Yang, Y., Gourinath, S., Kovács, M., Nyitray, L. , Reutzel, R., Himmel, D. M., O'Neall-Hennessey, E., Reshetnikova, L., Szent-Györgyi, A. G., Brown, J. H. & Cohen, C.

Rigor-like structures from muscle myosins reveal key mechanical elements in the transduction pathways of this allosteric motor.

Structure 15, 553-64.

Tóth, J., Varga, B., Kovács, M., Málnási-Csizmadia, A. & Vértessy, B. G.

Kinetic mechanism of human dUTPase, an essential nucleotide pyrophosphatase enzyme.

J Biol Chem 282, 33572-82.

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize gamma-(32)P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

Tóth, J., Simon, Z., Medveczky, P., Gombos, L., Jelinek, B., Szilágyi, L., Gráf, L. & Málnási-Csizmadia, A.

Site directed mutagenesis at position 193 of human trypsin 4 alters the rate of conformational change during activation: role of local internal viscosity in protein dynamics.

Proteins 67, 1119-27.V

Tóth, J., Siklódi, E., Medveczky, P., Gallatz, K., Németh, P., Szilágyi, L., Gráf, L. & Palkovits, M.

Regional distribution of human trypsinogen 4 in human brain at mRNA and protein level.

Neurochem Res 32, 1423-33.

Gene PRSS3 on chromosome 9 of the human genome encodes, due to alternative splicing, both mesotrypsinogen and trypsinogen 4. Mesotrypsinogen has long been known as a minor component of trypsinogens expressed in human pancreas, while the mRNA for trypsinogen 4 has recently been identified in brain and other human tissues. We measured the amount of trypsinogen 4 mRNA and the quantity of the protein as well in 17 selected areas of the human brain. Our data suggest that human trypsinogen 4 is widely but unevenly distributed in the human brain. By immunohistochemistry, here we show that this protease is localized in neurons and glial cells, predominantly in astrocytes. In addition to cellular immunoreactivity, human trypsinogen 4 immunopositive dots were detected in the extracellular matrix, supporting the view that human trypsinogen 4 might be released from the cells under special conditions.

Németh, A. L., Medveczky, P., Tóth, J., Siklódi, E., Schlett, K., Patthy, A., Palkovits, M., Ovádi, J., Tőkési, N., Németh, P., Szilágyi, L. & Gráf, L.

Unconventional translation initiation of human trypsinogen 4 at a CUG codon with an N-terminal leucine. A possible means to regulate gene expression.

Febs J 274, 1610-20

Chromosomal rearrangements apparently account for the presence of a primate-specific gene (protease serine 3) in chromosome 9. This gene encodes, as the result of alternative splicing, both mesotrypsinogen and trypsinogen 4. Whereas mesotrypsinogen is known to be a pancreatic protease, neither the chemical nature nor biological function of trypsinogen 4 has been explored previously. The trypsinogen 4 sequence contains two predicted translation initiation sites: an AUG site that codes for a 72-residue leader peptide on Isoform A, and a CUG site that codes for a 28-residue leader peptide on Isoform B. We report studies that provide evidence for the N-terminal amino acid sequence of trypsinogen 4 and the possible mechanism of expression of this protein in human brain and transiently transfected cells. We raised mAbs against a 28-amino acid synthetic peptide representing the leader sequence of Isoform B and against recombinant trypsin 4. By using these antibodies, we isolated and chemically identified trypsinogen 4 from extracts of both post mortem human brain and transiently transfected HeLa cells. Our results show that Isoform B, with a leucine N terminus, is the predominant (if not exclusive) form of the enzyme in post mortem human brain, but that both isoforms are expressed in transiently transfected cells. On the basis of our studies on the expression of a series of trypsinogen 4 constructs in two different cell lines, we propose that unconventional translation initiation at a CUG with a leucine, rather than a methionine, N terminus may serve as a means to regulate protein expression.

Marokházi, J., Mihala, N., Hudecz, F., Fodor, A., Gráf, L. & Venekei, I.

Cleavage site analysis of a serralysin-like protease, PrtA, from an insect pathogen Photorhabdus luminescens and development of a highly sensitive and specific substrate.

. Febs J 274, 1946-56.

Málnási-Csizmadia, A., Gyimesi, M., Song, L., Sen, I., Fajer, P.G.

Myosin cleft closure by double electron-electron resonance and dipolar EPR.

J Phys Condens Matter 19, 285208.

Málnási-Csizmadia, A., Tóth, J., Pearson, D. S., Hetényi, C., Nyitray, L. , Geeves, M. A., Bagshaw, C. R. & Kovács, M.

Selective perturbation of the myosin recovery stroke by point mutations at the base of the lever arm affects ATP hydrolysis and phosphate release.

J Biol Chem 282, 17658-64.

After ATP binding the myosin head undergoes a large structural rearrangement called the recovery stroke. This transition brings catalytic residues into place to enable ATP hydrolysis, and at the same time it causes a swing of the myosin lever arm into a primed state, which is a prerequisite for the power stroke. By introducing point mutations into a subdomain interface at the base of the myosin lever arm at positions Lys(84) and Arg(704), we caused modulatory changes in the equilibrium constant of the recovery stroke, which we could accurately resolve using the fluorescence signal of single tryptophan Dictyostelium myosin II constructs. Our results shed light on a novel role of the recovery stroke: fine-tuning of this reversible equilibrium influences the functional properties of myosin through controlling the effective rates of ATP hydrolysis and phosphate release.

Kovács, M., Thirumurugan, K., Knight, P. J. & Sellers, J. R.

Load-dependent mechanism of nonmuscle myosin 2.

Proc Natl Acad Sci U S A 104, 9994-9

Loads on molecular motors regulate and coordinate their function. In a study that directly measures properties of internally strained myosin 2 heads bound to actin, we find that human nonmuscle myosins 2A and 2B show marked load-dependent changes in kinetics of ADP release but not in nucleotide binding. We show that the ADP release rate constant is increased 4-fold by the assisting load on one head and decreased 5-fold (for 2A) or 12-fold (for 2B) by the resisting load on the other. Thus these myosins, especially 2B, have marked mechanosensitivity of product release. By regulating the actin attachment of myosin heads, this provides a basis for energy-efficient tension maintenance without obstructing cellular contractility driven by other motors such as smooth muscle myosin. Whereas forward load accelerates the cycle of interaction with actin, resistive load increases duty ratio to favor tension maintenance by two-headed attachment.

Kovács, B. M., Toussaint, M. J., Gruys, E., Fábián, I. B., Szilágyi, L., Janan, J. & Rudas, P.

Evaluation of goose serum amyloid a acute phase response by enzyme-linked immunosorbent assay.

Acta Vet Hung 55, 349-57.

Serum amyloid A (SAA) is of interest as the circulating precursor of amyloid A protein, the fibrillar component of AA (secondary) amyloid deposits, and also as an extremely sensitive and rapid major acute phase protein. Serum concentrations of acute phase proteins (APPs) provide valuable information about the diagnosis and prognosis of various diseases, and thus the relevance of APPs for monitoring the health status of domestic animals is widely accepted. More importantly, the measurement of SAA concentration assists in assessing the prognosis in secondary amyloidosis, which is a common disease of geese, affecting an increasing number of animals. In the present study we introduce a highly sensitive goose-specific ELISA method for measuring SAA concentration in goose serum or plasma samples. Samples were taken from geese of the Landes Grey and Hungarian White breeds, which were stimulated for an acute phase reaction by administration of a commercially available fowl cholera vaccine containing inactivated Pasteurella multocida. Strong and characteristically rapid acute phase responses were measured in both breeds, peaking at approximately 24 h after inoculation. The maximum SAA concentration was 1200 microg/ml. At 72 h postinoculation, the concentrations returned to pre-inoculation values. There was significantly (p = 0.004) less intense response in the control groups; however, a very mild increase of SAA levels was detected due to the stress inevitably caused by the sampling procedure.

Kintses, B., Gyimesi, M., Pearson, D. S., Geeves, M. A., Zeng, W., Bagshaw, C. R. & Málnási-Csizmadia, A.

Reversible movement of switch 1 loop of myosin determines actin interaction.

Embo J 26, 265-74.

The conserved switch 1 loop of P-loop NTPases is implicated as a central element that transmits information between the nucleotide-binding pocket and the binding site of the partner proteins. Recent structural studies have identified two states of switch 1 in G-proteins and myosin, but their role in the transduction mechanism has yet to be clarified. Single tryptophan residues were introduced into the switch 1 region of myosin II motor domain and studied by rapid reaction methods. We found that in the presence of MgADP, two states of switch 1 exist in dynamic equilibrium. Actin binding shifts the equilibrium towards one of the MgADP states, whereas ATP strongly favors the other. In the light of electron cryo-microscopic and X-ray crystallographic results, these findings lead to a specific structural model in which the equilibrium constant between the two states of switch 1 is coupled to the strength of the actin-myosin interaction. This has implications for the enzymatic mechanism of G-proteins and possibly P-loop NTPases in general.

Kawamichi, H., Zhang, Y., Hino, M., Nakamura, A., Tanaka, H., Farkas, L., Nyitray, L. & Kohama, K.

Calcium inhibition of Physarum myosin as examined by the recombinant heavy mero-myosin.

Adv Exp Med Biol 592, 265-72.

Jako, E., Ittzes, P., Szenes, Á., Kun, A., Szathmary, E. & Pál, G.

In silico detection of tRNA sequence features characteristic to aminoacyl-tRNA synthetase class membership.

Nucleic Acids Res 35, 5593-609.

Iwamoto, H., Oiwa, K., Kovács, M., Sellers, J. R., Suzuki, T., Wakayama, J., Tamura, T., Yagi, N. & Fujisawa, T.

Diversity of structural behavior in vertebrate conventional myosins complexed with actin.

J Mol Biol 369, 249-64.

Low-resolution three-dimensional structures of acto-myosin subfragment-1 (S1) complexes were retrieved from X-ray fiber diffraction patterns, recorded either in the presence or absence of ADP. The S1 was obtained from various myosin-II isoforms from vertebrates, including rabbit fast-skeletal and cardiac, chicken smooth and human non-muscle IIA and IIB species, and was diffused into an array of overstretched, skinned skeletal muscle fibers. The S1 attached to the exposed actin filaments according to their helical symmetry. Upon addition of ADP, the diffraction patterns from acto-S1 showed an increasing magnitude of response in the order as listed above, with features of a lateral compression of the whole diffraction pattern (indicative of increased radius of the acto-S1 complex) and an enhancement of the fifth layer-line reflection. The structure retrieval indicates that these changes are mainly due to the swing of the light chain (LC) domain in the direction consistent with the cryo-electron microscopic results. In the non-muscle isoforms, the swing is large enough to affect the manner of quasi-crystal packing of the S1-decorated actin filaments and their lattice dimension, with a small change in the twist of actin filaments. Variations also exist in the behavior of the 50K-cleft, which apparently opens upon addition of ADP to the non-muscle isoforms but not to other isoforms. The fast-skeletal S1 remains as the only isoform that does not clearly exhibit either of the structural changes. The results indicate that the "conventional" myosin-II isoforms exhibit a wide variety of structural behavior, possibly depending on their functions and/or the history of molecular evolution.

Gallatz, K., Medveczky, P., Németh, P., Szilágyi, L., Gráf, L., Palkovits, M.

Human trypsinogen 4-like immunoreactivity in the white matter of the cerebral cortex and the spinal cord.

Ideggyógyászati Szemle/Clinical Neuroscience 60, 118-123.

Eleftherianos, I., Gokcen, F., Felföldi, G., Millichap, P. J., Trenczek, T. E., ffrench-Constant, R. H. & Reynolds, S. E.

The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta.

Cell Microbiol 9, 1137-47.

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

Szenthe, B., Patthy, A., Gáspári, Z., Kékesi, A.K., Gráf, L., Pál, G.

When the surface tells what lies beneath: combinatorial phage-display mutagenesis reveals complex networks of surface-core interactions in the pacifastin protease inhibitor family.

J Mol Biol. 2007 Jun 29;370(1):63-79

J Mol Biol. 2007 Jun 29;370(1):63-79

Pacifastin protease inhibitors are small cysteine-rich motifs of approximately 35 residues that were discovered in arthropods. The family is divided into two related groups on the basis of the composition of their minimalist inner core. In group I, the core is governed by a Lys10-Trp26 interaction, while in group II it is organized around Phe10. Group I inhibitors exhibit intriguing taxon specificity: potent arthropod-trypsin inhibitors from this group are almost inactive against vertebrate enzymes. The group I member SGPI-1 and the group II member SGPI-2 are extensively studied inhibitors. SGPI-1 is taxon-selective, while SGPI-2 is not. Individual mutations failed to explain the causes underlying this difference. We deciphered this phenomenon using comprehensive combinatorial mutagenesis and phage display. We produced a complete chimeric SGPI-1 / SGPI-2 inhibitor-phage library, in which the two sequences were shuffled at the highest possible resolution of individual residues. The library was selected for binding to bovine trypsin and crayfish trypsin. Sequence analysis of the selectants revealed that taxon specificity is due to an intra-molecular functional coupling between a surface loop and the Lys10-Trp26 core. Five SGPI-2 surface residues transplanted into SGPI-1 resulted in a variant that retained the "taxon-specific" core, but potently inhibited both vertebrate and arthropod enzymes. An additional rational point mutation resulted in a picomolar inhibitor of both trypsins. Our results challenge the generally accepted view that surface residues are the exclusive source of selectivity for canonical inhibitors. Moreover, we provide important insights into general principles underlying the structure-function properties of small disulfide-rich polypeptides, molecules that exist at the borderline between peptides and proteins.