Fehérje-kölcsönhatások szerkezeti alapjainak feltárása és a kölcsönhatás szelektív gátlása irányított evolúciós eljárásokkal = Deciphering the structural basis of protein-protein interactions and their selective inhibition via directed evolutionary methods

Fő célom az volt, hogy meghonosítsam az irányított fehérjeevolúció szemléletét, és legsikeresebb technológiáját, a fágbemutatást. Egyrészt azt kutattuk, mi szabja meg a proteáz inhibitorok affinitását és specifitását. Három inhibitort vizsgáltunk: a kispeptid SFTI-t (Sunflower Trypsin Inhibitor), a kis fehérje SGPI-t (Schistocerca Gregaria Protease Inhibitor) és a homodimer Ecotint (E. Coli Trypsin Inhibitor). Másrészt inhibitorokat fejlesztettünk olyan humán proteázok ellen, amelyeknek nem volt ismert szelektív gátlószere. Mindhárom inhibitort sikerült evolválnunk. Feltártuk, hogy az SFTI mely csoportjai fontosak a molekula stabilitása és a proteázzal létesített specifikus kölcsönhatás szempontjából. Az SGPI családban feltártuk, hogy a hidrofób mag összetétele kihat a felszíni csoportokra és így a proteázzal alkotott komplex stabilitására. Egy rekord-felbontású SGPI variáns-tripszin komplex szerkezet által finomítottuk a szerin proteázok működési modelljét. Az Ecotint egyláncú formában fejeztük ki fágon, így a két monomer rész külön-külön evolválható lett. Szelektív inhibitorokat fejlesztettünk a hasnyálmirigy enzimek fő szabályzó proteáza, a kimotripszin C ellen, és a komplement rendszer lektin útjának MASP-1 és MASP-2 proteázai ellen. A MASP-gátlókkal létrehoztuk az első lektin út szelektív inhibitorokat, és korrigáltuk az útvonal-aktiválódás korábban hibásan leírt mechanizmusát. A projekt során 11 cikk, 3 szabadalom, 3 PhD dolgozat és tucatnyi tudományos díj született. | A major project goal was to establish in Hungary the principle and technology of directed protein evolution. Via phage display I aimed to decipher fundamental rules governing affinity and specificity of protease inhibitors focusing on 3 inhibitors: the small peptide SFTI (Sunflower Trypsin Inhibitor), the small protein SGPI (Schistocerca Gregaria Trypsin Inhibitor) and the homodimer Ecotin (E. Coli Trypsin Inhibitor). I also aimed to evolve selective inhibitors against human proteases lacking such blockers. We identified SFTI residues providing inhibitor stability and those involved in specific protease-binding. In the SGPI family we revealed that the hydrophobic inhibitor core greatly affects the function of surface residues and thereby the stability of the protease-inhibitor complex. Based on an ultrahigh resolution SGPI variant – trypsin structure we refined the model of serine protease catalysis. We displayed Ecotin on phage in a single-chain format such that the two monomers could be evolved independently. We developed selective inhibitors against a major regulatory pancreatic protease, human chymotrypsin C. We also evolved mono-specific inhibitors against the lectin pathway complement proteases MASP-1 and MASP-2. With these we developed the first lectin pathway selective complement inhibitors and corrected the hitherto incorrect pathway activation model. Altogether 11 papers, 3 patents, 3 PhD thesis and a dozen scientific awards indicate the success of the project

The application of Magnetic Resonance Imaging (MRI) to the examination of plant tissues and water barriers

The aim of the present study is to extend the applicability of MRI measurements similar to those used in human diagnostics to the examination of water barriers in living plants, thus broadening their use in natural sciences. The cucumber, Cucumis sativus, and Phillyrea angustifolia, or false olive, were chosen as test plants. The MRI measurements were carried out on three samples of each plant in the same position vis-a-vis the MRI apparatus using a Siemens Avanto MRI scanner. Two different relaxation times were employed, T1 capable of histological mapping, and T2, used for the examination of water content. In the course of the analysis, it was found that certain histological formations and branching cause modifications to the intensity detected with relaxation time T2. Furthermore, these positions can also be found in T1 measurements. A monotonic correlation (cucumber: ρ = 0.829; false olive: ρ = –0.84) was observed between the T1 and T2 measurements. In the course of the statistical analysis of the signal intensities of the xylems it was concluded that they cannot be regarded as independent in a statistical sense; these changes rather depend on the anatomic structure of the plant, as the intensity profile is modified by nodes, leaves and branches. This serves as a demonstration of the applicability of MRI to the measurement of well know plant physiological processes. The special parametrization required for this equipment, which is usually used in human diagnostics, is also documented in the present study

Globális regulátor mutációknak mint az attenuálás lehetőségének vizsgálata Escherichia coli-ban = Investigation on global regulator mutations as possible tools of attenuation in Escherichia coli

Az Escherichia coli globális regulátor génjeinek a virulencia funkciókra irányuló hatását vizsgálva megállapítottuk, hogy a húgyúti fertőzést és újszülöttkori meningitist okozó törzsek esetében a leuX és az rfaH gének kiesése (utóbbi Salmonella enterica esetében is) attenuált származékokat eredményez, a recA ás lrhA gének a virulenciát mérhetően nem befolyásolják. Tárgyaljuk a regulátoroknak az egyes virulencia faktorok expressziójára gyakorolt hatását. Az attenuálás olyan fontos virulencia funkciók kiesésére vezethető vissza, mint a haemolysin termelés, a tokantigén képzése, vagy a komplett "O" antigén szintézise. A származékok enterális kolonizáló képessége is csökken, ami a fenti gének szerepére utal abban, hogy az E. coli a kommenzális bélflóra részeként az extraintestinalis fertőzések forrásaként rezervoár szerepet töltsön be. A kutatások perspektivikusan vakcina jelölt származékok kifejlesztését eredményezhetik. | Investigations on the influence of global regulatory factors on Escherichia coli virulence have lead to the conclusion that leuX and rfaH (the latter also in the case of Salmonella enterica) play a pivotal role in pathogenic capacity. On the other hand knock out recA and lrhA mutants presented with equal virulence to the wild type. Effect of the regulators on the function of individual virulence factors is discussed: attenuation of virulence is due to downregulation of such important attributes like haemolysin production, capsule synthesis and expression of a complete 'O' antigen. Handicapped intestinal colonization capacity of the derivatives points to an important role for these regulators in sustaining the reservoir function of intestinal commensals as potential pathogens in subsequent extraintestinal manifestations. Our data imply a perspective for development of potential vaccine candidate derivatives

Periastron Precession Measurements in Transiting Extrasolar Planetary Systems at the Level of General Relativity

Transiting exoplanetary systems are surpassingly important among the planetary systems since they provide the widest spectrum of information for both the planet and the host star. If a transiting planet is on an eccentric orbit, the duration of transits T_D is sensitive to the orientation of the orbital ellipse relative to the line of sight. The precession of the orbit results in a systematic variation in both the duration of individual transit events and the observed period between successive transits, P_obs. The periastron of the ellipse slowly precesses due to general relativity and possibly the presence of other planets in the system. This secular precession can be detected through the long-term change in P_obs (transit timing variations, TTV) or in T_D (transit duration variations, TDV). We estimate the corresponding precession measurement precision for repeated future observations of the known eccentric transiting exoplanetary systems (XO-3b, HD 147506b, GJ 436b and HD 17156b) using existing or planned space-borne instruments. The TDV measurement improves the precession detection sensitivity by orders of magnitude over the TTV measurement. We find that TDV measurements over a ~4 year period can typically detect the precession rate to a precision well exceeding the level predicted by general relativity.Comment: Accepted for publication in MNRAS, 8+epsilon pages, 2 figure

Diabetes mellitus attenuates the repolarization reserve in mammalian heart

Objective: In diabetes mellitus several cardiac electrophysiological parameters are known to be affected. In rodent experimental diabetes models changes in these parameters were reported, but no such data are available in other mammalian species including the dog. The present study was designed to analyse the effects of experimental type I diabetes on ventricular repolarization and its underlying transmembrane ionic currents and channel proteins in canine hearts. Methods and results: Diabetes was induced by a single injection of alloxan, a subgroup of dogs received insulin substitution. After the development of diabetes (8 weeks) electrophysiological studies were performed using conventional microelectrodes, whole cell voltage clamp, and ECG. Expression of ion channel proteins was evaluated by Western blotting. The QT(c) interval and the ventricular action potential duration in diabetic dogs Were moderately prolonged. This was accompanied by significant reduction in the density of the transient outward K+ current (I-to) and the slow delayed rectifier K+ current (I-Ks), to 54.6% and 69.3% of control, respectively. No differences were observed in the density of the inward rectifier K+ current (I-K1), rapid delayed rectifier K+ current (I-Kr), and L-type Ca2+ current (I-Ca). Western blot analysis revealed a reduced expression of Kv4.3 and MinK (to 25 +/- 21% and 48 +/- 15% of control, respectively) in diabetic dogs, while other channel proteins were unchanged (HERG, MiRP1, alpha(1c)) or increased (Kv1.4, KChIP2, KvLQT1). Insulin substitution fully prevented the diabetes-induced changes in I-Ks, KvLQT1 and MinK, however, the changes in I-to, Kv4.3, and Kv1.4 were only partially diminished by insulin. Conclusion: It is concluded that type I diabetes mellitus, although only moderately, lengthens ventricular repolarization, attenuates the repolarization reserve by decreasing I-to and I-Ks currents, and thereby may markedly enhance the risk of sudden cardiac death

MASP-1 and MASP-2 Do Not Activate Pro-Factor D in Resting Human Blood, whereas MASP-3 Is a Potential Activator: Kinetic Analysis Involving Specific MASP-1 and MASP-2 Inhibitors.

It had been thought that complement factor D (FD) is activated at the site of synthesis, and only FD lacking a propeptide is present in blood. The serum of mannose-binding lectin-associated serine protease (MASP)-1/3(-/-) mice contains pro-FD and has markedly reduced alternative pathway activity. It was suggested that MASP-1 and MASP-3 directly activate pro-FD; however, other experiments contradicted this view. We decided to clarify the involvement of MASPs in pro-FD activation in normal, as opposed to deficient, human plasma and serum. Human pro-FD containing an APPRGR propeptide was produced in insect cells. We measured its activation kinetics using purified active MASP-1, MASP-2, MASP-3, as well as thrombin. We found all these enzymes to be efficient activators, whereas MASP proenzymes lacked such activity. Pro-FD cleavage in serum or plasma was quantified by a novel assay using fluorescently labeled pro-FD. Labeled pro-FD was processed with t1/2s of approximately 3 and 5 h in serum and plasma, respectively, showing that proteolytic activity capable of activating pro-FD exists in blood even in the absence of active coagulation enzymes. Our previously developed selective MASP-1 and MASP-2 inhibitors did not reduce pro-FD activation at reasonable concentration. In contrast, at very high concentration, the MASP-2 inhibitor, which is also a poor MASP-3 inhibitor, slowed down the activation. When recombinant MASPs were added to plasma, only MASP-3 could reduce the half-life of pro-FD. Combining our quantitative data, MASP-1 and MASP-2 can be ruled out as direct pro-FD activators in resting blood; however, active MASP-3 is a very likely physiological activator

Sustainable synthesis of azobenzenes, quinolines and quinoxalines via oxidative dehydrogenative couplings catalysed by reusable transition metal oxide–Bi( iii ) cooperative catalysts

Heterogeneous catalytic oxidative dehydrogenative processes for N-heterocycles are presented, which enable waste-minimized (additive-, oxidant-, base-free), efficient cyclisations/couplings via transition metal oxide–Bi( iii ) cooperative catalysis