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

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

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

Histone deacetylase inhibitor- and PMA-induced upregulation of PMCA4b enhances Ca2+ clearance from MCF-7 breast cancer cells

The expression of the plasma membrane Ca2+ ATPase (PMCA) isoforms is altered in several types of cancer cells suggesting that they are involved in cancer progression. In this study we induced differentiation of MCF-7 breast cancer cells by histone deacetylase inhibitors (HDACis) such as short chain fatty acids (SCFAs) or suberoylanilide hydroxamic acid (SAHA), and by phorbol 12-myristate 13-acetate (PMA) and found strong upregulation of PMCA4b protein expression in response to these treatments. Furthermore, combination of HDACis with PMA augmented cell differentiation and further enhanced PMCA4b expression both at mRNA and protein levels. Immunocytochemical analysis revealed that the upregulated protein was located mostly in the plasma membrane. To examine the functional consequences of elevated PMCA4b expression, the characteristics of intracellular Ca2+ signals were investigated before and after differentiation inducing treatments, and also in cells overexpressing PMCA4b. The increased PMCA4b expression – either by treatment or overexpression – led to enhanced Ca2+ clearance from the stimulated cells. We found pronounced PMCA4 protein expression in normal breast tissue samples highlighting the importance of this pump for the maintenance of mammary epithelial Ca2+ homeostasis. These results suggest that modulation of Ca2+ signaling by enhanced PMCA4b expression may contribute to normal development of breast epithelium and may be lost in cancer

ABC transzporterek vizsgálata emberi őssejtekben és a sejt-differenciálódás során = Expression and function of ABC transporters in human stem cells and during cell differentiation

A projektben az ABC (ATP-Binding Cassette) transzporterek kifejeződését és funkcióját vizsgáltuk emberi őssejtekben, valamint követtük a normális és daganatos sejtdifferenciálódás során bekövetkező molekuláris szintű változásokat. Elsősorban a gyógyszerek hatásában, anyagcseréjében, az ellenük kifejeződő rezisztenciában szereplő transzportereket, valamint a sejtmembrán lipid-anyagcseréjében szerepet játszó ABC transzportereket vizsgáltuk. A projekt fontos része volt az emberi embrionális őssejtek speciális tenyésztési és differenciálási módszereinek adaptálása, továbbfejlesztése. Az ABC transzporterek vizsgálata során igazoltuk, hogy a humán embrionális őssejtekben az ABCG2 a legjelentősebb xenobiotikum transzporter, a korai sejtdifferenciálódás során az ABCG2 kifejeződése először emelkedik, majd jelentősen csökken. Megállapítottuk, hogy az ABCG2 mRNS használata különbözik a tumorsejtekben, a differenciálódott szöveti sejtekben, illetve a nem-differenciálódott őssejtekben. Új, transzpozon-alapú módszereket dolgoztunk ki az őssejtek stabil genetikai módosítására, fluoreszcens riporter fehérjék bevitelére. Az ABCG2 és az ABCA1 fehérjék címkézett változatainak kifejezése lehetővé tette a transzporterek sejten belüli lokalizációjának követését. Részletesen elemeztük az ABCG2 multidrog transzporter szerkezet-funkció összefüggéseit, szubsztrátokkal jelentkező kölcsönhatásait, alkalmazásokat fejlesztettünk ki a gyógyszerhatások vizsgálatára. | In this project we have studied the expression and function of ABC (ATP-Binding Cassette) transporters in human embryonic and tumor stem cells and followed the changes during normal and tumor cell differentiation at a molecular level. We have focused on the investigation of ABC transporters involved in drug metabolism, cancer multidrug resistance and cellular lipid metabolism. An important part of the project was to establish and further develop proper laboratory conditions and methodologies for culturing and differentiating human pluripotent stem cells. When studying human ABC transporters in human embryonic stem cells we demonstrated the major role of the ABCG2 protein. We found that the expression of this transporter first increases, then greatly decreases during early stem cell differentiation. The use of mRNA was found to be different in the tumor cells, differentiated cells, and pluripotent stem cells, respectively. We have developed new, transposon-based methods for the stable genetic modification of pluripotent stem cells and for the expression of fluorescent reporter proteins. By using tagged versions of the ABCG2 and ABCA1 proteins we could follow the intracellular localization of these transporters. We have studied in detail the structure-function relationships and substrate interactions of the ABCG2 transporter and developed new assays for studying drug interactions

Multifaceted plasma membrane Ca2+ pumps: From structure to intracellular Ca2+ handling and cancer

Plasma membrane Ca2+ ATPases (PMCAs) are intimately involved in the control of intracellular Ca2+ concentration. They reduce Ca2+ in the cytosol not only by direct ejection, but also by controlling the formation of inositol-1,4,5-trisphosphate and decreasing Ca2+ release from the endoplasmic reticulum Ca2+ pool. In mammals four genes (PMCA1-4) are expressed, and alternative RNA splicing generates more than twenty variants. The variants differ in their regulatory characteristics. They localize into highly specialized membrane compartments and respond to the incoming Ca2+ with distinct temporal resolution. The expression pattern of variants depends on cell type; a change in this pattern can result in perturbed Ca2+ homeostasis and thus altered cell function. Indeed, PMCAs undergo remarkable changes in their expression pattern during tumorigenesis that might significantly contribute to the unbalanced Ca2+ homeostasis of cancer cells

Expression of calcium pumps is differentially regulated by histone deacetylase inhibitors and estrogen receptor alpha in breast cancer cells

Background: Remodeling of Ca2+ signaling is an important step in cancer progression, and altered expression of members of the Ca2+ signaling toolkit including the plasma membrane Ca2+ ATPases (PMCA proteins encoded by ATP2B genes) is common in tumors. Methods: In this study PMCAs were examined in breast cancer datasets and in a variety of breast cancer cell lines representing different subtypes. We investigated how estrogen receptor alpha (ER-α) and histone deacetylase (HDAC) inhibitors regulate the expression of these pumps. Results: Three distinct datasets displayed significantly lower ATP2B4 mRNA expression in invasive breast cancer tissue samples compared to normal breast tissue, whereas the expression of ATP2B1 and ATP2B2 was not altered. Studying the protein expression profiles of Ca2+ pumps in a variety of breast cancer cell lines revealed low PMCA4b expression in the ER-α positive cells, and its marked upregulation upon HDAC inhibitor treatments. PMCA4b expression was also positively regulated by the ER-α pathway in MCF-7 cells that led to enhanced Ca2+ extrusion capacity in response to 17β-estradiol (E2) treatment. E2-induced PMCA4b expression was further augmented by HDAC inhibitors. Surprisingly, E2 did not affect the expression of PMCA4b in other ER-α positive cells ZR-75-1, T-47D and BT-474. These findings were in good accordance with ChIP-seq data analysis that revealed an ER-α binding site in the ATP2B4 gene in MCF-7 cells but not in other ER-α positive tumor cells. In the triple negative cells PMCA4b expression was relatively high, and the effect of HDAC inhibitor treatment was less pronounced as compared to that of the ER-α positive cells. Although, the expression of PMCA4b was relatively high in the triple negative cells, a fraction of the protein was found in intracellular compartments that could interfere with the cellular function of the protein. Conclusions: Our results suggest that the expression of Ca2+ pumps is highly regulated in breast cancer cells in a subtype specific manner. Our results suggest that hormonal imbalances, epigenetic modifications and impaired protein trafficking could interfere with the expression and cellular function of PMCA4b in the course of breast cancer progression. © 2018 The Author(s)

The plasma membrane Ca2+ pump PMCA4b inhibits the migratory and metastatic activity of BRAF mutant melanoma cells

Oncogenic mutations of BRAF lead to constitutive ERK activity that supports melanoma cell growth and survival. While Ca2+ signaling is a well-known regulator of tumor progression, the crosstalk between Ca2+ signaling and the Ras-BRAF-MEK-ERK pathway is much less explored. Here we show that in BRAF mutant melanoma cells the abundance of the plasma membrane Ca2+ ATPase isoform 4b (PMCA4b, ATP2B4) is low at baseline but markedly elevated by treatment with the mutant BRAF specific inhibitor vemurafenib. In line with these findings gene expression microarray data also shows decreased PMCA4b expression in cutaneous melanoma when compared to benign nevi. The MEK inhibitor selumetinib-similarly to that of the BRAF-specific inhibitor-also increases PMCA4b levels in both BRAF and NRAS mutant melanoma cells suggesting that the MAPK pathway is involved in the regulation of PMCA4b expression. The increased abundance of PMCA4b in the plasma membrane enhances [Ca2+ ]i clearance from cells after Ca2+ entry. Moreover we show that both vemurafenib treatment and PMCA4b overexpression induce marked inhibition of migration of BRAF mutant melanoma cells. Importantly, reduced migration of PMCA4b expressing BRAF mutant cells is associated with a marked decrease in their metastatic potential in vivo. Taken together, our data reveal an important crosstalk between Ca2+ signaling and the MAPK pathway through the regulation of PMCA4b expression and suggest that PMCA4b is a previously unrecognized metastasis suppressor

Analysis of Intracellular Calcium Signaling in Human Embryonic Stem Cells

Measurement of changes in intracellular calcium concentration is one of the most common and useful tools for studying signal transduction pathways or cellular responses in basic research and drug screening purposes as well. Increasing number of such applications using human pluripotent stem cells and their derivatives requires development of calcium signal measurements for this special cell type. Here we describe a modified protocol for analysis of calcium signaling events in human embryonic stem cells, which can be used for other pluripotent cell types (such as iPSC) or their differentiated offspring as well

BRAF Modulates Stretch-Induced Intercellular Gap Formation through Localized Actin Reorganization

Mechanical forces acting on cell-cell adhesion modulate the barrier function of endothelial cells. The actively remodeled actin cytoskeleton impinges on cell-cell adhesion to counteract external forces. We applied stress on endothelial monolayers by mechanical stretch to uncover the role of BRAF in the stress-induced response. Control cells responded to external forces by organizing and stabilizing actin cables in the stretched cell junctions. This was accompanied by an increase in intercellular gap formation, which was prevented in BRAF knockdown monolayers. In the absence of BRAF, there was excess stress fiber formation due to the enhanced reorganization of actin fibers. Our findings suggest that stretch-induced intercellular gap formation, leading to a decrease in barrier function of blood vessels, can be reverted by BRAF RNAi. This is important when the endothelium experiences changes in external stresses caused by high blood pressure, leading to edema, or by immune or cancer cells in inflammation or metastasis