GTPáz aktiválo fehérjék (GAPok) élettani szerepe és szabályozása = Physiologcial role and regulation of GTPase activating proteins (GAPs)

Kísérleteinkben három, a Rho/Rac családba tartozó kis G-fehérjére ható GTPáz aktiváló fehérje (GAP) élettani szerepét vizsgáltuk 1.) A p50GAP-ról megállapítottuk, hogy jellegzetes, magkörüli elhelyezkedést mutat. Transzferrin- valamint EGF-receptorokkal végzett kolokalizációs vizsgálatok alapján azonosítottuk, hogy a p50GAP Sec-14 doménje felelős a Rab11-et tartalmazó késői endoszómákon történő lokalizációért valamint a transzferrin-felvétel gátlásáért. Először írtunk le kapcsolatot a Rho valamint a Rab családba tartozó kis G-fehérjék között a receptor-mediált endocitózis szabályozásában. 2.) A p190GAP fehérje GAP aktivitásában kimutattuk két különböző kináz által bekövetkező foszforiláció eltérő hatását. A GSK-3 foszforiláció egyaránt gátolja a p190 Rho- és RacGAP aktivitását. Ezzel szemben a PKC-foszforiláció önmagában nem befolyásolja a GAP-aktivitást, viszont hatásosan gátolja a savanyú foszfolipidekhez történő kötődést. A savanyú foszfolipidek egyedülálló módon megváltoztatják az enzim szubsztrát-specificitását: csökkentik a RhoGAP aktivitást és növelik a RacGAP aktivitást. 3.) Felfedeztünk egy eddig ismeretlen GAP-ot, ami in vitro körülmények között Rac-specifikusnak bizonyult és elsősorban hemopoetikus sejtekben fejeződik ki. siRNS-el történt csendesítése növelte PLB sejtekben az opszonizált részecskék fagocitózisát valamint az általuk kiváltott szuperoxid-termelést, viszont nem befolyásolta a PMA-val indukált választ. | Our experiments concentrated on the physiological role of three GTPase activating proteins (GAPs) acting on Rho/Rac family small GTPases. 1.) p50GAP showed a characteristic, perinuclear localization. On the basis of colocalization with transferrin- and EGF-receptors we demonstrated that the Sec14 domain of p50GAP was responsible both for localization on Rab11-containing late endosomes and for inhibition of transferrin uptake. We suggested that p50GAP provides a link between Rab and Rho family small GTPases in the regulation of receptor-mediated endocytosis. 2.) Investigating the regulation of p190GAP, we revealed the different effects of phosphorylation by different kinases. Phosphorylation by GSK-3 inhibits both the Rho- and the RacGAP activity of the protein. In contrast, phosphorylation by PKC does not directly affect the GAP activity, but it prevents binding of p190GAP to acidic phospholipids, which have a unique effect: they change the substrate preference of p190GAP inhibiting the RhoGAP and promoting the RacGAP activity. 3.) We revealed a new, hitherto unknown GAP that proved to be Rac-specific in in vitro assays, and seems to be specifically expressed in haemopoetic cells. Silencing of this new GAP in PLB cells resulted in an increase of phagocytosis of opsonized particles and of superoxide production induced by opsonized zymosan or bacteria. In contrast, responses induced by PMA were not altered

Molecular and Functional Characterization of Hv1 Proton Channel in Human Granulocytes

Voltage-gated proton current (IHv) has been characterized in several cell types, but the majority of the data was collected in phagocytes, especially in human granulocytes. The prevailing view about the role of IHv in phagocytes is that it is an essential supporter of the intense and sustained activity of Nox2 (the core enzyme of the phagocyte NADPH oxidase complex) during respiratory burst. Recently Hv1, a voltage-gated proton channel, was cloned, and leukocytes from Hv1 knockout mice display impaired respiratory burst. On the other hand, hardly anything is known about Hv1 in human granulocytes. Using qPCR and a self made antibody, we detected a significant amount of Hv1 in human eosinophil and neutrophil granulocytes and in PLB-985 leukemia cells. Using different crosslinking agents and detergents in reducing and non-reducing PAGE, significant expression of Hv1 homodimers, but not that of higher-order multimers, could be detected in granulocytes. Results of subcellular fractionation and confocal imaging indicate that Hv1 is resident in both plasmalemmal and granular membrane compartments of resting neutrophils. Furthermore, it is also demonstrated that Hv1 accumulates in phagosome wall during zymosan engulfment together with, but independently of Nox2. During granulocytic differentiation early and parallel upregulation of Hv1 and Nox2 expression was observed in PLB-985 cells. The upregulation of Hv1 or Nox2 expression did not require the normal expression of the other molecule. Using RNA interference, we obtained strong correlation between Hv1 expression and IHv density in PLB-985 cells. It is also demonstrated that a massive reduction in Hv1 expression can limit the Nox2 mediated superoxide production of PLB-985 granulocytes. In summary, beside monomers native Hv1 forms stable proton channel dimer in resting and activated human granulocytes. The expression pattern of Hv1 in granulocytes is optimized to support intense NADPH oxidase activity

The Homolog of the Five SH3-Domain Protein (HOFI/SH3PXD2B) Regulates Lamellipodia Formation and Cell Spreading

Motility of normal and transformed cells within and across tissues requires specialized subcellular structures, e.g. membrane ruffles, lamellipodia and podosomes, which are generated by dynamic rearrangements of the actin cytoskeleton. Because the formation of these sub-cellular structures is complex and relatively poorly understood, we evaluated the role of the adapter protein SH3PXD2B [HOFI, fad49, Tks4], which plays a role in the development of the eye, skeleton and adipose tissue. Surprisingly, we find that SH3PXD2B is requisite for the development of EGF-induced membrane ruffles and lamellipodia, as well as for efficient cellular attachment and spreading of HeLa cells. Furthermore, SH3PXD2B is present in a complex with the non-receptor protein tyrosine kinase Src, phosphorylated by Src, which is consistent with SH3PXD2B accumulating in Src-induced podosomes. Furthermore, SH3PXD2B closely follows the subcellular relocalization of cortactin to Src-induced podosomes, EGF-induced membrane ruffles and lamellipodia. Because SH3PXD2B also forms a complex with the C-terminal region of cortactin, we propose that SH3PXD2B is a scaffold protein that plays a key role in regulating the actin cytoskeleton via Src and cortactin

The Blackberry Image: Self-identified Perceptions and Motivations Associated with College Student BlackBerry Use

We report the results of a qualitative research study conducted with university students regarding their phenomenological perspectives of BlackBerry use. Three key themes inductively emerged throughout the interview and analysis process regarding self-perceptions college students reported regarding their own BlackBerry use. First, students offered practical motivations that influenced their decisions to purchase and use BlackBerrys, including the relatively convenient and instant access to email and Internet that BlackBerry offers. Second, all students in our study intentionally compared and contrasted their own BlackBerrys with competing Apple products on the market, specifically the iPhone. In comparison with the iPhone, BlackBerry users in our sample viewed their own phones as only moderately ‘cool’ or prestigious. Additionally, BlackBerry users also seemed to view their phones as less technologically advanced than iPhone technology. Third, students reported particular images they associated with BlackBerry use. These included both a financial ‘stigma’ as well as that of a ‘business person’ stereotype

Urothelial cells produce hydrogen peroxide through the activation of Duox1.

Hydrogen peroxide (H(2)O(2)) has important messenger and effector functions in the plant and animal kingdom. Phagocytes produce H(2)O(2) to kill pathogens, and epithelial cells of large airways have also been reported to produce H(2)O(2) for signaling and host defense purposes. In this report, we show for the first time that urothelial cells produce H(2)O(2) in response to a calcium signal. Using a gene-deficient mouse model we also demonstrate that H(2)O(2) is produced by the NADPH oxidase Duox1, which is expressed in the mouse urothelium. In contrast, we found no evidence for the expression of lactoperoxidase, an enzyme that has been shown to cooperate with Duox enzymes. We also found that specific activation of TRPV4 calcium channels elicits a calcium signal and stimulates H(2)O(2) production in urothelial cells. Furthermore, we detected altered pressure responses in the urinary bladders of Duox1 knockout animals. Our results raise the possibility that mechanosensing in epithelial cells involves calcium-dependent H(2)O(2) production similar to that observed in plants.JOURNAL ARTICLESCOPUS: ar.jinfo:eu-repo/semantics/publishe