Publications (9)22.51 Total impact
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Article: ABCC6 Expression Is Regulated by CCAAT/Enhancer-Binding Protein Activating a Primate-Specific Sequence Located in the First Intron of the Gene.
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ABSTRACT: Pseudoxanthoma elasticum (PXE), a rare recessive genetic disease causing skin, eye, and cardiovascular lesions, is characterized by the calcification of elastic fibers. The disorder is due to loss-of-function mutations of the ABCC6 gene, but the pathophysiology of the disease is still not understood. Here we investigated the transcriptional regulation of the gene, using DNase I hypersensitivity assay followed by luciferase reporter gene assay. We identified three DNase I hypersensitive sites (HSs) specific to cell lines expressing ABCC6. These HSs are located in the proximal promoter and in the first intron of the gene. We further characterized the role of the HSs by luciferase assay and demonstrated the transcriptional activity of the intronic HS. We identified the CCAAT/enhancer-binding protein β (C/EBPβ) as a factor binding the second intronic HS by chromatin immunoprecipitation and corroborated this finding by luciferase assays. We also showed that C/EBPβ interacts with the proximal promoter of the gene. We propose that C/EBPβ forms a complex with other regulatory proteins including the previously identified regulatory factor hepatocyte nuclear factor 4α (HNF4α). This complex would account for the tissue-specific expression of the gene and might serve as a metabolic sensor. Our results point toward a better understanding of the physiological role of ABCC6.Journal of Investigative Dermatology advance online publication, 5 July 2012; doi:10.1038/jid.2012.218.Journal of Investigative Dermatology 07/2012; · 6.31 Impact Factor -
Article: Expression of sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes.
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ABSTRACT: The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.Biochimica et Biophysica Acta 01/2009; 1788(3):587-99. · 4.66 Impact Factor -
Article: External cell control polymerase chain reaction: replacing internal standards with an unbiased strategy for quantitative polymerase chain reaction normalization.
Analytical Biochemistry 02/2008; 372(2):261-3. · 3.00 Impact Factor -
Article: Isoform-specific up-regulation of plasma membrane Ca2+ATPase expression during colon and gastric cancer cell differentiation.
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ABSTRACT: In this work we demonstrate a differentiation-induced up-regulation of the expression of plasma membrane Ca2+ATPase (PMCA) isoforms being present in various gastric/colon cancer cell types. We found PMCA1b as the major isoform in non-differentiated cancer cell lines, whereas the expression level of PMCA4b was significantly lower. Cell differentiation initiated with short chain fatty acids (SCFAs) and trichostatin A, or spontaneous differentiation of post-confluent cell cultures resulted in a marked induction of PMCA4b expression, while only moderately increased PMCA1b levels. Up-regulation of PMCA4b expression was demonstrated both at the protein and mRNA levels, and closely correlated with the induction of established differentiation markers. In contrast, the expression level of the Na+/K+-ATPase or that of the sarco/endoplasmic reticulum Ca2+ATPase 2 protein did not change significantly under these conditions. In membrane vesicles obtained from SCFA-treated gastric/colon cancer cells a marked increase in the PMCA-dependent Ca2+ transport activity was observed, indicating a general increase of PMCA function during the differentiation of these cancer cells. Because various PMCA isoforms display distinct functional characteristics, we suggest that up-regulated PMCA expression, together with a major switch in PMCA isoform pattern may significantly contribute to the differentiation of gastric/colon cancer cells. The analysis of PMCA expression may provide a new diagnostic tool for monitoring the tumor phenotype.Cell Calcium 01/2008; 42(6):590-605. · 3.77 Impact Factor -
Article: Identification, expression, function, and localization of a novel (sixth) isoform of the human sarco/endoplasmic reticulum Ca2+ATPase 3 gene.
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ABSTRACT: Understanding of Ca(2+) signaling requires the knowledge of proteins involved in this process. Among these proteins are sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) that pump Ca(2+) into the endoplasmic reticulum (ER). Recently, the human SERCA3 gene was shown to give rise to five isoforms (SERCA3a-e (h3a-h3e)). Here we demonstrate the existence of an additional new member, termed SERCA3f (h3f). By reverse transcriptase-PCR using monocytic U937 cell RNA, h3f mRNA was found to exclude the antepenultimate exon 21. h3f mRNA expression appeared as a human-specific splice variant. It was not found in rats or mice. h3f mRNA gave rise to an h3f protein differing in its C terminus from h3a-h3e. Of particular interest, h3f diverged in the first amino acids after the first splice site but presented the same last 21 amino acids as h3b. Consequently, we further investigated the structure-function-location relationships of the h3b and h3f isoforms. Comparative functional study of h3b and h3f recombinant proteins in intact HEK-293 cells and in fractionated membranes showed the following distinct characteristics: (i) resting cytosolic Ca(2+) concentration ([Ca(2+)](c)) and (ii) ER Ca(2+) content ([Ca(2+)](er)); similar characteristics were shown for the following: (i) the effects of the SERCA inhibitor, thapsigargin, on Ca(2+) release ([Ca(2+)](Tg)) and subsequent Ca(2+) entry ([Ca(2+)](e)) and (ii) the low apparent Ca(2+) affinity and the enhanced rate of dephosphorylation of the E(2)P phosphoenzyme intermediate. Subcellular location of h3b and h3f by immunofluorescence and/or confocal microscopy using the h3b- and h3f-specific polyclonal and the pan-h3 monoclonal (PL/IM430) antibodies suggested overlapping but distinct ER location. The endogenous expression of h3f protein was also proved in U937 cells. Altogether these data suggest that the SERCA3 isoforms have a more widespread role in cellular Ca(2+) signaling than previously appreciated.Journal of Biological Chemistry 07/2004; 279(23):24297-306. · 4.77 Impact Factor -
Article: A szarko/endoplazmatikus retikulum Ca2+ ATPáz 3 szerkezet-elemzése és a nem-izom típusú sejtek Ca2+ -transzport fehérje készletének változásai sejtdifferenciáció során = Structural study of the sarco/endoplasmic reticulum Ca2+ ATPase 3 proteins and the modulation of the machinery of Ca2+-transport proteins during the differentiation of non-muscle cells
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ABSTRACT: A Ca2+ függő sejtélettani folyamatok szabályozásában fontos szerepet betöltő szarko/endoplazmás retikulum (SERCA) és plazmamembrán (PMCA) Ca2+ATPázokat vizsgáltuk. Endogén, valamint vad típusú és mutáns rekombináns SERCA3 fehérjék tripszines proteolízisét elemezve kimutattuk két, kinetikailag eltérő fragmentációs profil párhuzamos megjelenését. Irányított mutagenezissel azonosítottunk néhány tripszines hasítási helyet. Eredményeink arra utalnak, hogy natív membránkörnyezetben a fragmentációs profilok a SERCA3 fehérje eltérő konformációs állapotaihoz rendelhetőek. Korábbi munkáinkhoz kapcsolódva elemeztük a gyomor-/béltumorsejtek differenciációja, és a SERCA és PMCA fehérjék expressziója, funkciója közötti összefüggéseket. Számos differenciációs modellben igazoltuk, hogy az éretlen sejtekben alacsony szinten kifejeződő SERCA3 és PMCA4b izoformák expressziója erősen indukálódik a tumorsejtek differenciációja során. A SERCA fehérjék funkcionális gátlása elősegítette egyes béltumorsejtek differenciációját. A béltumorok kialakulásában fontos APC/b-katenin/TCF4 jelátviteli út gátlása indukálta a SERCA3 expressziót. Különböző stádiumú béltumorokból nyert szöveti metszetek immunhisztokémiai vizsgálatai szerint a SERCA3 expresszió defektusa már a tumorok kialakulásának korai szakaszában jelentkezik. Eredményeink alapján felvetjük annak lehetőségét, hogy a gyomor-/béltraktusban egyes Ca2+ transzporterek, mint markerfehérjék, segíthetik malignus elváltozások kialakulásának és fenotípusának meghatározását, és diagnosztikai fejlesztések potenciális célpontjai lehetnek. | We studied the sarco/endoplasmic reticulum (SERCA) and plasma membrane (PMCA) Ca2+ATPases. The proper functions of these proteins are essential in the regulation of Ca2+-dependent cellular processes. Limited tryptic digestion of endogenously expressed or wild-type and mutant recombinant SERCA3 proteins resulted in two distinct fragmentation profiles. Using site-directed mutagenesis approach some of the tryptic sites were determined. Our data indicated that the SERCA3 fragmentation profiles were related to different conformational states of the enzyme. To further explore our previous work, we investigated the expression and function of various SERCA and PMCA isoforms during the differentiation of gastric/colon cancer cells. Using a wide range of model cells and differentiation protocols, strong induction of SERCA3 and PMCA4b expression were detected in differentiating cancer cells. Inhibition of SERCA function induced the maturation of colon cancer cells. Inhibition of the APC/b-catenin/TCF4 signaling pathway, essential during colon carcinogenesis, resulted in up-regulated SERCA3 expression. Immunohystochemical analysis of various tissue sections from colonic lesions, adenomas and adenocarcinomas showed that loss of SERCA3 expression is an early event during colon carcinogenesis. Our data suggest that some Ca2+ transport proteins could serve as new biomarkers for the analysis of the formation and phenotype of gastric/colon tumors, and should help in novel diagnostic development. -
Article: 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
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ABSTRACT: 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. -
Article: Expression of hPMCA4b, the major form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in mature human platelets
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ABSTRACT: Antibodies 5F10 and JA3 (raised against the erythrocyte Ca2+ pump) were used to identify hPMCA4b as the major form of the plasma membrane Ca2+ pump in human platelets and in three human megakaryoblastoid cell lines, MEG 01, DAMI and CHRF 288-11. 5F10 was used because it has been shown to recognize all known isoforms of the hPMCA and JA3 because it reacts exclusively with hPMCA4b [Caride A.J., Filoteo A.G., Enyedi A., Verma A.K., Penniston J.T Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies. Biochem J 1996; 316: 353–359]. In addition to hPMCA4b, hPMCA1b was also detected in the megakaryoblastoid cells by using isoform-specific polyclonal antibodies. The apparent size of this isoform, however, was smaller than that seen in HeLa and COS-7 cell membranes indicating the presence of a modified form of hPMCA1 b. In platelets, no evidence of the expression of hPMCA1 b could be found. The amount of PMCA in these cells was compared with that of the constitutive form of the sarco/endoplasmic reticulum Ca2+ pump in non-muscle cells (SERCA2b) and also with the amount of PMCA in human erythrocytes. A very low level of the plasma membrane Ca2+ pump was found in platelets while in their precursor cells the expression of this Ca2+ pump was much more abundant. Whereas the expression level of PMCA decreased dramatically in mature human platelets, the expression of SERCA2b did not change substantially upon megakaryocytic differentiation.Cell Calcium. -
Article: Expression of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes
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ABSTRACT: The SERCA family includes 3 genes (SERCA1–3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg334, Arg396 and Arg638 were directly assigned to the ETF and ii) Arg198 was assigned as the only tryptic site to the LTF. Arg671, Lys712/Lys713 and Lys728 were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl2, EGTA or AlF4− strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b–3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg1002 as an additional tryptic site in SERCA3b–3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.Biochimica et Biophysica Acta (BBA) - Biomembranes.
