EGF regulates tyrosine phosphorylation and membrane-translocation of the scaffold protein Tks5

Background: Tks5/FISH is a scaffold protein comprising of five SH3 domains and one PX domain. Tks5 is a substrate of the tyrosine kinase Src and is required for the organization of podosomes/invadopodia implicated in invasion of tumor cells. Recent data have suggested that a close homologue of Tks5, Tks4, is implicated in the EGF signaling.Results: Here, we report that Tks5 is a component of the EGF signaling pathway. In EGF-treated cells, Tks5 is tyrosine phosphorylated within minutes and the level of phosphorylation is sustained for at least 2 hours. Using specific kinase inhibitors, we demonstrate that tyrosine phosphorylation of Tks5 is catalyzed by Src tyrosine kinase. We show that treatment of cells with EGF results in plasma membrane translocation of Tks5. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutation of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks5.Conclusions: Our results identify Tks5 as a novel component of the EGF signaling pathway. © 2013 Fekete et al.; licensee BioMed Central Ltd

A Renox szuperoxid-termelő enzim vizsgálata emlős sejtekben = Characterization of the Renox superoxide-producing enzyme in mammalian cells

1. Kifejlesztettünk egy Nox4 (Renox)-deficiens egérmodellt. Az eddigi vizsgálataink szerint a Nox4 jelenléte nem nélkülözhetetlen az élethez és az állatok konstitutív vérképzése is normálisnak tűnik. 2. Kimutattuk, hogy az eddig ismeretlen funkciójú p50RhoGAP fehérje fontos szerepet játszhaz a Rho és a Rab G-fehérjék jelátviteli útvonalainak összekapcsolásában. 3. Dr Thomas Leto laboratóriumával együttműködésben megállapítottuk, hogy a Rac1 kismólsúlyú GTP-kötő fehérje fontos szerepet játszik a Nox1-et és Nox3-at tartalmazó NADPH oxidázok szabályozásában. 4. A Nox4 élettani funkcióját kutatva sikerült azonosítanunk egy olyan új emberi peroxidázt amely fontos szerepet játszhat a reaktív oxigénszármazékok hatásának közvetítésében. | 1. We have developed a Nox4 (Renox)-deficient mouse model. Preliminary analysis of this model indicates that the function of Nox4 is not essential for life and Nox4 has no role in the regulation of constitutive haematopoiesis. 2. We have shown that the p50 Rho-GAP protein provides a link between Rho- and Rab GTPases. 3. In collaboration with Dr Thomas Leto we have shown that the small GTPas Rac1 has an important role in the regulation of Nox1- and Nox3-based NADPH oxidases. 4. While searching for the physiological function of Nox4 we have identified a novel human peroxidase which might have an important role in the mediation of the effects of reactive oxygen species

Composition of the redox environment of the endoplasmic reticulum and sources of hydrogen peroxide

The endoplasmic reticulum (ER) is a metabolically active organelle, which has a central role in proteostasis by translating, modifying, folding, and occasionally degrading secretory and membrane proteins. The lumen of the ER represents a separate compartment of the eukaryotic cell, with a characteristic proteome and metabolome. Although the redox metabolome and proteome of the compartment have not been holistically explored, it is evident that proper redox conditions are necessary for the functioning of many luminal pathways. These redox conditions are defined by local oxidoreductases and the membrane transport of electron donors and acceptors. The main electron carriers of the compartment are identical with those of the other organelles: glutathione, pyridine and flavin nucleotides, ascorbate, and others. However, their composition, concentration, and redox state in the ER lumen can be different from those observed in other compartments. The terminal oxidases of oxidative protein folding generate and maintain an "oxidative environment" by oxidizing protein thiols and producing hydrogen peroxide. ER-specific mechanisms reutilize hydrogen peroxide as an electron acceptor of oxidative folding. These mechanisms, together with membrane and kinetic barriers, guarantee that redox systems in the reduced or oxidized state can be present simultaneously in the lumen. The present knowledge on the in vivo conditions of ER redox is rather limited; development of new genetically encoded targetable sensors for the measurement of the luminal state of redox systems other than thiol/disulfide will contribute to a better understanding of ER redox homeostasis

Nox/Duox Family of NADPH Oxidases: Lessons from Knockout Mouse Models

Nox/Duox NADPH oxidases are now considered the primary, regulated sources of reactive oxygen species (ROS). These enzymes are expressed in diverse cells and tissues, and their products are essential in several physiological settings. Knockout mouse models are instrumental in identifying the physiological functions of Nox/Duox enzymes as well as in exploring the impact of their pharmacological targeting on disease progression. The currently available data from experiments on knockout animals suggest that the lack of non-phagocytic Nox/Duox enzymes often modifies the course and phenotype in many disease models. Nevertheless, as illustrated by studies on Nox4-deficient animals, the absence of Nox-derived ROS can also lead to aggravated disease manifestation, reinforcing the need for a more balanced view on the role of ROS in health and disease. Members of the Nox/Duox NADPH oxidase family produce ROS in a regulated manner in several different cells and tissues.Pharmacological inhibition of non-phagocytic Nox/Duox enzymes might have therapeutic potential.Several studies have described the disease-modifying phenotypes of Nox1 and Nox4 knockouts.The lack of Nox4-derived ROS can lead to aggravated disease development, which is in contrast to the prevailing dogma that considers ROS to be generally harmful. © 2016 Elsevier Ltd

Reaktív oxigén származékok szerepe a fibrózis kialakulásában = Reactive oxygen species in the development of organ fibrosis

Kutatásaink legfontosabb eredményei a következők: 1. Kimutattuk, hogy a peroxidazin fehérje expressziója fokozódik a miofibroblasztok differenciálódása folyamán és a fehérje szekretálódik a sejtek közötti térbe. Azt is kimutattuk, hogy a vese fibrotikus átalakulása során a peroxidazin felszaporodik a tubulus hámsejtek közötti térben. A peroxidazin sejtek közötti térbe történő szekréciója fontos, eddig ismeretlen eleme lehet a szöveti fibrózisnak. 2. Kimutattuk, hogy az emlős peroxidázok családjába tartozó laktoperoxidáz enzim hatékonyan katalizálja tirozin aminosavak összekapcsolását. Az emlős peroxidázok ditirozin-képző aktivitásának szerepe lehat a sejtek közötti állomány módosításában. 3. A NADPH oxidáz enzimcsalád Duox1 tagjáról kimutattuk, hogy szerepet játszhat a húgyhólyag hámsejtjeinek jelátviteli folyamataiban. 4. Elsőként mutattuk ki élő sejtekben, hogy az endoplazmás retikulum lumenében magas a H2O2 szintje, ami elsősorban az Ero-1L enzim aktivitásának köszönhető és független a Nox enzimek aktivitásától. 5. Genetikai modellekkel alátámasztva kimutattuk, hogy a fibroblaszt-miofibroblaszt átalakulás közben megfigyelhető H2O2 termelés a Nox4-p22phox enzimkomplex aktivitásának köszönhető. | The most important results of the research project are the followings: 1. We demonstrated the increased expression and secretion of peroxidasin during myofibroblastic differentiation. We showed that during the course of kidney fibrosis, peroxidasin accumulates in the peritubular space. The secretion of peroxidasin represents a previously unknown mechanism in tissue fibrosis. 2. We showed that lactoperoxidase, a member of the mammalian peroxidase family, efficiently catalyzes the formation of dityrosine residues. Dityrosine formation by mammalian peroxidases may play a role in the modification of the extracellular matrix. 3. We showed that the NADPH oxidase Duox1 has a role in the signaling mechanisms of urothelial cells. 4. We were the first to show in live cells that lumen of the mammalian endoplasmic reticulum is highly oxidative. The high level of H2O2 in the lumen is mainly due to Ero-1L activity and seems to be independent of Nox enzymes. 5. Using genetic models we showed that H2O2 production during myofibroblastic differentiation is due to the activity of the Nox4-p22phox enzyme complex

CGD: Less is more

In this issue of Blood, Song et al demonstrate that alteration of calcium trafficking in neutrophilic granulocytes due to lack of superoxide generation results in excessive production of leukotriene B4 (LTB4), and they show its key role in the pathogenesis of lung inflammation in chronic granulomatous disease (CGD). © 2020 by The American Society of Hematolog

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