159 research outputs found
Protein foszfatáz 2A szerepe a human endothelium barrier funkciójának szabályozásában = Role of protein phosphatase 2A in the regulation of human endothelial barrier function
TĂĽdĹ‘ artĂ©ria endothel modell rendszerben tanulmányoztuk a protein foszfatáz 2A (PP2A) szerepĂ©t. EredmĂ©nyeink arra utalnak, hogy a PP2A aktĂvan rĂ©szt vesz az endothelium barrier funkciĂłjának citoszkeletális fehĂ©rjĂ©ken keresztĂĽl törtĂ©nĹ‘ reguláciĂłjában. Overexpresszált PP2A a barrier diszfunkciĂłt kiváltĂł ágensek (thrombin, nokodazol) hatását az aktin citoszkeletonra Ă©s a mikrotubulusokra mĂ©rsĂ©kli/megszĂĽnteti. KĂ©t potenciális citoszkeletális szubsztrátot azonosĂtottunk, a tau Ă©s a Hsp27 fehĂ©rjĂ©t, amelyeknek szerepe lehet az agonista-indukált citoszkeleton átrendezĹ‘dĂ©sben Ă©s permeabilitás változásban. A PP2A aktivitás ezen fehĂ©rjĂ©k foszforiláciĂłs szintjĂ©t befolyásolja Ă©s ezáltal rĂ©sze lehet az endothel sejtek citoszkeletonjában a mikrotubulusok Ă©s a mikrofilamentumok közötti párbeszĂ©d Ă©s a barrier funkciĂł szabályozásának. Továbbá kimutattuk, hogy a MYPT fehĂ©rje család egyik kevĂ©sbĂ© jellemzett tagja, a TIMAP, ami egy membrán-asszociált fehĂ©rje, specifikusan kölcsönhat a protein foszfatáz 1 (PP1) bĂ©ta izoformájával, ami valĂłszĂnűsĂti, hogy a PP1 regulátora. A TIMAP a barrier funkciĂłt pozitĂvan szabályozza, ezt az RNS interferenciával TIMAP-depletált sejtek vizsgálatával mutattuk ki. EredmĂ©nyeink alapján feltĂ©telezhetĹ‘, hogy a TIMAP az ERM (ezrin/radixin/moezin) fehĂ©rjĂ©k PKA indukált szabályozásában vesz rĂ©szt Ă©s ez a TIMAP-mediált endothel barrier vĂ©dĹ‘ hatás rĂ©sze lehet. | The role of protein phosphatase 2A (PP2A) in pulmonary artery endothelial cells was studied. Our results suggest the direct involvement of PP2A in the regulation of cytoskeleton proteins and endothelial barrier maintenance. PP2A overexpression attenuated or diminished thrombin- or nocodazole-induced effects on microfilaments and microtubule. We identified two potential substrates of PP2A, tau and Hsp27, which potentially could be involved in agonist-induced cytoskeleton rearrangement and change in permeability. PP2A regulates the phosphorylation level of these proteins, and as a consequence, it is involved in the regulation of the crosstalk between microfilaments and microtubule, and barrier function. Further, we found specific interaction between the beta isoform of protein phosphatase 1 (PP1) and TIMAP, a less characterized member of the MYPT family, implying its regulatory role in PP1 activity. Using siRNA technique we showed that TIMAP is a positive regulator of the endothelial barrier function. Our results also suggest the involvement of TIMAP in PKA-mediated ERM (ezrin/radixin/moesin) dephosphorylation and the connection of this dephosphorylation in TIMAP-mediated EC barrier protection
Protein foszfatázok szabályozása kölcsönható fehérjékkel = Regulation of protein phosphatases by protein-protein interactions
A pályázatban a miozin foszfatáz foszforiláciĂłval törtĂ©nĹ‘ szabályozását Ă©s az enzim fehĂ©rje-fehĂ©rje kölcsönhatásait tanulmányoztuk. A holoenzimben protein foszfatáz-1 katalitikus alegysĂ©g (PP1c) kapcsolĂłdik a miozinhoz is kötĹ‘dĹ‘ szabályozĂł alegysĂ©ghez (MYPT). A MYPT Rho-kináz általi foszforiláciĂłja gátolta a miozin foszfatáz aktivitását Ă©s simaizom kontrakciĂłt indukált alacsony Ca2+-koncentráciĂłnál. Ez a gátlĂł foszforiláciĂł nem-izom sejtek esetĂ©n stressz-rostok kĂ©pzĹ‘dĂ©sĂ©t Ă©s a sejtek migráciĂłjának gátlását eredmĂ©nyezte. A MYPT protein kináz G általi foszforiláciĂłja viszont csökkentette a gátlĂł foszforiláciĂł mĂ©rtĂ©kĂ©t, elĹ‘segĂtve ezzel a simaizom relaxáciĂłt. A miozin foszfatáz nem-izom sejtekben számos, a kontraktilitástĂłl eltĂ©rĹ‘ sejtfolyamatot is szabályozhat. Erre a PP1c Ă©s a MYPT neuronális, sejtmag Ă©s mitokondriális fehĂ©rjĂ©kkel törtĂ©nĹ‘ kölcsönhatásábĂłl következtettĂĽnk. A miozin foszfatáz változatos lokalizáciĂłja Ă©s kölcsönhatásai az idegrendszerben a neurotranszmissziĂłt szabályozĂł szerepĂ©t sejteti. JellemeztĂĽk egy Ăşj, elsĹ‘sorban idegszövetben elĹ‘fordulĂł fehĂ©rje foszforiláciĂłját Ă©s kimutattuk miozin foszfatázt gátlĂł kĂ©pessĂ©gĂ©t. Az enzimnek a retinoblasztĂłma fehĂ©rje defoszforiláciĂłjában játszott szerepe pedig arra utal, hogy a miozin foszfatáz a sejtciklust is szabályozhatja. További kutatásaink olyan molekulák azonosĂtására irányulnak, amelyek az enzim szelektĂv gátlásával/aktiválásával elĹ‘segĂthetik sejtfolyamatokat szabályozĂł szerepĂ©nek mĂ©lyebb megĂ©rtĂ©sĂ©t. | In this project the regulation of myosin phosphatase by phosphorylation and by protein-protein interactions were studied. The holoenzyme is composed of protein phosphatase-1 catalytic subunit (PP1c) and myosin-binding regulatory subunit (MYPT). Phosphorylation of MYPT by Rho-kinase resulted in the inhibition of the activity of myosin phosphatase and induced smooth muscle contraction at low Ca2+-concentration. In non-muscle cells this inhibitory phosphorylation increased stress-fiber formation and supressed cell migration. Phosphorylation of MYPT by protein kinase G decreased the extent of inhibitory phosphorylation and resulted in smooth muscle relaxation. In non-muscle cells the myosin phosphatase may regulate cellular events distinct from the contractile processes suggested by the interaction of PP1c and MYPT with neuronal, nuclear and mitochondrial proteins. The widespread localization of myosin phosphatase in neuronal cells implicates this enzyme in the regulation of neuronal transmission. We characterized the phosphorylation and myosin phosphatase inhibitory potency of a novel protein which is mainly enriched in neuronal tissue. The myosin phosphatase dephosphorylate also the retinoblastoma protein implying its regulatory role in the cell cycle. Our further studies aim to identify molecules able to inhibit/activate the myosin phosphatase selectively; therefore they may help to clarify the regulatory role of this enzyme in the various cellular processes
Thrombocyta aktiváció során bekövetkező változások biokémiai és sejtbiológiai jellemzés = Biochemical and cell biological changes during platelet activation
Ex vivo mintákon vĂ©gzett analĂzisek közĂĽl elvĂ©geztĂĽk 30 db stent beĂĽltett beteg esetĂ©ben perifáriás vĂ©rminták analĂzisĂ©t az alábbi thrombocyta aktiváciĂłs markerekre: thrombocyta P-selectin, solubilis P-selectin, CD63 expressziĂł, heterotipikus aggregátumok aránya, mikropartikula mennyisĂ©g. Fenti eredmĂ©nyeket korreláltattuk a betegek elĹ‘zetes kezelĂ©si protokolljával (csak ASA, vagy ASA+clopidogrel). A XIII-as faktorral (F XIII) kapcsolatos vizsgálatok során megállapĂtást nyert, hogy thrombocyták TRAP aktiválása, az FXIII expressziĂł fokozĂłdásával jár. Azonban kimutattuk, hogy ez a plazmában lĂ©vĹ‘ FXIII kötĹ‘dĂ©se Ă©s nem az intracellluláris FXIII felszĂni megjelenĂ©se miatt jön lĂ©tre. Specifikus fibrinogĂ©nek felhasználásával tisztáztuk az FXIII kötĹ‘dĂ©s biokĂ©miai jellemzĹ‘it. A thrombocyta foszfatáz inhibitorokkal vĂ©gzett kĂsĂ©rletek során PRP-ben bizonyĂtottuk, hogy az 1 Ă©s 2A tĂpusĂş foszfatázoknak csak egyĂĽttes gátlása hozza lĂ©tre a thrombocyta aktiválás során Ă©szlelhetĹ‘ hatást. TRAP aktivált mintákon bizonyĂtottuk, hogy mind az aggregáciĂł, mind a P-selectin expressziĂł dĂłzisfĂĽggĹ‘ mĂłdon gátlĂłdik calyculin (CLA) hatására. Ugyanakkor nem aktivált mintákon azt találtuk, hogy a CLA dĂłzisfĂĽggĹ‘ mĂłdon alakváltozást hoz lĂ©tre a thrombocytákban, mely aggregometriával, valamint áramlási citometriai fĂ©nyszĂłrási Ă©s antigĂ©n expressziĂłs vizsgálatokkal bizonyĂthatĂł volt. | On ex vivo samples we examined 30 cases of stent implanted patients where in peripheral blood samples the following platelet activation markers were investigated: paletelt P-selectin, soluble P-selectin, CD63, heterotypic aggregates, microparticles. The above data were correlated with previous treatment protocol of the patients (ASA only or ASA and clopidogrel). Regarding studies with factor XIII we identified that in TRAP-activated platelets FXIII is elevated on the platelet surface. However, we proved, that FXIII is not derived from intracellular source but is attached from the plasma. By using specific fibrinogens we identified the biochemical requirements for FXIII binding to platelets. The manuscript was submitted to Thrombosis and Haemostasis where a minor revision was required. Regarding phosphatase inhibitors we found that phosphatase 1 and 2A types are both involved in platelet activation, since their simultaneous inhibition by calyculin is required to prevent TRAP-elicited P-selectin axpression and platelet aggregation. At the same time on non-activated platelet samples we found that calyculin dose-dependently inhibited platelet shape-change as measured by aggregometry and flow cytometry
Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles
Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles
Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a moonlighting protein
A miozin foszfatáz új szabályozó funkciói nem-izom sejtekben = Novel regulatory functions of myosin phosphatase in non-muscle cells
A protein foszfatáz-1 katalitikus alegysĂ©get (PP1c) Ă©s a PP1c-t „cĂ©lra irányĂtó” regulátor alegysĂ©get (MYPT1) tartalmazĂł miozin foszfatáz (MP) szerepĂ©t tanulmányoztuk nem-izom sejtek sejtfolyamataiban. PP1c specifikus inhibitorok alkalmazásával, valamint a MYPT1-el kölcsönhatĂł Ăşj fehĂ©rje partnerek azonosĂtásával kĂvántuk feltárni a MP Ăşj funkciĂłt. Tanulmányoztuk a MYPT-családba tartozĂł TIMAP fehĂ©rje Ă©s a PP1c kölcsönhatását is Ă©s kimutattuk a PP1c-TIMAP komplex szabályozĂł szerepĂ©t a moezin defoszforiláciĂłjában. FelismertĂĽk, hogy tanninokban Ă©s zöld teában elĹ‘fordulĂł polifenolok PP1c szelektĂv inhibitorok Ă©s csökkentik a daganatos sejtek Ă©letkĂ©pessĂ©gĂ©t. A MYPT1-el kölcsönhatĂł fehĂ©rjekĂ©nt Ă©s/vagy a MP lehetsĂ©ges szubsztrátjaikĂ©nt azonosĂtottuk a retinoblasztĂłma fehĂ©rjĂ©t (Rb), a nitrogĂ©n-monoxid szintetázt (NOS) Ă©s preszinaptikus neuronális fehĂ©rjĂ©ket (szinapszin, szintaxin, SNAP-25, CaM-kináz-II, kalcineurin). Kimutattuk, hogy a Rb MP által törtĂ©nĹ‘ defoszforiláciĂłja a leukĂ©miás sejtek kemoszenzitivitásának, a neuronális fehĂ©rjĂ©k (szintaxin, szinapszin) Rho-kináz/MP által törtĂ©nĹ‘ foszforiláciĂłja/defoszforiláciĂłja pedig a neurotranszmitter felszabadulás fontos szabályozĂł folyamatai. JellemeztĂĽk a MYPT1 Ă©s a kalcineurin kölcsönhatását Ă©s kimutattuk, hogy a MYPT1 kalcineurin által törtĂ©nĹ‘ defoszforiláciĂłja az endotĂ©l sejtek „barrier” funkciĂłját szabályozza. Ă–sszefoglalva: a MP Ăşj szabályozĂł funkciĂłinak felismerĂ©se bĹ‘vĂti e sejtfolyamatok farmakolĂłgiai befolyásolásának lehetĹ‘sĂ©geit. | We studied the role of myosin phosphatase (MP) composed of protein phosphatase-1 catalytic subunit and PP1c-targeting regulatory subunit (MYPT1) in cellular processes of non-muscle cells. We wished to uncover novel functions of MP by using PP1c specific inhibitors as well as by identification of novel MYPT1-interacting proteins. The interaction of TIMAP, a MYPT-family protein, with PP1c was also studied and it was shown that the PP1c-TIMAP complex controlled the dephosphorylation of moesin. We identified the polyphenols of tannins and green tea as PP1c selective inhibitors which were implicated in decreasing the viability of malignant cells. The retinoblastoma protein (Rb), the nitrogen-monoxide (NO) synthase (NOS) and presynaptic neuronal proteins (synapsin, syntaxin, CaM-kinase-II,) were identified as MYPT1-interacting proteins and/or possible substrates for MP. We proved that the dephosphorylation of Rb by MP influenced chemosensitivity of leukemic cells, while the phosphorylation/dephosphorylation of neuronal proteins (synapsin, syntaxin) by Rho-kinase/MP had important regulatory roles in the neurotransmitter release. We characterized the interaction of MYPT1 with calcineurin and showed that dephosphorylation of MYPT1 by calcineurin mediated the barrier function of endothelial cells. In summary, the novel regulatory functions of MP recognized here extend the possibilities to pharmacologically influence the above cellular processes
Dissection of the regulatory role for the N-terminal domain in Candida albicans protein phosphatase Z1
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Molecular Insights into the Fungus-Specific Serine/Threonine Protein Phosphatase Z1 in Candida albicans
Inhibition of protein phosphatase-1 and -2A by ellagitannins: structure-inhibitory potency relationships and influences on cellular systems
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