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

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

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1.

Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H2O2) production. Understanding the significance of this response is limited by the fact that the source of EGF-induced H2O2 production is unknown. Here we show that EGF-induced H2O2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1). Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H2O2 production in both cell lines. We also demonstrate that H2O2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1

Lrig1 marks a population of gastric epithelial cells capable of long-term tissue maintenance and growth in vitro

The processes involved in renewal of the epithelium that lines the mouse stomach remain unclear. Apart from the cells in the isthmus, several other populations located deeper in the gastric glands have been suggested to contribute to the maintenance of the gastric epithelium. Here, we reveal that Lrig1 is expressed in the basal layer of the forestomach and the lower part of glands in the corpus and pylorus. In the glandular epithelium of the stomach, Lrig1 marks a heterogeneous population comprising mainly non-proliferative cells. Yet, fate-mapping experiments using a knock-in mouse line expressing Cre specifically in Lrig1(+) cells demonstrate that these cells are able to contribute to the long-term maintenance of the gastric epithelium. Moreover, when cultured in vitro, cells expressing high level of Lrig1 have much higher organoid forming potential than the corresponding cellular populations expressing lower levels of Lrig1. Taken together, these observations show that Lrig1 is expressed primarily by differentiated cells, but that these cells can be recruited to contribute to the maintenance of the gastric epithelium. This confirms previous observations that cells located in the lower segments of gastric glands can participate in tissue replenishment

Structure-function analysis of peroxidasin provides insight into the mechanism of collagen IV crosslinking

Basement membranes provide structural support and convey regulatory signals to cells in diverse tissues. Assembly of collagen IV into a sheet-like network is a fundamental mechanism during the formation of basement membranes. Peroxidasin (PXDN) was recently described to catalyze crosslinking of collagen IV through the formation of sulfilimine bonds. Despite the significance of this pathway in tissue genesis, our understanding of PXDN function is far from complete. In this work we demonstrate that collagen IV crosslinking is a physiological function of mammalian PXDN. Moreover, we carried out structure-function analysis of PXDN to gain a better insight into its role in collagen IV synthesis. We identify conserved cysteines in PXDN that mediate the oligomerization of the protein into a trimeric complex. We also demonstrate that oligomerization is not an absolute requirement for enzymatic activity, but optimal collagen IV coupling is only catalyzed by the PXDN trimers. Localization experiments of different PXDN mutants in two different cell models revealed that PXDN oligomers, but not monomers, adhere on the cell surface in "hot spots," which represent previously unknown locations of collagen IV crosslinking. ©2015 Published by Elsevier Inc

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

Peroxidasin-like protein: A novel peroxidase homologue in the human heart

AimsPeroxidases serve diverse biological functions including well-characterized activities in host defence and hormone biosynthesis. More recently, peroxidasin (PXDN) was found to be involved in collagen IV cross-linking in the extracellular matrix (ECM). The aim of this study was to characterize the expression and function of peroxidasin-like protein (PXDNL), a previously unknown peroxidase homologue.Methods and resultsWe cloned the PXDNL cDNA from the human heart and identified its expression pattern by northern blot, in situ hybridization, and immunohistochemistry. PXDNL is expressed exclusively in the heart and it has evolved to lose its peroxidase activity. The protein is produced by cardiomyocytes and localizes to cell-cell junctions. We also demonstrate that PXDNL can form a complex with PXDN and antagonizes its peroxidase activity. Furthermore, we show an increased expression of PXDNL in the failing myocardium.ConclusionPXDNL is a unique component of the heart with a recently evolved inactivation of peroxidase function. The elevation of PXDNL levels in the failing heart may contribute to ECM dysregulation due to its antagonism of PXDN function. © 2013 The Author