással bír, ezért az ezt a változatot hordozó egyedek bizonyos gyógyszerekkel szemben érzékenyebbek lehetnek. 3. Kimutattuk, hogy az ABCG2 kölcsönhatásba lép egyes, a klinikumban használt tirozin kináz gátló gyógyszerekkel, tehát befolyásolhatja ezek hatékonyságát a kemoterápia során. 4. Elvégeztük egy ABCG2-specifikus antitest jellemzését, amely hasznosítható lehet tumoros mintákban lévő ABCG2 kimutatására, vagy akár őssejtek kiválogatására. 5. Kimutattuk, hogy a membrán koleszterin tartalma befolyásolja az ABCG2 transzporter aktivitását, tehát a fehérje működése különböző membrán kompartmentekbe történő lokalizációja illetve internalizációja által szabályozódhat. 6. Fluoreszcens ?címkével? ellátott ABCG2 fehérjét hoztunk létre, amellyel pl. ABCG2 szubsztrátok tesztelhetők élő sejtekben vagy lokalizációs vizsgálatok végezhetők. | Human ABCG2 is a plasma membrane glycoprotein that protects our body against toxic compounds. ABCG2 is also able to confer resistance against several drugs used in cancer chemotherapy. ABCG2 is a marker protein of stem cells. The main findings of our research are the following: 1) We have generated ABCG2 mutants having altered substrate specificity and transport activity by inducing single amino acid change at position 482. These mutants are ideal candidates to be used for protection of modified cells in medical gene therapy. 2) We have shown that the Q141K polymorphism of ABCG2 has decreased activity, therefore may result in altered pharmacokinetics of ABCG2 substrates and increase chemosensitivity in clinical oncology. 3) We have demonstrated that ABCG2 interacts with several clinically relevant tyrosine kinase inhibitors; therefore this protein may influence their effectiveness. 4) We have characterised an ABCG2-specific antibody that can be an important tool in detection of ABCG2 in clinical samples or in stem cell research. 5) We have shown that membrane cholesterol level influences the activity of ABCG2, therefore the localisation of ABCG2 e.g. in cholesterol rich rafts may have major impact on the activity of this transporter. 6) We have generated a cell line expressing functionally active, green fluorescence protein tagged ABCG2 that may be a useful tool for e.g. localisation studies or drug-screening in living cells

The importance of steroid uptake and intracrine action in endometrial and ovarian cancers

Endometrial and ovarian cancers predominately affect women after menopause, and are more frequently observed in developed countries. These are considered to be hormone-dependent cancers, as steroid hormones, and estrogens in particular, have roles in their onset and progression. After the production of estrogens in the ovary has ceased, estrogen synthesis occurs in peripheral tissues. This depends on the cellular uptake of estrone-sulfate and dehydroepiandrosterone-sulfate, as the most important steroid precursors in the plasma of postmenopausal women. The uptake through transporter proteins, such as those of the organic anion-transporting polypeptide (OATP) and organic anion-transporter (OAT) families, is followed by the synthesis and action of estradiol E2. Here, we provide an overview of the current understanding of this intracrine action of steroid hormones, which depends on the availability of the steroid precursors and transmembrane transporters for precursor uptake, along with the enzymes for the synthesis of E2. The data is also provided relating to the selected transmembrane transporters from the OATP, OAT, SLC51, and ABC-transporter families, and the enzymes involved in the E2-generating pathways in cancers of the endometrium and ovary. Finally, we discuss these transporters and enzymes as potential drug targets. © 2017 Rižner, Thalhammer and özvegy-Laczka

Az ABCA1 membránfehérje funkciójának és fehérje-kölcsönhatásainak vizsgálata = Investigation of the function and protein interactions of the ABCA1 membrane protein

A kutatás célja az ABCA1 membránfehérje működésének és fehérje-kölcsönhatásainak jellemzése volt. Új modellrendszereket alakítottunk ki Sf9 rovarsejt-bakulovírus és retrovirális expressziós rendszerek segítségével, részletesen vizsgáltuk a vad-típusú és mutáns ABCA1 fehérjék sejten belüli lokalizációját, működését és PDZ fehérjékkel való kölcsönhatását. Bizonyítottuk, hogy az ABCA1 fehérje mind az ApoA1-függő koleszterin kiáramlás, mind a Ca2+-aktivált sejtfelszíni foszfatidilszerin expozíció folyamatában fontos szerepet játszik. Elsőként mutattunk ki összefüggést egy vérzékenységi betegség és az ABCA1 működése között. Elemeztük az ABCA1 mutációnak hatását a betegségre jellemző hibás foszfatidilszerin expozícióban. Vizsgáltuk a lipidanyagcserére ható vegyületek hatását az ABCA1-hez köthető funkciókra, azonosítottunk két új gátló vegyületet. Megállapítottuk, hogy egy speciális PDZ fehérje a vizsgált ABC fehérjék közül egyedül az ABCA1 fehérjével lép kölcsönhatásba, más ABC transzporterekhez kötő egyéb PDZ fehérjék nem kötődtek az ABCA1-hez. Kimutattuk polarizált sejtekben az ABCA1, a b2-syntrophin és az utrophin bazolaterális ko-lokalizációját. A kidolgozott mérési módszereket más ABC transzporterek működésének vizsgálatára is eredményesen alkalmaztuk. Megkezdtük a foszfolipid-transzportért felelős ABC fehérjék azonosítását trombocitákban. | The aims of this project were the functional characterization of the ABCA1 protein and identification of its potential interactions with intracellular proteins. We installed new assay systems to analyse the function, subcellular localization and protein interactions of the wild-type and mutant ABCA1 versions, by using two expression systems: the baculovirus-Sf9 insect cell system and retrovirus based expression system for mammalian cells. We proved that ABCA1 plays a key role both in cellular ApoAI-mediated cholesterol removal pathway, and in the exofacial translocation of phosphatidylserine. Our results provided the first link between a defect in a transbilayer phospholipid transport pathway, that of ABCA1, and the bleeding phenotype. We analysed the effects of various mutations of ABCA1 on the Ca2+-stimulated PS exposition. We screened the influence of potential inhibitors on the ABCA1-dependent processes and identified new inhibitors of the PS exposition. We demonstrated that among the examined ABC transporters only ABCA1 binds b2-syntrophin. A diverse group of PDZ proteins that interacts with other ABC proteins does not bind to ABCA1. We showed basolateral colocalization of ABCA1 protein with b2-syntrophin and utrophin. The assays for ABCA1 characterization were applied for studying other ABC proteins successfully. We started the identification of ABC proteins involved in phospholipid translocation in platelets

Lipid Regulation of the ABCB1 and ABCG2 Multidrug Transporters

This chapter deals with the interactions of two medically important multidrug ABC transporters (MDR-ABC), ABCB1 and ABCG2, with lipid molecules. Both ABCB1 and ABCG2 are capable of transporting a wide range of hydrophobic drugs and xenobiotics and are involved in cancer chemotherapy resistance. Therefore, the exploration of their mechanism of action has major therapeutic consequences. As discussed here in detail, both ABCB1 and ABCG2 are significantly affected by various lipid compounds especially those residing in their close proximity in the plasma membrane. ABCB1 is capable of transporting lipids and lipid derivatives, and thus may alter the general membrane composition by "flopping" membrane lipid constituents, while there is no such information regarding ABCG2. Still, both ABCB1 and ABCG2 show complex interactions with a variety of lipid molecules, and the transporters are significantly modulated by cholesterol and cholesterol derivatives at the posttranslational level. In this chapter, we explore the molecular details of the direct transporter-lipid interactions, the potential role of lipid-sensor domains within the proteins, as well as the application of experimental site-directed mutagenesis, detailed structural studies, and in silico modeling for examining these interactions. We also discuss the regulation of ABCB1 and ABCG2 expression at the transcriptional level, occurring through nuclear receptors involved in lipid sensing. The better understanding of lipid interactions with these medically important MDR-ABC transporters may significantly improve further drug development and clinical treatment options

Mutations of the central tyrosines of putative cholesterol recognition amino acid consensus (CRAC) sequences modify folding, activity, and sterol-sensing of the human ABCG2 multidrug transporter

Human ABCG2 is a plasma membrane glycoprotein causing multidrug resistance in cancer. Membrane cholesterol and bile acids are efficient regulators of ABCG2 function, while the molecular nature of the sterol-sensing sites has not been elucidated. The cholesterol recognition amino acid consensus (CRAC, L/V-(X)(1-5)-Y-(X)(1-5)-R/K) sequence is one of the conserved motifs involved in cholesterol binding in several proteins. We have identified five potential CRAC motifs in the transmembrane domain of the human ABCG2 protein. In order to define their roles in sterol-sensing, the central tyrosines of these CRACs (Y413, 459, 469, 570 and 645) were mutated to S or F and the mutants were expressed both in insect and mammalian cells. We found that mutation in Y459 prevented protein expression; the Y469S and Y645S mutants lost their activity; while the Y570S, Y469F, and Y645F mutants retained function as well as cholesterol and bile acid sensitivity. We found that in the case of the Y413S mutant, drug transport was efficient, while modulation of the ATPase activity by cholesterol and bile acids was significantly altered. We suggest that the Y413 residue within a putative CRAC motif has a role in sterol-sensing and the ATPase/drug transport coupling in the ABCG2 multidrug transporter

The role of organic anion transporting polypeptides in drug absorption, distribution, excretion and drug-drug interactions

INTRODUCTION: The in vivo fate and effectiveness of a drug depends highly on its absorption, distribution, metabolism, excretion and toxicity (ADME-Tox). Organic anion transporting polypeptides (OATPs) are membrane proteins involved in the cellular uptake of various organic compounds, including clinically used drugs. Since OATPs are significant players in drug absorption and distribution, modulation of OATP function via pharmacotherapy with OATP substrates/inhibitors, or modulation of their expression, affects drug pharmacokinetics. Given their cancer-specific expression, OATPs may also be considered anticancer drug targets. Areas covered: We describe the human OATP family, discussing clinically relevant consequences of altered OATP function. We offer a critical analysis of published data on the role of OATPs in ADME and in drug-drug interactions, especially focusing on OATP1A2, 1B1, 1B3 and 2B1. Expert opinion: Four members of the OATP family, 1A2, 1B1, 1B3 and 2B1, have been characterized in detail. As biochemical and pharmacological knowledge on the other OATPs is lacking, it seems timely to direct research efforts towards developing the experimental framework needed to investigate the transport mechanism and substrate specificity of the poorly described OATPs. In addition, elucidating the role of OATPs in tumor development and therapy response are critical avenues for further research

Selective antiproliferative effect of C-2 halogenated 13α-estrones on cells expressing Organic anion-transporting polypeptide 2B1 (OATP2B1)

Organic anion-transporting polypeptide 2B1 (OATP2B1) is a multispecific transporter mediating the cellular uptake of steroids and numerous drugs. OATP2B1 is abundantly expressed in the intestine and is also present in various tumors. Increased steroid hormone uptake by OATP2B1 has been suggested to promote progression of hormone dependent tumors. 13 alpha-estrones are effective inhibitors of endogenous estrogen formation and are potential candidates to inhibit proliferation of hormone dependent cancers. Recently, we have identified a variety of 13 alpha/beta-estrone-based inhibitors of OATP2B1. However, the nature of this interaction, whether these inhibitors are potential transported substrates of OATP2B1 and hence may be enriched in OATP2B1overexpressing cells, has not yet been investigated. In the current study we explored the antiproliferative effect of the most effective OATP2B1 inhibitor 13 alpha/beta-estrones in control and OATP2B1-overexpressing A431 carcinoma cells. We found an increased antiproliferative effect of 3-O-benzyl 13 alpha/beta-estrones in both mock transfected and OATP2B1-overexpressing cells. However, C-2 halogenated 13 alpha-estrones had a selective OATP2B1-mediated cell growth inhibitory effect. In order to demonstrate that increased sensitization can be attributed to OATP2B1-mediated cellular uptake, tritium labeled 2-bromo-13 alpha-estrone was synthesized for direct transport measurements. These experiments revealed increased accumulation of [H-3]2-bromo-13 alpha-estrone due to OATP2B1 function. Our results indicate that C-2 halogenated 13 alpha-estrones are good candidates in the design of anti-cancer drugs targeting OATP2B1

Az ABCG2 multidrog transzporter fehérje szerkezetének és működésének vizsgálata = Structure and function of the multidrug transporter ABCG2

Az ABCG2 multidrog transzporternek fontos szerepe van mind a daganatok kemoterápia-rezisztenciájában, mind a fiziológiás xenobiotikum transzportban. A projektben előállítottuk az ABCG2 fehérje különböző mutáns és polimorf változatait, elvégeztük ezek részletes funkcionális vizsgálatát. Egy sejtfelszínen reagáló, konformáció-érzékeny anti-ABCG2 monoklonális antitest alkalmazásával felderítettük a transzporter funkcionális állapotait, kémiai módosítások és mutációk segítségével elvégeztük az epitópok jellemzését és molekuláris szintű modellezését. Részletesen elemeztük az ABCG2 transzporter és a membrán lipidek kölcsönhatásait, megállapítottuk a membrán koleszterin jelentős szabályozó szerepét. Az ABCG2 transzporter és célzott hatású rákellenes vegyületek kölcsönhatásainak vizsgálata során klinikailag is alkalmazott gyógyszerekre vonatkozóan kaptunk új információkat. Új módszereket fejlesztettünk ki az ABCG2 szabályozásának, lokalizációjának és funkciójának vizsgálatára, elemeztük a transzporter expresszióját humán embrionális őssejtekben. Több, magas impakt faktorú nemzetközi folyóiratban közöltünk a témáról review cikkeket. | The human ABCG2 multidrug transporter plays a key role in the chemotherapy resistance of malignant tumors, as well as in the physiological elimination of xenobiotics. In this project we have prepared and expressed various mutant and polymorphic variants of the transporter, performed their detailed functional characterization. By using a cell-surface reacting, conformation-sensitive monoclonal antibody against ABCG2, we mapped the functional states of the transporter. In these experiments we applied specific chemical modifications and generated site-directed mutations to characterize the extracellular loop epitope region of ABCG2 and constructed a molecular model for this part of the transporter. We have investigated the modulation of ABCG2 by membrane lipids and found a major role for cholesterol in regulating the transport activity of this protein. By examining a number of new targeted anticancer agents we found that ABCG2 interacts with several of these compounds and may be involved in the resistance against clinically applied molecules. We have developed new methods for studying the regulation, localization and function of the ABCG2 protein, examined the expression profile of this transporter in human embryonic stem cells. During this project we have published several review articles in high-impact international journals