Öt lipid transzporter (ABCC6, ABCG5, ABCG8, ABCG1 és ABCG4) vizsgálata: szerkezet, transzport mechanizmus, fiziológiai szerepük és sejten belüli lokalizációjuk = Structure, transport mechanism, physiological role and subcellular localization of five lipid transporters (ABCC6, ABCG5, ABCG8, ABCG1 and ABCG4).

Elsőként sikerült feltárnunk a humán ABCC6 gén transzkripciós szabályozásának fő vonásait. Modelt állítottunk fel, amely segítségével megmagyarázható, hogy a különös evoluciós események révén keletkezett ABCC6 pseudogének hogyan járulnak hozzá a génben keletkező mutációkhoz. Feldolgoztuk és meghatároztuk a humán ABC-fehérjék membrán-topológiáját. Létrehoztuk a humán ABCC6 fehérje homológiai modelljét. Első sikerült biokémiai bizonyítékkal szolgálnunk az ABCG5-G8 heterodimérként való működésére. Kimutattuk, hogy az ABCG2 transzporter aktivitását specifikus módon és jelentős mértékben fokozza a membrán koleszterin-telítése. A transzporter koleszterin-érzékenysége egy merőben újszerű szabalyozómechanizmust feltételez. Feltártuk az ABCG2 és a tirozinkináz gátló tipusú, klinikailag kiemelkedő fontosságú rákellenes szerek kölcsönhatását. Az ABC-fehérjéknek a chemo-immunitást biztosító hálózatban betöltött szerepét leíró hipotézist dolgoztunk ki, és azt egy terjedelmes review-cikk kereteiben az élettudományok egyik legrangosabb, igen magas impaktfaktorú folyóiratában fejtettük ki. Húsz közleményt jelentettünk meg az adott időszakban, amelyek összesített impakt faktora 117. | We have revealed, the very first time, the basics of the transcriptional regulation of the human ABCC6 gene. We have a developed a model expaining how the evolutionary scenario of the human ABCC6 gene contributed to the disease-causing mutations of the gene. We have determined the mebrane topology of the human ABC protein family members. We have constructed a homology model of the ABCC6 protein. We showed that the human ABCG5 and ABCG8 works as a heterodimer providing the first biochemical proof of its activity. We have found that the transport activity of the human ABCG2 protein can be significatly increased by uploading the membrane with cholesterol. This finding raises the possibility of a novel regulatory mechanism of the transporter's activity. We have described the details of the interactions between the new-type tyrosinekinase inhibitor anticancer drugs and the multidrug transporter, ABCG2. We have developed a hypothesis describing the specific role of ABC transporter in a defense mechanism, called 'chemo-immunity'. We have published this new idea in a leading journal with outstandig reputation and very high impact factor. We have published twenty papers during the given period with total impact factor of 117

Az ABC-fehérjék Tudományos Iskolája: a gének regulációjától a transzport-mechanizmusig = The School of ABC-proteins: From Gene Regulation to Transport Mechanism

Megállapítottuk, hogy az ABCC6 gén duplikációi a low-copy repeat 16a elemekhez kötődnek, és ilyen duplikációk több alkalommal is bekövetkeztek különböző főemlős fajokban. Populációgenetikai vizsgálatunkban kimutattuk, hogy egy inaktív ABCC6 allél növeli a koronáriás artéria betegség (CAD) kialakulásának esélyét. Az ABCC6 expressziójánk szabályozását is vizsgáltuk, megállapítottuk, hogy az ERK1/2 szignál-útvonal és a HNF4 transzkripciós faktor felelős az ABCC6 szövet-specifikus szabályozásáért. Megalkottuk az ABCC6 transzporter homológia modelljét, és tanulmányoztuk az ismert 119 misszensz PXE-t okozó mutáció eloszlását. A komplex domén-domén határfelületeken a misszensz mutációk jelentős feldúsúlását figyeltük meg, ami ezen kapcsolatok fontosságának genetikai bizonyítéka. Az ABCC6 viszgálatára alkalmas új állat modellt fejlesztettünk ki: a zebrahal (Danio rerio) modell-rendszert. Bemutattuk, hogy az ecetmuslica MRP az ortológ humán ABCC fehérjékhez hasonló biokémiai tulajdonságokkal rendelkezik, így azok magas turnover-rel rendelkező modellje. Megfigyeltük, hogy a koleszterin szelektíven módosítja az ABCG2 aktivitását. Tanulmányoztuk az ABCG2 katalitikus ciklusa során fellépő intramolekuláris átrendeződéseket. Kifejlesztettünk egy sejtes modell-rendszert az ABCA1, egy fontos koleszterin transzporter tanulmányozására. Új módszert fejlesztettünk ki a kvantitatív PCR reprodukálhatóságának javítására. 15 közleményt publikáltunk szakmailag lektorált nemzetközi folyóiratokban. | We established that duplications of ABCC6 are associated to low-copy repeat 16a and such duplications have occurred several times in different primates. Our population genetic study revealed that one inactive allele of ABCC6 increases the risk of coronary artery disease (CAD) significantly. Th signal transduction pathways leading to the modulation of ABCC6 expression have also been deciphered: the ERK1/2 pathway and HNF4 are responsible for the tissue-specific regulation of ABCC6. We have built a homology model of this transporter, and analyzed the distribution of the known 119 missense PXE-associated mutations within the structure. Significant clustering of the missense mutations has been found at domain-domain interfaces providing a genetic proof of the importance of these domain-domain interactions. A novel animal model to investigate ABCC6 has been developed: the zebrafish (Danio rerio) model system. We have demonstrated that the Drosophila MRP shares the biochemical features of its human ABCC orthologues and serves as a high turn-over model protein of human ABCC-type transporters. We have found that cholesterol selectively modulates the activity of ABCG2. We have studied the intramolecular rearrangments during the catalytic cycle of ABCG2. We have developed a cellular model system to study ABCA1, an important cholesterol transporter. We have developed a novel method to improve the quantitative PCR technique. We have published 15 papers in peer-reviewed international journals

FHR-5 Serum Levels and CFHR5 Genetic Variations in Patients With Immune Complex-Mediated Membranoproliferative Glomerulonephritis and C3-Glomerulopathy

Factor H-related protein 5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in the pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement, and follow-up data.A total of 120 patients with a histologically proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger sequencing, and selected variants were studied as recombinant proteins in ELISA and surface plasmon resonance (SPR).Eight exonic CFHR5 variations in 14 patients (12.6%) were observed. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period; furthermore, it showed clear association with signs of complement overactivation and clinically meaningful clusters.Our observations raise the possibility that the FHR-5 protein plays a fine-tuning role in the pathogenesis of IC-MPGN/C3G

Expression and In Vivo Rescue of Human ABCC6 Disease-Causing Mutants in Mouse Liver

Loss-of-function mutations in ABCC6 can cause chronic or acute forms of dystrophic mineralization described in disease models such as pseudoxanthoma elasticum (OMIM 26480) in human and dystrophic cardiac calcification in mice. The ABCC6 protein is a large membrane-embedded organic anion transporter primarily found in the plasma membrane of hepatocytes. We have established a complex experimental strategy to determine the structural and functional consequences of disease-causing mutations in the human ABCC6. The major aim of our study was to identify mutants with preserved transport activity but failure in intracellular targeting. Five missense mutations were investigated: R1138Q, V1298F, R1314W, G1321S and R1339C. Using in vitro assays, we have identified two variants; R1138Q and R1314W that retained significant transport activity. All mutants were transiently expressed in vivo, in mouse liver via hydrodynamic tail vein injections. The inactive V1298F was the only mutant that showed normal cellular localization in liver hepatocytes while the other mutants showed mostly intracellular accumulation indicating abnormal trafficking. As both R1138Q and R1314W displayed endoplasmic reticulum localization, we tested whether 4-phenylbutyrate (4-PBA), a drug approved for clinical use, could restore their intracellular trafficking to the plasma membrane in MDCKII and mouse liver. The cellular localization of R1314W was significantly improved by 4-PBA treatment, thus potentially rescuing its physiological function. Our work demonstrates the feasibility of the in vivo rescue of cellular maturation of some ABCC6 mutants in physiological conditions very similar to the biology of the fully differentiated human liver and could have future human therapeutic application

FcRn and IgG residues known to be involved in FcRn–IgG interaction.

<p>FcRn and IgG residues known to be involved in FcRn–IgG interaction.</p

pH-dependent binding of rbIgG and hIgG1 by rbFcRn.

<p>Soluble rbFcRn (Panel A and B), as well as rbIgG (Panel C) and hIgG1 (Panel D) were immobilized on a GLC chip at densities of 1200, 1900 and 2100 RU, respectively. rbIgG (Panel A) and hIgG1 (Panel B) at 133 nM, while soluble rbFcRn (Panel C and D) at 600 nM concentration in PBS-T buffer were injected at pH 6.0, as well as at pH 7.4 and interactions were monitored at 25°C. The curves were corrected by subtracting the non-specific binding responses obtained from control channel (n = 2–3, one representative figure is shown in each cases).</p

Kinetic parameters of the interaction of immobilized rbFcRn and hFcRn with rbIgG and hIgG isotypes at pH 6.0.

<p>Kinetic parameters of the interaction of immobilized rbFcRn and hFcRn with rbIgG and hIgG isotypes at pH 6.0.</p

Kinetic parameters of the interaction of immobilized rbIgG and hIgG1 with rbFcRn and hFcRn at pH 6.0.

<p>Kinetic parameters of the interaction of immobilized rbIgG and hIgG1 with rbFcRn and hFcRn at pH 6.0.</p

FcRn-mediated recycling studies of rbIgG and hIgG1 in rabbit peritoneal macrophages.

<p>Cells were pulsed with Alexa 488-conjugated rbIgG and hIgG1 for 20 minutes and chased for 30 minutes. Confocal images were collected at chase starting point (rbIgG on Panel A and hIgG1 on Panel C) and after 30 minutes (rbIgG on Panel B and hIgG1 on Panel D) and were processed using Fiji software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185662#pone.0185662.ref030" target="_blank">30</a>]. Cells were drawn around, and the median level of pixel intensity were determined, and for comparison, relative fluorescent intensity of the cells were calculated in all cases (Panel E). Values shown are the means ± SEM (**, p< 0.01; ***, p<0.001).</p