ABC TRANSZPORTEREK ÚTON ÚTFÉLEN

A különböző ABC transzporterek igen fontos szerepet látnak el a szervezet határ- felületei anyagforgalmának szabályozásában. Ezek az élettani határolók polari- zált sejtekből állnak, melyek szoros sejtkapcsolattal kapcsolódnak egymáshoz, megakadályozva az anyagok szabad áramlását. A polarizált sejtekben megfelelő rend szerint helyezkednek el a transzporter fehérjék, és funkciójuk révén megha- tározzák a különböző anyagok áthaladását a határfelületeken. Kutatásainkban arra keresünk választ, hogy az egyes ABC transzporterek milyen úton kerülnek el a feladatuk ellátásához szükséges cél-kompartmentbe, milyen tényezők ha- tározzák meg ott a sorsukat, és milyen szabályozó mechanizmusok működtetik a transzporter fehérjék sejten belüli vándorlását. Munkánk során elsősorban a májat alkotó poláris sejtekre: hepatocitákra és epevezetéksejtekre összponto- sítjuk figyelmünket, de reményeink szerint ezekkel a vizsgálatokkal általánosabb érvényű összefüggésekre tudunk majd fény deríteni

Functional Cooperativity between ABCG4 and ABCG1 Isoforms

ABCG4 belongs to the ABCG subfamily, the members of which are half transporters composed of a single transmembrane and a single nucleotide-binding domain. ABCG proteins have a reverse domain topology as compared to other mammalian ABC transporters, and have to form functional dimers, since the catalytic sites for ATP binding and hydrolysis, as well as the transmembrane domains are composed of distinct parts of the monomers. Here we demonstrate that ABCG4 can form homodimers, but also heterodimers with its closest relative, ABCG1. Both the full-length and the short isoforms of ABCG1 can dimerize with ABCG4, whereas the ABCG2 multidrug transporter is unable to form a heterodimer with ABCG4. We also show that contrary to that reported in some previous studies, ABCG4 is predominantly localized to the plasma membrane. While both ABCG1 and ABCG4 have been suggested to be involved in lipid transport or regulation, in accordance with our previous results regarding the long version of ABCG1, here we document that the expression of both the short isoform of ABCG1 as well as ABCG4 induce apoptosis in various cell types. This apoptotic effect, as a functional read-out, allowed us to demonstrate that the dimerization between these half transporters is not only a physical interaction but functional cooperativity. Given that ABCG4 is predominantly expressed in microglial-like cells and endothelial cells in the brain, our finding of ABCG4-induced apoptosis may implicate a new role for this protein in the clearance mechanisms within the central nervous system

Recent advances in the exploration of the bile salt export pump (BSEP/ABCB11) function

ntroduction: The bile salt export pump (BSEP/ABCB11), residing in the apical membrane of hepatocyte, mediates the secretion of bile salts into the bile. A range of human diseases is associated with the malfunction of BSEP, including fatal hereditary liver disorders and mild cholestatic conditions. Manifestation of these diseases primarily depends on the mutation type; however, other factors such as hormonal changes and drug interactions can also trigger or influence the related diseases. Areas covered: Here, we summarize the recent knowledge on BSEP by covering its transport properties, cellular localization, regulation and major mutations/polymorphisms, as well as the hereditary and acquired diseases associated with BSEP dysfunction. We discuss the different model expression systems employed to understand the function of the BSEP variants, their drug interactions and the contemporary therapeutic interventions. Expert opinion: The limitations of the available model expression systems for BSEP result in controversial conclusions, and obstruct our deeper insight into BSEP deficiencies and BSEP-related drug interactions. The knowledge originating from different methodologies, such as clinical studies, molecular genetics, as well as in vitro and in silico modeling, should be integrated and harmonized. Increasing availability of robust molecular biological tools and our better understanding of the mechanism of BSEP deficiencies should make the personalized, mutation-based therapeutic interventions more attainable

A plazmamembrán Ca2+ ATPáz 4b izoforma apoptotikus fragmentjét reprezentáló mutáns sejten belüli lokalizációja, stabilitása, hatásai a sejtek Ca2+ háztartására és szerepe az apoptózis folyamatban = Intracellular localization and stability of a mutant representing the apoptotic fragment of the plasma membrane Ca2+ ATPase 4b and its role in cellular Ca2+ homeostasis and apoptosis

A jelen kutatási periódus alatt egy fontos Ca2+ transzport fehérje, a plazma membrán Ca2+ ATPáz (PMCA4b) struktúra/funkció változásait tanulmányoztuk az apoptózis valamint a nekrózis folyamatai alatt. Eredményeink azt bizonyítják, hogy a PMCA4b fehérjét - függetlenül az apoptózist kiváltó októl - a kaszpáz-3 proteáz hasítja és egy 120 kDa molekulatömegű fragment képződik. A fehérje közben elveszíti C-terminális regulátor régióját, és -szuper aktívvá - válik. A "szuper aktív" PMCA fragmentnek megfelelő mutáns a plazmamembránban lokalizálódik és képes a citoszólikus Ca2+ szint szabályozására. Ezzel szemben oxidatív stresszt kiváltó szerek hatására a PMCA jelentős mértékű internalizációja és a citoszólikus Ca2+ szint hosszan tartó megemelkedése figyelhető meg. A C-terminális régió meghatározónak bizonyult a PMCA4b plazmamembránban történő eloszlásának szempontjából is. Kimutattuk, hogy a PSD-95 állványfehérje a PMCA C-terminálisán található PDZ-kötő motívumon keresztül elősegíti a PMCA kijutását a plazmamembránba, és itt a képződő fehérje-komplex szigetszerű csoportokba rendeződik. Vizsgálataink elősegíthetik a Ca2+ homeosztázis felborulására visszavezethető egyes malignus elváltozások illetve degeneratív betegségek közötti összefüggések jobb megismerését. | During the present research period we studied structural and functional changes of an essential Ca2+ extrusion protein, the plasma membrane Ca2+ pump (PMCA), during apoptosis and necrosis. We followed truncation of PMCA4b during apoptosis induced by mitochondrial or receptor-mediated pathways and found that a similar fragment of 120 kDa was formed and remained intact for several hours after treatment. We constructed a C-terminally truncated mutant that corresponded to this 120 kDa fragment and showed that it was fully and constitutively active, and targeted properly to the plasma membrane. In contrast, arsenate or excitotoxic concentration of glutamate induced PMCA internalization and consequently, resulted in an impaired Ca2+ clearance from the cytoplasm. We also showed that interaction with the postsynaptic?density-95 (PSD-95) scaffolding protein increased the plasma membrane expression of PMCA4b and redistributed the pump into clusters. The clustering of PMCA4b was fully dependent on the presence of its C-terminus. We suggest that loss of function internalization of PMCAs and/or disruption of specific Ca2+ signaling microdomains may contribute to the Ca2+ dysregulation that accompanies a number of degenerative diseases

Comparative study of CYP2B1/2 induction and the transport of bilirubin and taurocholate in rat hepatocyte-mono- and hepatocyte-Kupffer cell co-cultures

Introduction Hepatocyte-Kupffer cell (KC) co-cultures represent a promising approach for in vitro modeling of complex interactions between parenchymal and non-parenchymal cells in the liver, responsible for drug-induced liver injury (DILI). In this study we aimed to compare hepatocyte monocultures with hepatocyte-KC co-cultures regarding some basic liver functions associated with the chemical defense system. These pathways involve transporters and enzymes the function of which is highly sensitive towards hepatotoxic events. Methods CYP2B1/2 induction and the biliary and sinusoidal elimination of bilirubin (B) and taurocholate (TC) were studied in rat hepatocyte sandwich cultures compared with rat hepatocyte-KC sandwich co-cultures of 1:0, 6:1, 2:1 and 1:1 cell combinations representing the physiologic and pathologic conditions of the liver. Results KCs decreased phenobarbital inducibility of CYP2B1/2 in a cell ratio dependent manner and activation of KCs by lipopolisacharide (LPS) amplified this effect. Similarly, KCs decreased the transport of B and its glucuronides (BG) in both sinusoidal and canalicular directions resulting in its intracellular accumulation. In contrast, the uptake and the efflux of TC were greater in the co-cultures than in the hepatocyte monocultures. Immuno-labelling of sodium-dependent taurocholate transporter (Ntcp) revealed increased expression of the transporter in the presence of KCs. Discussion Here we presented that KCs have a direct impact on some hepatocyte functions suggesting that the co-culture model may be more suitable for drug related hepatotoxicity studies than hepatocyte monocultures. Abbreviations B, bilirubin; Bsep, bile salt export pump; CYP, cytochrome P-450; GdCl3, gadolinium(III) chloride; H/KC, hepatocyte-Kupffer-cell co-culture; HBSS, Hanks' balanced salt solution; HPLC, high-performance liquid chromatography; KC, Kupffer-cell; LPS, lipopolisacharide; Mrp, multidrug resistance-associated protein; Ntcp, sodium-dependent taurocholate transporter; PB, phenobarbital; PBS, phosphate-buffered saline; PTX, pentoxyphylline; TC, taurocholat

Generation of multidrug resistant human tissues by overexpression of the ABCG2 multidrug transporter in embryonic stem cells

The ABCG2 multidrug transporter provides resistance against various endo- and xenobiotics, and protects the stem cells against toxins and stress conditions. We have shown earlier that a GFP-tagged version of ABCG2 is fully functional and may be used to follow the expression, localization and function of this transporter in living cells. In the present work we have overexpressed GFP-ABCG2, driven by a constitutive (CAG) promoter, in HUES9 human embryonic stem cells. Stem cell clones were generated to express the wild-type and a substrate-mutant (R482G) GFP-ABCG2 variant, by using the Sleeping Beauty transposon system. We found that the stable overexpression of these transgenes did not change the pluripotency and growth properties of the stem cells, nor their differentiation capacity to hepatocytes or cardiomyocytes. ABCG2 overexpression provided increased toxin resistance in the stem cells, and protected the derived cardiomyocytes against doxorubicin toxicity. These studies document the potential of a stable ABCG2 expression for engineering toxin-resistant human pluripotent stem cells and selected stem cell derived tissues

Ezrin interacts with S100A4 via both its N- and C-terminal domains

Ezrin belongs to the ERM (ezrin, radixin, moesin) protein family that has a role in cell morphology changes, adhesion and migration as an organizer of the cortical cytoskeleton by linking actin filaments to the apical membrane of epithelial cells. It is highly expressed in a variety of human cancers and promotes metastasis. Members of the Ca2+-binding EF-hand containing S100 proteins have similar pathological properties; they are overexpressed in cancer cells and involved in metastatic processes. In this study, using tryptophan fluorescence and stopped-flow kinetics, we show that S100A4 binds to the N-terminal ERM domain (N-ERMAD) of ezrin with a micromolar affinity. The binding involves the F2 lobe of the N-ERMAD and follows an induced fit kinetic mechanism. Interestingly, S100A4 binds also to the unstructured C-terminal actin binding domain (C-ERMAD) with similar affinity. Using NMR spectroscopy, we characterized the complex of S100A4 with the C-ERMAD and demonstrate that no ternary complex is simultaneously formed with the two ezrin domains. Furthermore, we show that S100A4 co-localizes with ezrin in HEK-293T cells. However, S100A4 very weakly binds to full-length ezrin in vitro indicating that the interaction of S100A4 with ezrin requires other regulatory events such as protein phosphorylation and/or membrane binding, shifting the conformational equilibrium of ezrin towards the open state. As both proteins play an important role in promoting metastasis, the characterization of their interaction could shed more light on the molecular events contributing to this pathological process

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

Expression of Tight Junction Components in Hepatocyte-Like Cells Differentiated from Human Embryonic Stem Cells

Human embryonic stem cells can be differentiated in vitro into a wide variety of progeny cells by addition of different morphogens and growth factors. Our aim was to monitor the expression pattern of tight junction (TJ) components and various cellular markers during differentiation of stem cell lines toward the hepatic lineage. Human embryonic stem cell lines (HUES1, HUES9) were differentiated into endoderm-like cells, and further differentiated to hepatocyte-like cells. Gene expressions of Oct3/4, Nanog, alpha-fetoprotein, albumin, cytokeratins (CK-7, CK-8, CK-18, CK-19), ATP-binding cassette (ABC) transporters (ABCC2, ABCC7, ABCG2), and various TJ components, including claudin-1, claudin-4, claudin-5, claudin-7, and tricellulin, as well as an extracellular matrix component, agrin were monitored during hepatic differentiation by real-time quantitative PCR. The differentiated cells exhibit epithelial morphology and functional assessments similar to that of hepatocytes. The expression level of stem cell marker genes (Oct3/4 and Nanog) significantly and gradually decreased, while liver-associated genes (alpha-fetoprotein, albumin) reached their highest expression at the end of the differentiation. The endoderm-like cells expressed claudin-1, which declined eventually. The expression levels of cholangiocyte markers including claudin-4, CK-7, CK-19, and agrin gradually increased and reached their highest level at the final stage of differentiation. In contrast, these cells did not express notable level of claudin-7, CK-8 and tricellulin. The marker set used for monitoring differentiation revealed both hepatocyte and cholangiocyte characteristics of the differentiated cells at the final stage. This is the first report describing the expression level changes of various TJ components, and underlining their importance in hepatic differentiation. © 2015 Arányi Lajos Foundatio