Publikációk

2013

Kovács D, Simon Z, Hári P, Málnási-Csizmadia A, Hegedűs C, Drimba L, Németh J, Sári R, Szilvássy Z, Peitl B.

Identification of PPARγ ligands with One-dimensional Drug Profile Matching.

Drug Des Devel Ther. 2013 Sep 2;7:917-28.

INTRODUCTION: Computational molecular database screening helps to decrease the time and resources needed for drug development. Reintroduction of generic drugs by second medical use patents also contributes to cheaper and faster drug development processes. We screened, in silico, the Food and Drug Administration-approved generic drug database by means of the One-dimensional Drug Profile Matching (oDPM) method in order to find potential peroxisome proliferator-activated receptor gamma (PPARγ) agonists. The PPARγ action of the selected generics was also investigated by in vitro and in vivo experiments. MATERIALS AND METHODS: The in silico oDPM method was used to determine the binding potency of 1,255 generics to 149 proteins collected. In vitro PPARγ activation was determined by measuring fatty acid-binding protein 4/adipocyte protein gene expression in a Mono Mac 6 cell line. The in vivo insulin sensitizing effect of the selected compound (nitazoxanide; 50-200 mg/kg/day over 8 days; n = 8) was established in type 2 diabetic rats by hyperinsulinemic euglycemic glucose clamping. RESULTS: After examining the closest neighbors of each of the reference sets members and counting their most abundant neighbors, ten generic drugs were selected with oDPM. Among them, four enhanced fatty acid-binding protein/adipocyte protein gene expression in the Mono Mac 6 cell line, but only bromfenac and nitazoxanide showed dose-dependent actions. Induction by nitazoxanide was higher than by bromfenac. Nitazoxanide lowered fasting blood glucose levels and improved insulin sensitivity in type 2 diabetic rats. CONCLUSION: We demonstrated that the oDPM method can predict previously unknown therapeutic effects of generic drugs. Nitazoxanide can be the prototype chemical structure of the new generation of insulin sensitizers. KEYWORDS: PPARγ, computer-aided prediction of receptor-ligand interaction, in silico lead selection, insulin sensitizers, one-dimensional drug profile matching, peroxisome proliferator activated receptor gamma, type two diabetes

Végner L, Peragovics A, Tombor L, Jelinek B, Czobor P, Bender A, Simon Z, Málnási-Csizmadia A.

Experimental confirmation of new drug-target interactions predicted by drug profile matching.

J Med Chem. 2013 Nov 14;56(21):8377-88.

We recently introduced Drug Profile Matching (DPM), a novel affinity fingerprinting-based in silico drug repositioning approach. DPM is able to quantitatively predict the complete effect profiles of compounds via probability scores. In the present work, in order to investigate the predictive power of DPM, three effect categories, namely, angiotensin-converting enzyme inhibitor, cyclooxygenase inhibitor, and dopamine agent, were selected and predictions were verified by literature analysis as well as experimentally. A total of 72% of the newly predicted and tested dopaminergic compounds were confirmed by tests on D1 and D2 expressing cell cultures. 33% and 23% of the ACE and COX inhibitory predictions were confirmed by in vitro tests, respectively. Dose-dependent inhibition curves were measured for seven drugs, and their inhibitory constants (Ki) were determined. Our study overall demonstrates that DPM is an effective approach to reveal novel drug-target pairs that may result in repositioning these drugs.

Gógl G, Törő I, Reményi A.

Protein-peptide complex crystallization: a case study on the ERK2 mitogen-activated protein kinase.

Acta Crystallogr D Biol Crystallogr. 2013 Mar;69(Pt 3):486-9. doi: 10.1107/S0907444912051062.

Linear motifs normally bind with only medium binding affinity (Kd of ∼0.1-10 µM) to shallow protein-interaction surfaces on their binding partners. The crystallization of proteins in complex with linear motif-containing peptides is often challenging because the energy gained upon crystal packing between symmetry mates in the crystal may be on a par with the binding energy of the protein-peptide complex. Furthermore, for extracellular signal-regulated kinase 2 (ERK2) the protein-peptide docking surface is comprised of a small hydrophobic surface patch that is often engaged in the crystal packing of apo ERK2 crystals. Here, a rational surface-engineering approach is presented that involves mutating protein surface residues that are distant from the peptide-binding ERK2 docking groove to alanines. These ERK2 surface mutations decrease the chance of `unwanted crystal packing of ERK2 and the approach led to the structure determination of ERK2 in complex with new docking peptides. These findings highlight the importance of negative selection in crystal engineering for weakly binding protein-peptide complexes.

Masoudi N, Fancsalszky L, Pourkarimi E, Vellai T, Alexa A, Reményi A, Gartner A, Mehta A, Takács-Vellai K.

The NM23-H1/H2 homolog NDK-1 is required for full activation of Ras signaling in C. elegans.

Development 2013 Aug;140(16):3486-95. doi: 10.1242/dev.094011.

The group I members of the Nm23 (non-metastatic) gene family encode nucleoside diphosphate kinases (NDPKs) that have been implicated in the regulation of cell migration, proliferation and differentiation. Despite their developmental and medical significance, the molecular functions of these NDPKs remain ill defined. To minimize confounding effects of functional compensation between closely related Nm23 family members, we studied ndk-1, the sole Caenorhabditis elegans ortholog of group I NDPKs, and focused on its role in Ras/mitogen-activated protein kinase (MAPK)-mediated signaling events during development. ndk-1 inactivation leads to a protruding vulva phenotype and affects vulval cell fate specification through the Ras/MAPK cascade. ndk-1 mutant worms show severe reduction of activated, diphosphorylated MAPK in somatic tissues, indicative of compromised Ras/MAPK signaling. A genetic epistasis analysis using the vulval induction system revealed that NDK-1 acts downstream of LIN-45/Raf, but upstream of MPK-1/MAPK, at the level of the kinase suppressors of ras (KSR-1/2). KSR proteins act as scaffolds facilitating Ras signaling events by tethering signaling components, and we suggest that NDK-1 modulates KSR activity through direct physical interaction. Our study reveals that C. elegans NDK-1/Nm23 influences differentiation by enhancing the level of Ras/MAPK signaling. These results might help to better understand how dysregulated Nm23 in humans contributes to tumorigenesis.

Megyeri M, Harmat V, Major B, Végh Á, Balczer J, Héja D, Szilágyi K, Datz D, Pál G, Závodszky P, Gál P, Dobó J.

Quantitative characterization of the activation steps of mannan-binding lectin (MBL)-associated serine proteases (MASPs) points to the central role of MASP-1 in the initiation of the complement lectin pathway.

J Biol Chem. 2013 Mar 29;288(13):8922-34

Mannan-binding lectin (MBL)-associated serine proteases, MASP-1 and MASP-2, have been thought to autoactivate when MBL/ficolin·MASP complexes bind to pathogens triggering the complement lectin pathway. Autoactivation of MASPs occurs in two steps: 1) zymogen autoactivation, when one proenzyme cleaves another proenzyme molecule of the same protease, and 2) autocatalytic activation, when the activated protease cleaves its own zymogen. Using recombinant catalytic fragments, we demonstrated that a stable proenzyme MASP-1 variant (R448Q) cleaved the inactive, catalytic site Ser-to-Ala variant (S646A). The autoactivation steps of MASP-1 were separately quantified using these mutants and the wild type enzyme. Analogous mutants were made for MASP-2, and rate constants of the autoactivation steps as well as the possible cross-activation steps between MASP-1 and MASP-2 were determined. Based on the rate constants, a kinetic model of lectin pathway activation was outlined. The zymogen autoactivation rate of MASP-1 is ∼3000-fold higher, and the autocatalytic activation of MASP-1 is about 140-fold faster than those of MASP-2. Moreover, both activated and proenzyme MASP-1 can effectively cleave proenzyme MASP-2. MASP-3, which does not autoactivate, is also cleaved by MASP-1 quite efficiently. The structure of the catalytic region of proenzyme MASP-1 R448Q was solved at 2.5 Å. Proenzyme MASP-1 R448Q readily cleaves synthetic substrates, and it is inhibited by a specific canonical inhibitor developed against active MASP-1, indicating that zymogen MASP-1 fluctuates between an inactive and an active-like conformation. The determined structure provides a feasible explanation for this phenomenon. In summary, autoactivation of MASP-1 is crucial for the activation of MBL/ficolin·MASP complexes, and in the proenzymic phase zymogen MASP-1 controls the process.

Frank Odei-Addo, Carminita Frost, Nanette Smith, Tomohisa Ogawa, Koji Muramoto, Maria Luiza Vilela Oliva, Lászlo´ Gráf, and Ryno Naude

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

J Enzyme Inhib Med Chem. 2013 Oct 3.

Abstract One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Molnár T, Vörös J, Szeder B, Takáts K, Kardos J, Katona G, Gráf L.

Comparison of complexes formed by a crustacean and a vertebrate trypsin with bovine pancreatic trypsin inhibitor - the key to achieving extreme stability?

FEBS J. 2013 Nov;280(22):5750-63.

This paper provides evidence for the extremely high resistance of a complex of crayfish trypsin (CFT) and bovine pancreatic trypsin inhibitor (BPTI) against heating and chemical denaturing agents such as sodium dodecyl sulfate (SDS) and urea. To dissociate this complex, 15 min boiling in SDS was necessary, compared to a complex of bovine trypsin (BT) (EC 3.4.21.4) and BPTI, which dissociates in SDS without boiling. The CFT-BPTI complex remained stable even in 9 m urea, while the BT-BPTI complex started to dissociate at concentrations of approximately 4 m urea. The melting temperatures of the BT-BPTI and CFT-BPTI complexes, as determined by differential scanning calorimetry, were found to be 79.6 and 100.1 °C, respectively. The behaviour of the apo-enzymes - CFT was found to have a less stable structure compared to BT - did not provide a definite indication regarding the differential effects on their stabilities. To explore the structural features responsible for this extreme stability, we crystallized CFT in complex with BPTI, and identified extended contacts compared to the BT-BPTI complex. Comparison of the B-factors of similar trypsin-trypsin inhibitor complexes suggests that molecular flexibility of the components is also required for the strong protein-protein interaction. Although the structural reason for the extreme stability of the CFT-BPTI complex is not yet fully understood, our study may be a starting point for the development of new protein complexes with enhanced stability.

Kocsis S. Zsuzsa, Haracska Lajos, Szüts Dávid, Kovács Mihály

DNS-hibajavítás megkettőződéskor.

Természet Világa 144(6): 271-3.

Mihály Kovács profile interview

Newsletter of the Biophysical Society (USA), January

Gyimesi, M., Pires, R. H., Billington, N., Sarlós, K., Kocsis, Z. S., Módos, K., Kellermayer, M. S., Kovács, M.

Visualization of human Blooms syndrome helicase molecules bound to homologous recombination

FASEB J. 27: 4954-64.

Homologous recombination (HR) is a key process in the repair of double-stranded DNA breaks (DSBs) that can initiate cancer or cell death. Human Blooms syndrome RecQ-family DNA helicase (BLM) exerts complex activities to promote DSB repair while avoiding illegitimate HR. The oligomeric assembly state of BLM has been a key unresolved aspect of its activities. In this study we assessed the structure and oligomeric state of BLM, in the absence and presence of key HR-intermediate DNA structures, by using single-molecule visualization (electron microscopic and atomic force microscopic single-particle analysis) and solution biophysical (dynamic light scattering, kinetic and equilibrium binding) techniques. Besides full-length BLM, we used a previously characterized truncated construct (BLM(642-1290)) as a monomeric control. Contrary to previous models proposing a ring-forming oligomer, we found the majority of BLM molecules to be monomeric in all examined conditions. However, BLM showed a tendency to form dimers when bound to branched HR intermediates. Our results suggest that HR activities requiring single-stranded DNA translocation are performed by monomeric BLM, while complex DNA structures encountered and dissolved by BLM in later stages of HR induce partial oligomerization of the helicase.

Harami, G.M., Gyimesi, M., Kovács, M.

From keys to bulldozers: expanding roles for winged helix domains in nucleic acid-binding proteins.

Trends Biochem. Sci. 38: 364-71.

The winged helix domain (WHD) is a widespread nucleic-acid-binding protein structural element found in all kingdoms of life. Although the overall structure of the WHD is conserved, its functional properties and interaction profiles are extremely versatile. WHD-containing proteins can exploit nearly the full spectrum of nucleic acid structural features for recognition and even covalent modification or noncovalent rearrangement of target molecules. WHD functions range from sequence-recognizing keys in transcription factors and bulldozer-like strand-separating wedges in helicases to mediators of protein-protein interactions (PPIs). Further investigations are needed to understand the contribution of WHD structural dynamics to nucleic-acid-modifying enzymatic functions.

Nagy, N. T., Chakraborty,S., Harami, G.M., Sellers, J. R., Sakamoto, T., Kovács, M.

A subdomain interaction at the base of the lever allosterically tunes the mechanochemical mechanism of myosin 5a.

PLoS One 8: e62640

The motor domain of myosin is the core element performing mechanochemical energy transduction. This domain contains the actin and ATP binding sites and the base of the force-transducing lever. Coordinated subdomain movements within the motor are essential in linking the ATPase chemical cycle to translocation along actin filaments. A dynamic subdomain interface located at the base of the lever was previously shown to exert an allosteric influence on ATP hydrolysis in the non-processive myosin 2 motor. By solution kinetic, spectroscopic and ensemble and single-molecule motility experiments, we determined the role of a class-specific adaptation of this interface in the mechanochemical mechanism of myosin 5a, a processive intracellular transporter. We found that the introduction of a myosin 2-specific repulsive interaction into myosin 5a via the I67K mutation perturbs the strong-binding interaction of myosin 5a with actin, influences the mechanism of ATP binding and facilitates ATP hydrolysis. At the same time, the mutation abolishes the actin-induced activation of ADP release and, in turn, slows down processive motility, especially when myosin experiences mechanical drag exerted by the action of multiple motor molecules bound to the same actin filament. The results highlight that subtle structural adaptations of the common structural scaffold of the myosin motor enable specific allosteric tuning of motor activity shaped by widely differing physiological demands.

Bibó, A., Kovács, M., Károlyi, G.

Overdamped mechanical model of myosin II.

Periodica Polytechnica Civil Engineering 57: 11-19.

Gál P, Dobó J, Beinrohr L, Pál G, Závodszky P.

Inhibition of the serine proteases of the complement system.

Adv Exp Med Biol. 2013;735:23-40.

Proteases play important roles in human physiology and pathology. The complement system is a proteolytic cascade, where serine proteases activate each other by limited proteolysis in a strictly ordered manner. Serine proteases are essential in both the initiation and the amplification of the cascade. Since uncontrolled complement activation contributes to the development of serious disease conditions, inhibition of the complement serine proteases could be an attractive therapeutic approach. In this chapter, we give a brief overview of the major types of natural serine protease inhibitors and their role in controlling the complement cascade. A special emphasis is laid on C1-inhibitor, a natural complement protease inhibitor, which is approved for clinical use in hereditary angioedema (HAE). We also examine the potential of developing artificial complement protease inhibitors. Synthetic small-molecule drugs can be very efficient serine protease inhibitors, but they usually lack sufficient specificity. A promising approach to yield more specific compounds is the alteration of natural protease inhibitors through engineering or directed evolution resulting in new variants with fine-tuned specificity and enhanced affinity.

Rauscher A, Derényi I, Gráf L, Málnási-Csizmadia A.

Internal friction in enzyme reactions.

IUBMB Life. 2013 Jan;65(1):35-42. doi: 10.1002/iub.1101.

The empirical concept of internal friction was introduced 20 years ago. This review summarizes the results of experimental and theoretical studies that help to uncover the nature of internal friction. After the history of the concept, we describe the experimental challenges in measuring and interpreting internal friction based on the viscosity dependence of enzyme reactions. We also present speculations about the structural background of this viscosity dependence. Finally, some models about the relationship between the energy landscape and internal friction are outlined. Alternative concepts regarding the viscosity dependence of enzyme reactions are also discussed.

Peragovics A, Simon Z, Tombor L, Jelinek B, Hári P, Czobor P, Málnási-Csizmadia A.

Virtual Affinity Fingerprints for Target Fishing: A New Application of Drug Profile Matching.

J. Chem. Inf. Model., 2013, 53 (1), pp 103-113

We recently introduced Drug Profile Matching (DPM), a novel virtual affinity fingerprinting bioactivity prediction method. DPM is based on the docking profiles of ca. 1200 FDA-approved small-molecule drugs against a set of nontarget proteins and creates bioactivity predictions based on this pattern. The effectiveness of this approach was previously demonstrated for therapeutic effect prediction of drug molecules. In the current work, we investigated the applicability of DPM for target fishing, i.e. for the prediction of biological targets for compounds. Predictions were made for 77 targets, and their accuracy was measured by Receiver Operating Characteristic (ROC) analysis. Robustness was tested by a rigorous 10-fold cross-validation procedure. This procedure identified targets (N = 45) with high reliability based on DPM performance. These 45 categories were used in a subsequent study which aimed at predicting the off-target profiles of currently approved FDA drugs. In this data set, 79% of the known drug-target interactions were correctly predicted by DPM, and additionally 1074 new drug-target interactions were suggested. We focused our further investigation on the suggested interactions of antipsychotic molecules and confirmed several interactions by a review of the literature.

Elleuche S, Fodor K, Klippel B, von der Heyde A, Wilmanns M, Antranikian G.

Structural and biochemical characterization of a NAD+-dependent alcohol dehydrogenase from O. oeni as a new model molecule for bioindustrial applications.

Appl. Micorbiol. Biotechnol.

Alcohol dehydrogenases are highly diverse enzymes catalysing the interconversion of alcohols and aldehydes or ketones. Due to their versatile specificities, these biocatalysts are of great interest for industrial applications. The adh3-gene encoding a group III alcohol dehydrogenase was isolated from the gram-positive bacterium Oenococcus oeni and was characterised after expression in the heterologous host Escherichia coli. Adh3 has been identified by genome BLASTP analyses using the amino acid sequence of 1,3-propanediol dehydrogenase DhaT from Klebsiella pneumoniae and group III alcohol dehydrogenases with known activity towards 1,3-propanediol as target sequences. The recombinant protein was purified in a two-step column chromatography approach. Crystal structure determination and biochemical characterisation confirmed that Adh3 forms a Ni(2+)-containing homodimer in its active form. Adh3 catalyses the interconversion of ethanol and its corresponding aldehyde acetaldyhyde and is also capable of using other alcoholic compounds as substrates, such as 1,3-propanediol, 1,2-propanediol and 1-propanol. In the presence of Ni(2+), activity increases towards 1,3-propanediol and 1,2-propanediol. Adh3 is strictly dependent on NAD(+)/NADH, whereas no activity has been observed with NADP(+)/NADPH as co-factor. The enzyme exhibits a specific activity of 1.1 U/mg using EtOH as substrate with an optimal pH value of 9.0 for ethanol oxidation and 8.0 for aldehyde reduction. Moreover, Adh3 exhibits tolerance to several metal ions and organic solvents, but is completely inhibited in the presence of Zn(2+). The present study demonstrates that O. oeni is a group III alcohol dehydrogenase with versatile substrate specificity, including Ni(2+)-dependent activity towards 1,3-propanediol.

Rovó P, Stráner P, Láng A, Bartha I, Huszár K, Nyitray L, Perczel A.

Structural insights into the trp-cage folding intermediate formation

IF:5,925

Chemistry. 2013 Feb 18;19(8):2628-40

The 20 residue long Trp-cage is the smallest protein known, and thus has been the subject of several in vitro and in silico folding studies. Here, we report the multistate folding scenario of the miniprotein in atomic detail. We detected and characterized different intermediate states by temperature dependent NMR measurements of the (15) N and (13) C/(15) N labeled protein, both at neutral and acidic pH values. We developed a deconvolution technique to characterize the invisible-fully folded, unfolded and intermediate-fast exchanging states. Using nonlinear fitting methods we can obtain both the thermodynamic parameters (ΔH(F-I) , T(m) (F-I) , ΔC(p) (F-I) and ΔH(I-U) , T(m) (I-U) , ΔC(p) (I-U) ) and the NMR chemical shifts of the conformers of the multistate unfolding process. During the unfolding of Trp-cage distinct intermediates evolve: a fast-exchanging intermediate is present under neutral conditions, whereas a slow-exchanging intermediate-pair emerges at acidic pH. The fast-exchanging intermediate has a native-like structure with a short α-helix in the G(11) -G(15) segment, whereas the slow-exchanging intermediate-pair presents elevated dynamics, with no detectable native-like residue contacts in which the G(11) P(12) peptide bond has either cis or trans conformation. Heteronuclear relaxation studies combined with MD simulations revealed the source of backbone mobility and the nature of structural rearrangements during these transitions. The ability to detect structural and dynamic information about folding intermediates in vitro provides an excellent opportunity to gain new insights into the energetic aspects of the energy landscape of protein folding. Our new experimental data offer exceptional testing ground for further computational simulations.

Venekei, I. and Marokházi, J.

PrtA Peptidases.

In Neil D. Rawlings and Guy S. Salvesen, editors: Handbook of Proteolytic Enzymes, Oxford: Academic Press, pp. 877 - 882.