NADPH oxidáz szabályozása és élettani szerepe = Regulation and physiological role of NADPH oxidase

A kutatási program célja a neutrofil granulociták O2.--termeléséért felelős NADPH oxidáz enzim szabályozásának valamint a baktériumölésben játszott szerepének vizsgálata volt. Megállapítottuk, hogy az oxidáz-komplexben részt vevő Rac monomer G-fehérje GTP-kötött állapota elengedhetetlen a folyamatos enzimaktivitás fenntartásához. A GTPáz aktiváló fehérjék (GAPok) hatásosan és folyamatosan gátolják a O2- termelést. Két különböző, granulocitákban előforduló GAP esetén mutattunk ki eddig ismeretlen szabályozó mechanizmust. A p190GAP-nál egyes foszfolipidek a szubsztrát specificitást változtatják meg: a Rho-GAP aktivitást gátolják, míg a Rac-GAP aktivitást fokozzák. A p50GAP nativ állapotában viszont molekulán belüli interakciók egyaránt gátolják a Rho- és Rac-GAP aktivitást; a G-fehérje prenil csoportja szükséges a p50 megnyílásához. Intakt sejten a NADPH oxidáz elektrogén működése a plazma membrán depolarizációján keresztül gátolja a Ca2+ belépést. Kvantitatív méréseinkkel kimutattuk a baktériumölési képesség korrelációját egyrészt a O2.- termelés intenzitásával, másrészt a depolarizációval és a K+ leadás mértékével. Tehát a NADPH oxidáz kettős szerepet játszik a baktériumölésben: mind az elektrogén működése következtében létrejövő ionvándorlások, mind az enzimreakció végterméke, a szuperoxid kémiai hatása érvényesül. A kutatások adatokat szolgáltattak két emberi megbetegedés (CGD, Gaucher kór) kialakulásához, és 8 hallgató doktori értekezésének elkészítését támogatták. | NADPH oxidase is responsible for superoxide (O2.-) production by neutrophilic granulocytes. The aim of the project was to investigate the regulation of the enzyme and its role in killing of microorganisms. We demonstrated that sustained enzyme activity depends on the GTP-bound state of Rac, an essential subunit of the assembled enzyme. We revealed that GTPase activating proteins (GAPs) effectively and continuously down-regulate O2.- production. We showed novel regulatory mechanisms for two GAPs prevalent in granulocytes. In case of p190GAP, substrate specificity is altered by specific phospholipids: Rho-GAP activity is decreased whereas Rac-GAP activity is enhanced. In p50GAP, intramolecular interactions inhibit both Rac-GAP and Rho-GAP activity, but the prenyl group of the small GTPase is able to open up the GAP molecule. In intact cells, NADPH oxidase function is electrogenic and we showed that the resulting depolarization of the plasma membrane blocks Ca2+ entry. In a fine quantitative analysis we found correlation between killing of S. aureus and O2.- production resp. K+ efflux. We conclude that NADPH oxidase plays dual role in bacterial killing: both the initiated ion movements and the chemical product (O2.-) are vital for efficient elimination of some microorganisms. Our experiments provided new data on the pathomechanism of two human diseases (CGD and Gaucher) and supported the completion of the thesis of 8 PhD students

Development of Pancreatic Cancer: Targets for Early Detection and Treatment

Background: Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer death worldwide and compared to other malignancies its share in cancer mortality is expected to rise further. This is due to a lack of sensitive diagnostic tools that would permit earlier detection in a potentially curable stage and the very slow progress in finding effective drug treatments for pancreatic cancer. Key Messages: Aside from genetic predispositions and environmental agents, chronic pancreatitis is by far the greatest risk factor for PDAC. It also shares several etiological factors with pancreatic cancer and represents its most challenging differential diagnosis. Biomarkers that can distinguish between chronic pancreatitis and PDAC may therefore be suitable for the latter's early detection. Moreover, targeting the natural history of chronic pancreatitis would be one approach to prevent PDAC. Targeting tumor-cell signaling directly by interfering with receptor tyrosine kinases has shown some efficacy, although the results in clinical trials were less encouraging than for other cancers. Other compounds developed have targeted the formation of extracellular matrix around the tumor, the proteolytic activity in the tumor environment, histone deacetylases, hedgehog signaling and heat shock proteins, but none has yet found its way into routine patient care. Attempts to individualize treatment according to the tumor's somatic mutation profile are novel but so far impractical. Conclusions: Progress in the treatment of pancreatic cancer has been exceedingly slow and mostly dependent on improved pharmaceutical preparations or combinations of established chemotherapeutic agents. The promise of major breakthroughs implied in targeting tumor signal transduction events has so far not materialized

The p400 ATPase regulates nucleosome stability and chromatin ubiquitination during DNA repair

p400 unwinds chromatin from nucleosomes flanking double-strand breaks to facilitate recruitment of the DNA repair components brca1 and 53BP1

Autoinhibition of p50 Rho GTPase-activating protein (GAP) is released by prenylated small GTPases.

International audienceInteraction of p50 Rho GTPase-activating protein (p50RhoGAP) with Rho family small GTPases was investigated in a yeast two-hybrid system, by radioactive GAP assay, and in a Rac-regulated enzymatic reaction, through superoxide production by the phagocytic NADPH oxidase. The yeast two-hybrid system revealed an interaction between the C-terminal GAP domain and the N-terminal part of p50RhoGAP. The first 48 amino acids play a special role both in the stabilization of the intramolecular interaction and in recognition of the prenyl tail of small GTPases. The GAP assay and the NADPH oxidase activity indicate that the GTPase-activating effect of full-length p50RhoGAP is lower on non-prenylated than on prenylated small GTPase. Removal of amino acids 1-48 and 169-197 of p50RhoGAP increases the GAP effect on non-prenylated Rac, whereas prenylated Rac reacts equally well with the full-length and the truncated proteins. We suggest that p50RhoGAP is in an autoinhibited conformation stabilized by the stretches 1-48 and 169-197 and the prenyl group of the small GTPase plays a role in releasing this intramolecular restraint

Participation of Rac GTPase activating proteins in the deactivation of the phagocytic NADPH oxidase.

International audienceThe aim of the present study was to investigate possible mechanisms that could be involved in the deactivation of the assembled, catalytically active NADPH oxidase of phagocytic cells and thereby lead to termination of O(2)(.-) production. Our major findings are the following: (1) Addition of GDP to the active oxidase is able to reduce O(2)(.-) production both in the fully purified and in a semi-recombinant cell-free activation system. (2) p67(phox) inhibits GTP hydrolysis on Rac whereas p47(phox) has no effect on Rac GTPase activity. (3) Soluble regulatory proteins (GTPase activating protein, guanine nucleotide dissociation inhibitor, and the Rac-binding domain of the target protein p21-activated kinase) inhibit activation of the NADPH oxidase but have no effect on electron transfer via the assembled enzyme complex. (4) Membrane-associated GTPase activating proteins (GAPs) have access also to the assembled, catalytically active oxidase. Taken together, we propose that the GTP-bound active form of Rac is required for sustained enzyme activity and that membrane-localized GAPs have a role in the deactivation of NADPH oxidase

MiR-502 is the first reported miRNA simultaneously targeting two components of the classical non-homologous end joining (C-NHEJ) in pancreatic cell lines.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Acquired inherited and/or somaticmutations drive its development. In order to prevent the formation of these mutations, precise and immediaterepair of any DNA damage is indispensable. Non-homologous end-joining (NHEJ) is the key mechanism of DNAdouble-strand break repair. Here, we report that miR-502 targets two components in pancreatic cell lines, Ku70and XLF of the C-NHEJ. Interestingly, we also observed an attenuated cell cycle response to gamma ionizingradiation (γ-IR) via diminished phosphorylation of checkpoint kinase 1 (Chk1) on serine 345 in these cell lines.Altogether, pancreatic cells showed increased susceptibility toγ-IR via direct inhibition of DNA double-strandbreak repair and attenuation of the cell cycle response

Glucocerebroside inhibits NADPH oxidase activation in cell-free system.

International audienceWe reported earlier that monocytes and macrophages from patients with type I Gaucher disease have a decreased capacity to generate superoxide anion (O(2)(-)) on stimulation with opsonized S. aureus or formyl-methionyl-leucyl-phenylalanine. In this study, various forms of the cell-free assay system were used to probe the hypothesis that glucocerebroside (GC) accumulating in Gaucher patients' phagocytes may interfere with the activation of NADPH oxidase. Xanthine/xanthine oxidase assay was applied to explore the possibility that GC may scavenge O(2)(-). We found that addition of GC to the crude, semirecombinant or fully purified cell-free systems inhibited activation of NADPH oxidase in a concentration-dependent manner. The inhibitory effect of GC could be overcome by increased concentrations of p47(phox) and p67(phox). In contrast, O(2)(-) generation was not decreased by GC added to the assembled, catalytically active enzyme complex. In the xanthine/xanthine oxidase system, GC had no effect on the generation of O(2)(-). These data indicate that assembly of the respiratory burst oxidase of phagocytic cells may be a possible target of the pathologic actions of GC

Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5)

NADPH oxidase 5 (NOX5) is a homologue of the gp91(phox) subunit of the phagocyte NADPH oxidase. NOX5 is expressed in lymphoid organs and testis and distinguished from the other NADPH oxidases by its unique N terminus, which contains three canonical EF-hands, Ca(2+)-binding domains. Upon heterologous expression, NOX5 was shown to generate superoxide in response to intracellular Ca(2+) elevations. In this study, we have analyzed the mechanism of Ca(2+) activation of NOX5. In a cell-free system, Ca(2+) elevations triggered superoxide production by NOX5 (K(m) = 1.06 microm) in an NADPH- and FAD-dependent but cytosol-independent manner. That result indicated a role for the N-terminal EF-hands in NOX5 activation. Therefore, we generated recombinant proteins of NOX5 N terminus and investigated their interactions with Ca(2+). Flow dialysis experiments showed that NOX5 N terminus contained four Ca(2+)-binding sites and allowed us to define the hitherto unidentified fourth, non-canonical EF-hand. The EF-hands of NOX5 formed two pairs: the very N-terminal pair had relatively low affinity for Ca(2+), whereas the more C-terminal pair bound Ca(2+) with high affinity. Ca(2+) binding caused a marked conformation change in the N terminus, which exposed its hydrophobic core, and became able to bind melittin, a model peptide for calmodulin targets. Using a pull-down assay, we demonstrate that the regulatory N terminus and the catalytic C terminus of NOX5 interact in a Ca(2+)-dependent way. Our results indicate that the Ca(2+)-induced conformation change of NOX5 N terminus led to enzyme activation through an intra-molecular interaction. That represents a novel mechanism of activation among NAD(P)H oxidases and Ca(2+)-activated enzymes