A cirkadián óramű molekuláris jellemzése: Az oszcillátor fehérje Frequency kifejeződésének transzkripciós és poszttranszkripciós szinten történő szabályozása = Molecular characterization of the circadian clockwork: Regulation of the expression of the oscillator protein on the transcriptional and posttranscriptional level

Kutatási munkánk célja a cirkadián oszcillátor működését biztosító, illetve módosító faktorok megismerése és funkcionális jellemzése volt a Neurospora crassa modellorganizmusban. Eredményeink: 1. Létrehoztunk egy olyan számítógépes programot, amely a Neurospora crassa genomjában képes szekvencia-mintázatokat keresni. A cirkadián óra pozitív komponensének ismert kötőhelyeihez hasonló struktúrákat kerestünk és így azonosítottunk egy új, a cirkadián oszcillátor működését befolyásoló faktort, egy feltehetően RasGEF aktivitású fehérjét. 2. A cirkadián óra egyik pozitív komponensének, a White Collar-1 fehérjének a vizsgálata kapcsán azonosítottunk két, a fehérje foszforilációját és ezen keresztül működését alapvetően meghatározó fehérjerégiót. Az egyik régión belül szekvencia homológia alapján valószínűleg egy MAP kináz foszforilációs hely található. 3. Kimutattuk, hogy a VIVID nevű másodlagos fényreceptor a környezeti fényintenzitásra vonatkozó molekuláris memóriaként működik. A cirkadián óra pozitív faktorával kölcsönhatásba lépve gátolja annak fényfüggő aktiválódását és ezáltal természetes fényperiódusok mellett stabilizálja a cirkadián óra működését. 4. Eredményeink szerint a reaktív oxigén származékok (ROS) szintjének változása fontos tényezője a cirkadián óra szabályozásának. A ROS-szint a molekuláris oszcillátorra hat, emelkedése korábbra helyezi a fázist és rövidíti a periódust. A ROS szint változásának hatását valószínűleg a protein foszfatáz 2A közvetíti. | Aim of our work was to functionally characterize factors involved in the regulation of the circadian clockwork of the model organism Neurospora crassa. Our most important results are summarized below: 1. We designed a computer program that enables one to search the genome of Neurospora for special sequence patterns. By searching for possible binding sites of the positive factor of the Neurospora clock we found a gene coding for a putative RasGEF protein. Our observations on a RasGEF mutant suggest that this protein is a modulator of the molecular clockwork. 2. We characterized two regulatory regions of the White Collar-1 protein. Deletion of these regions alters the phosphorylation of the protein and results in severe circadian phenotypes. One of these regions is a putative binding site of the MAP kinase. 3. We showed that the VIVID protein acting as a molecular memory interacts with and inhibits the positive component of the circadian clock and thus stabilizes the circadian rhythm even in naturally ambiguous photoperiods. 4. We found that reactive oxygen species (ROS) are important factors controlling the circadian clock. Increased ROS production advances the phase and shortens the circadian period. We suggest that the effect of ROS on the molecular oscillator is mediated by the protein phosphatase 2A

Az egyes fehérjesejt-integrinek élettani szerepének vizsgálata génhiányos (knockout) egerek segítségével = the physiological role of individual leukocyte integrins studies using genetificient (knockout) mice

A támogatott kutatások során a neutrofil granulociták működését vizsgáltuk genetikai, biokémiai és farmakológiai megközelítésekkel. Legfontosabb tudományos eredményeink alábbiak voltak: 1) A neutrofil granulociták integrinjei önmagukban nem képesek a sejtek teljes aktiválódását létrehozni (Jakus et al., J Immunol 2004; IF: 6.70) 2) A humán genom a korábban feltételezettnél lényegesen több ITAM-tartalmú molekulát kódol (Fodor et al., Immunol Lett 2006; IF: 2.14) 3) Az integrinek jelátvitele ITAM-függő mechanizmusokon keresztül jön létre (Mócsai et al., Nat Immunol 2006; IF: 27.60) 4) Egy új integrin-jelátviteli modell felállítása (Jakus et al., Trends Cell Biol 2007;IF: 12.43) 5) Egér neutrofilek immunkomplex-mediált aktiválódása az FcgammaRIII és az FcgammaRIV együttműködésével jön létre (Jakus et al., J Immunol 2008; IF: 6.29) Ezek az eredmények nagyban járulnak hozzá a neutrofilek működésének és az autoimmun gyulladásos folyamatok patomechanizmusának megértéséhez. | The sponsored research activity focused on the analysis of neutrophil functions by genetic, biochemical and pharmacological approaches. Our most important observations were the following: 1) Neutrophil integrins by themselves are not able to induce full activation of the cells (Jakus et al., J Immunol 2004; IF: 6.70) 2) The human genome contains significantly more ITAM-containing adapters than previousy thought (Fodor et al., Immunol Lett 2006; IF: 2.14) 3) Integrins utilize an ITAM-based signal transduction mechanism (Mócsai et al., Nat Immunol 2006; IF: 27.60) 4) Development of a new integrin signal transduction model (Jakus et al., Trends Cell Biol 2007;IF: 12.43) 5) Activation of murine neutrophils by immune complexes proceeds through a cooperative action of FcgammaRIII and FcgammaRIV (Jakus et al., J Immunol 2008; IF: 6.29) These results will strongly contribute to the understanding of neutrophil functions and the pathomechanism of autoimmune inflammatory diseases

Arachidonic acid activatable electrogenic H+ transport in the absence of cytochrome b558 in human T lymphocytes

AbstractTo test the suggested structural relationship between the electrogenic H+ transporting system and the NADPH oxidase of phagocytes, the existence of the enzyme and the transport process was investigated in human tonsillar T lymphocytes. It is shown that tonsillar T cells possess an arachidonic acid activatable, Cd2+- and Zn2+-sensitive electrogenic H+ efflux pathway with similar properties as reported earlier in various phagocytic cells. The presence of cytochrome b558, the membrane component of the oxidase, could not be detected in tonsillar T lymphocytes either by immunoblot or by flow cytometric analysis. It is suggested that the electrogenic H+ transporting pathway is structurally independent of the NADPH oxidase complex

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

Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3'UTR intron-mediated NMD in Neurospora crassa

Eukaryotic release factor 1 (eRF1) is a translation termination factor that binds to the ribosome at stop codons. The expression of eRF1 is strictly controlled, since its concentration defines termination efficiency and frequency of translational readthrough. Here, we show that eRF1 expression in Neurospora crassa is controlled by an autoregulatory circuit that depends on the specific 3'UTR structure of erf1 mRNA. The stop codon context of erf1 promotes readthrough that protects the mRNA from its 3'UTR-induced nonsense-mediated mRNA decay (NMD). High eRF1 concentration leads to inefficient readthrough, thereby allowing NMD-mediated erf1 degradation. We propose that eRF1 expression is controlled by similar autoregulatory circuits in many fungi and seed plants and discuss the evolution of autoregulatory systems of different translation termination factors

Interconnections of Reactive Oxygen Species Homeostasis and Circadian Rhythm in Neurospora crassa.

Abstract Significance: Both circadian rhythm and the production of reactive oxygen species (ROS) are fundamental features of aerobic eukaryotic cells. The circadian clock enhances the fitness of organisms by enabling them to anticipate cycling changes in the surroundings. ROS generation in the cell is often altered in response to environmental changes, but oscillations in ROS levels may also reflect endogenous metabolic fluctuations governed by the circadian clock. On the other hand, an effective regulation and timing of antioxidant mechanisms may be crucial in the defense of cellular integrity. Thus, an interaction between the circadian timekeeping machinery and ROS homeostasis or signaling in both directions may be of advantage at all phylogenetic levels. Recent Advances: The Frequency-White Collar-1 and White Collar-2 oscillator (FWO) of the filamentous fungus Neurospora crassa is well characterized at the molecular level. Several members of the ROS homeostasis were found to be controlled by the circadian clock, and ROS levels display circadian rhythm in Neurospora. On the other hand, multiple data indicate that ROS affect the molecular oscillator. Critical Issues: Increasing evidence suggests the interplay between ROS homeostasis and oscillators that may be partially or fully independent of the FWO. In addition, ROS may be part of a complex cellular network synchronizing non-transcriptional oscillators with timekeeping machineries based on the classical transcription-translation feedback mechanism. Future Directions: Further investigations are needed to clarify how the different layers of the bidirectional interactions between ROS homeostasis and circadian regulation are interconnected. Antioxid. Redox Signal. 00, 000-000

The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa.

Accumulating evidence from both experimental and clinical investigations indicate a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and therefore is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in wild type (wt), in Deltaras2 operation of the circadian clock was affected by glucose: compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Whereas glucose did not affect the subcellular distribution of FRQ in wt, highly elevated FRQ levels were detected in the nucleus in Deltaras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-Br-cAMP partially rescued the circadian phenotype in vivo. We propose therefore that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock

Perivascular Expression and Potent Vasoconstrictor Effect of Dynorphin A in Cerebral Arteries

BACKGROUND: Numerous literary data indicate that dynorphin A (DYN-A) has a significant impact on cerebral circulation, especially under pathophysiological conditions, but its potential direct influence on the tone of cerebral vessels is obscure. The aim of the present study was threefold: 1) to clarify if DYN-A is present in cerebral vessels, 2) to determine if it exerts any direct effect on cerebrovascular tone, and if so, 3) to analyze the role of κ-opiate receptors in mediating the effect. METHODOLOGY/PRINCIPAL FINDINGS: Immunohistochemical analysis revealed the expression of DYN-A in perivascular nerves of rat pial arteries as well as in both rat and human intraparenchymal vessels of the cerebral cortex. In isolated rat basilar and middle cerebral arteries (BAs and MCAs) DYN-A (1-13) and DYN-A (1-17) but not DYN-A (1-8) or dynorphin B (DYN-B) induced strong vasoconstriction in micromolar concentrations. The maximal effects, compared to a reference contraction induced by 124 mM K(+), were 115±6% and 104±10% in BAs and 113±3% and 125±9% in MCAs for 10 µM of DYN-A (1-13) and DYN-A (1-17), respectively. The vasoconstrictor effects of DYN-A (1-13) could be inhibited but not abolished by both the κ-opiate receptor antagonist nor-Binaltorphimine dihydrochloride (NORBI) and blockade of G(i/o)-protein mediated signaling by pertussis toxin. Finally, des-Tyr(1) DYN-A (2-13), which reportedly fails to activate κ-opiate receptors, induced vasoconstriction of 45±11% in BAs and 50±5% in MCAs at 10 µM, which effects were resistant to NORBI. CONCLUSION/SIGNIFICANCE: DYN-A is present in rat and human cerebral perivascular nerves and induces sustained contraction of rat cerebral arteries. This vasoconstrictor effect is only partly mediated by κ-opiate receptors and heterotrimeric G(i/o)-proteins. To our knowledge our present findings are the first to indicate that DYN-A has a direct cerebral vasoconstrictor effect and that a dynorphin-induced vascular action may be, at least in part, independent of κ-opiate receptors