Biological and molecular characterization of P101 isolate, a tobamoviral pepper strain from Bulgaria

A tobamovirus isolated from pepper crops in Bulgaria has been characterized, and is referred to below as P101. It was closely related to Paprika mild mottle virus (PaMMV) (Dutch isolate), based upon the serological relationship of its coat protein, and the nucleotide sequence analysis of the gene encoding the coat protein and the 3′ non-coding region of the viral RNA. The coat proteins of the two isolates differ by two amino acids, and these substitutions may be responsible for the different reactivity of the isolates towards a polyclonal antiserum raised against the virion of the Dutch isolate. The biological behaviour of both isolates was similar in îhe hosts tested, except in pepper plants where P101 induced delayed and milder symptoms compared with PaMMV, although their accumulation levels were similar. In addition, we investigated the infection pattern of the two isolates in tomato plants. Both isolates accumulated in protoplasts as well as in inoculated leaves, although systemic invasion was limited. This limited spread was not due to activation of defense mechanism(s) in the plant, since the upper uninoculated leaves from P101 infected tomato plants were fully susceptible to challenge inoculation with the virus. Instead, it appears due to a restriction of long-distance movement, that could be overcome in tomato plants co-infected with Tobacco mosaic virus (TMV), but not with either Cucumber mosaic virus or Pepino mosaic virus. The ability of P101 to move systemically in the presence of TMV was not linked to enhanced accumulation of P101 at the cellular level. Thus, a tobamovirus but not the viruses tested from other genera could complement, in trans, the function(s) required for PaMMV to invade the upper uninoculated leaves. Paprika mild mottle virus strain B is proposed as the name for this new isolate.The work was supported by grants from the CICYT (AGF95-0696) and from the CAM (07B/0034/1997). P. G. is the recipient of a CAM predoctoral fellowshi

Nuclear DNA polymerase beta from Leishmania infantum. Cloning, molecular analysis and developmental regulation

13 p.-8 fig.We have identified a novel polymerase beta (Pol β)-like enzyme from Leishmania infantum, a parasite protozoon causing disease in humans. This protein, named Li Pol β, shows a nuclear localization that contrasts with the mitochondrial localization of Pol β from Crithidia fasciculata, a closely related parasite, the only polymerase β described so far in Trypanosomatidae. Li Pol β, that belongs to the DNA polymerase X family, displays an evolutionarily conserved Pol β-type DNA polymerase core, in which most of the key residues involved in DNA binding, nucleotide binding, dRPase and polymerization catalysis are conserved. In agreement with this, Li Pol β, overproduced in Escherichia coli, displayed intrinsic DNA polymerase activity. Cell synchronization experiments showed a correlation between both Li Pol β mRNA and protein levels along the parasite cell cycle. Analysis of these parameters at the different growth phases of the parasite, from the proliferative (non-infective) logarithmic phase to the non-dividing (highly infectious) stationary phase, showed high levels of Li Pol β at the infective phase of the parasite. The data suggest a role of Li Pol β in base excision repair in L.infantum, a parasite usually affected by oxygen stress environments into the macrophage host cells.This work was supported by a grant 97/0492 from the Fondo de Investigación Sanitaria of the Spanish Health Ministry, a grant BIO1999-0853 from the Ministry of Science and Tecnology and partially by grants 08.20037/1999 and 08.1/0044.1/1998 from the CAM.Peer reviewe

Evolutionary distinct residues in the uncoupling protein UCP1 are essential for its characteristic basal proton conductance

13 páginas, 6 figuras, 2 tablas -- PAGS nros. 1010-1022The uncoupling proteins (UCPs) are mitochondrial transporters that modulate the efficiency of oxidative phosphorylation. Members of this family have been described in many phyla within the animal and plant kingdoms, as well as in fungi. The mammalian uncoupling protein UCP1 is activated by fatty acids and inhibited by nucleotides. In the absence of both regulators, UCP1 presents a high ohmic proton conductance that is a unique property of this carrier. The increasing number of protein sequences available has enabled us to apply a sequence analysis approach to investigate transporter function. We reconstructed a robust phylogeny of UCPs and used comparative sequence analysis to search for phylogenetically shared derived sequence features that may confer distinct properties on UCP1. We assessed the functional relevance of shared derived UCP1 residues by substituting them with their counterparts in UCP2, and expressing the protein chimeras in yeast. We found that substitution of both Glu134 and Met140 abolishes the basal proton permeability of UCP1 while preserving fatty acid activation and its nucleotide inhibitionThe authors thank Dr Antonio Romero and Dr Federico Abascal for their help and advice. The work was supported by grants from the Spanish Ministry of Education and Science (BIO2002-00142) to E.R. and (CGL2004-00401) to R.Z. A.L. was supported by a grant from the Comunidad de MadridPeer reviewe

Diversity of trypsins in the Mediterranean corn borer Sesamia nonagrioides (Lepidoptera: Noctuidae), revealed by nucleic acid sequences and enzyme purification

Acknowledgments We thank “Servicio de secuenciación de DNA” and “Servicio de Química de proteínas y Síntesis de oligonucleótidos” at CIB-CSIC, Madrid, and “Unidad de Proteómica” at CNIC-ISCIII, Madrid for the kind assistance of their staff. This work was supported by grants from European Commission (QLRT-2001-01969), and Ministerio de Educación y Ciencia (CICYT BIO2003-03428).The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed.Depto. de Bioquímica y Biología MolecularFac. de Ciencias BiológicasTRUEpu

Solution structure of Taxotere-induced microtubules to 3-nm resolution. The change in protofilament number is linked to the binding of the taxol side chain

8 p.-6 fig.-1 tab.The synchrotron x-ray solution scattering profiles of microtubules assembled from purified GDP- or GTP-tubulin with the antitumor drug docetaxel (Taxotere) are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known surface lattice of microtubules, with a center to center lateral spacing of 5.7 +/- 0.1 nm. The higher angle part of the scattering profile, and therefore the substructure of the microtubule wall is identical in Taxotere- and Taxol-induced microtubules, to the resolution of the measurements. However, Taxotere-induced microtubules have a mean diameter of 24.2 +/- 0.4 nm, which is 1.12 +/- 0.01 times larger than that of paclitaxel (Taxol) induced microtubules. The population of Taxotere microtubules has on average 13.4 protofilaments, which is similar to control microtubules assembled with glycerol but is in marked contrast with Taxol-induced microtubules, which have on average 12 protofilaments under identical solution conditions. Model populations of Taxotere and Taxol microtubules with the distributions of protofilament numbers determined by electron microscopy reproduce the positions and approximate intensities of the experimental x-ray scattering data. Comparison of the structures and activities of both taxoids strongly suggests that the change of the more frequent lateral bond angle between tubulin molecules from 152.3 degrees (13-protofilament microtubules) to 150 degrees (12-protofilament microtubules) is linked to the binding of the side chain of Taxol. Optimal microtubule formation is obtained with unitary Taxotere to tubulin heterodimer ratio; however, ligand molecules in excess over tubulin dimers cause a loss of cylindrical scattering features, consistent with microtubule opening. The results are compatible with the observed biochemical and thermodynamic properties of this ligand-induced microtubule assembly system and also with the simple working hypothesis that taxoids would bind between adjacent microtubule protofilaments.This work was supported in part by Dirección General de Investigación Científica y Técnica Grant PB 92007, European Community Science Contract SC1-CT91-0658, and the Consejo Superior de Investigaciones Científicas program Acciones Especiales de Estructura y Función de Proteinas. Access to the Daresbury Laboratory Synchrotron Radiation Source was obtained through the European Community Large Scale Facilities Program.Peer reviewe

The C-terminus of H-ras as a target for the covalent binding of reactive compounds modulating Ras-dependent pathways

Ras proteins are crucial players in differentiation and oncogenesis and constitute important drug targets. The localization and activity of Ras proteins are highly dependent on posttranslational modifications at their C-termini. In addition to an isoprenylated cysteine, H-Ras, but not other Ras proteins, possesses two cysteine residues (C181 and C184) in the C-terminal hypervariable domain that act as palmitoylation sites in cells. Cyclopentenone prostaglandins (cyPG) are reactive lipidic mediators that covalently bind to H-Ras and activate H-Ras dependent pathways. Dienone cyPG, such as 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) and Δ12-PGJ2 selectively bind to the H-Ras hypervariable domain. Here we show that these cyPG bind simultaneously C181 and C184 of H-Ras, thus potentially altering the conformational tendencies of the hypervariable domain. Based on these results, we have explored the capacity of several bifunctional cysteine reactive small molecules to bind to the hypervariable domain of H-Ras proteins. Interestingly, phenylarsine oxide (PAO), a widely used tyrosine phosphatase inhibitor, and dibromobimane, a cross-linking agent used for cysteine mapping, effectively bind H-Ras hypervariable domain. The interaction of PAO with H-Ras takes place in vitro and in cells and blocks modification of H-Ras by 15d-PGJ2. Moreover, PAO treatment selectively alters H-Ras membrane partition and the pattern of H-Ras activation in cells, from the plasma membrane to endomembranes. These results identify H-Ras as a novel target for PAO. More importantly, these observations reveal that small molecules or reactive intermediates interacting with spatially vicinal cysteines induce intramolecular cross-linking of H-Ras C-terminus potentially contributing to the modulation of Ras-dependent pathways.This work was supported by European Union Cooperation in the field of Scientific and Technical research (COST) Action CM1001, grant SAF2009-11642 from Ministerio de Ciencia e Innovación and Red Temática de Investigación Cooperativa from Instituto de Salud Carlos II (Spain) RD07/0064/0007 to DP-S and RD07/0064/0000 to EP-I. Work at Universitat Pompeu Fabra was supported by grants BIO2005-07592-CO2-02 and BIO2008-04487-CO3-02 from Ministerio de Ciencia e Innovación (Spain) to DA, and by the regional government of Catalunya (SGR2005-00494). JMR’s work is supported by Fondo de Investigaciones Sanitarias-Intrasalud (PS09/00562) and Red Temática de Investigación Cooperativa RD06/0020/0003 from Instituto de Salud Carlos III, and the Spanish Association Against Cancer (AECC). CLO is the recipient of a predoctoral fellowship from the Formación de Personal Investigador (FPI) program (Ministerio de Ciencia e Innovación, Spain, BES-2010-033718). CAG-D is the recipient of a fellowship from Fondo de Investigaciones Sanitarias-Beca de Formación en Investigación. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscri

Modification of the K170-K185 peptide from the C-terminal region of H-Ras by cyPG.

<p>(A) The synthetic K170-K185 peptide was incubated with the indicated cyPG and the resulting adducts analyzed by MALDI-TOF MS. When indicated, incubation mixtures were treated with 50 mM iodoacetamide after addition of 10 mM DTT. Spectra presented are representative from at least three independent assays per experimental condition. Structures of the cyPG used are shown in insets. Electrophilic carbons are marked by asterisks. (B) Summary of the theoretical peptide adducts for which compatible peaks were observed experimentally. +CAM indicates that the peak is compatible with the formation of carbamidomethyl cysteine subsequent to iodoacetamide treatment. (C) Ribbon diagram for the theoretical backbone structure of the H-Ras K170-K185 peptide (in green) modified by addition of 15d-PGJ₂ to cysteines 181 and 184. The side-chains of the cysteine residues and the 15d-PGJ₂ ligand are displayed in yellow and a default atom-type color scheme, respectively.</p

Modification of the K170-K185 peptide by PAO.

<p>(A) The K170-K185 peptide was incubated with PAO and analyzed by MALDI-TOF MS. Results are representative from at least four assays. (B) Scheme of the formation of a PAO adduct with two thiol groups. (C) Analysis of the PAO-peptide adduct by MALDI-TOF-TOF MS/MS analysis. The sequence of the peptide with the fragments generated is shown for reference. (D) Ribbon diagram for the theoretical backbone structure of H-Ras K170-K185 peptide model (in green) modified by PAO addition. The side-chains of the cysteine residues and PAO are displayed in yellow and a default atom-type color scheme, respectively.</p