Natural Resources for Therapeutic Use: Evidence from Brazil.

Identify plants used for insect stings in selfcare practices in the situation of suffering by people living in a rural location of Santa Maria municipality, Southern Brazil

The two kinases, AbrC1 and AbrC2, of the atypical two-component system AbrC are needed to regulate antibiotic production and differentiation in Streptomyces coelicolor

Two-component systems (TCSs) are the most important sensing mechanisms in bacteria. In Streptomyces, TCSs-mediated responses to environmental stimuli are involved in the regulation of antibiotic production. This study examines the individual role of two histidine kinases (HKs), AbrC1 and AbrC2, which form part of an atypical TCS in Streptomyces coelicolor. qRT-PCR analysis of the expression of both kinases demonstrated that both are expressed at similar levels in NB and NMMP media. Single deletion of abrC1 elicited a significant increase in antibiotic production, while deletion of abrC2 did not have any clear effect. The origin of this phenotype, probably related to the differential phosphorylation ability of the two kinases, was also explored indirectly, analyzing the toxic phenotypes associated with high levels of phosphorylated RR. The higher the AbrC3 regulator phosphorylation rate, the greater the cell toxicity. For the first time, the present work shows in Streptomyces the combined involvement of two different HKs in the response of a regulator to environmental signals. Regarding the possible applications of this research, the fact that an abrC1 deletion mutant overproduces three of the S. coelicolor antibiotics makes this strain an excellent candidate as a host for the heterologous production of secondary metabolites.Our research was funded by grants CSI099A12-1 from the Junta de Castilla y León to RS and BFU2010-17551 from the MICINN to MD. SR had a JAE-PreDoc grant from the CSIC. HR had a postdoctoral fellowship from the Botín Foundation.Peer Reviewe

Crystallization and Preliminary X-Ray Diffraction Analysis of a Mammal Inositol 1,3,4,5,6-Pentakisphosphate 2-Kinase.

Inositol 1,3,4,5,6-pentakisphosphate 2-kinase (IP5 2-K) is an enzyme that catalyses the formation of phytic acid (IP6) from IP5 and ATP. In mammals, IP6 is involved in multiple events such as DNA repair and mRNA edit and it is the precursor of inositol pyrophosphates, emerging compounds shown to have an essential role in apoptosis. In addition, IP5 2-K have functions in cells independently of its catalytic activity, for example in rRNA biogenesis. We pursue the structure determination of a mammal IP5 2-K by Protein Crystallography. For this purpose, we have designed protocols for recombinant expression and purification of Mus musculus IP5 2-K (mIP5 2-K). The recombinant protein has been expressed in two different hosts, E. coli and insect cells using the LSLt and GST fusion proteins, respectively. Both macromolecule preparations yielded crystals of similar quality. Best crystals diffracted to 4.3 Å (E. coli expression) and 4.0 Å (insect cells expression) maximum resolution. Both type of crystals belong to space group P212121 with an estimated solvent content compatible with the presence of two molecules per asymmetric unit. Gel filtration experiments are in agreement with this enzyme being a monomer. Crystallographic data analysis is currently undergoing.Peer Reviewe

Caracterización funcional de las quinasas AbrC1 y AbrC2 del sistema atípico de dos componentes AbrC de Streptomyces coelicolor

Resumen del trabajo presentado a la X Reunión de Microbiología Molecular celebrada en Segovia del 9 al 11 de junio de 2014.[Introducción]: Los sistemas de dos componentes (SDC) constituyen mecanismos de respuesta a estímulos ambientales ycontrolan la expresión de genes pueden afectar a la síntesis de antibióticos en Streptomyces. En el trabajo presentado se estudió el papel de dos histidína quinasas: AbrC1 (codificada por el gen SCO4598) y AbrC2 (codificada por el gen SCO4597) que junto con el regulador de respuesta AbrC3 (codificado por el genSCO4596) constituyen un SDC atípico que interviene en la regulación de la síntesis de antibióticos. Estos genes están codificados en la cadena negativa del genoma y entre cada gen existe una región intergénica con tamaño suficiente para contener un promotor independiente para cada uno de ellos. [Métodos]: Con el fin de estudiar la participación de ambas quinasas en la regulación de la producción de antibióticos se han obtenido mutantes individuales de cada quinasa así como un mutante de deleción de ambas y se han estudiado sus fenotipos en diferentes medios. Además, se han realizado estudios de expresión de ambos genes en diferentes condiciones mediante q-RT-PCR. Las capacidades fosforilativas de ambas proteínas se ha estudiado indirectamente sobreexpresando el regulador en diferentes cepas (la toxicidad producida por el regulador depende de su nivel de fosforilación) y también fosforilaciones in vitro utilizando para ello fósforo marcado radiactivamente.[Resultados]: La deleción ΔabrC1 genera una cepa que super produce actinorhodina (ACT) en los medios NB, NMMP y LB y de CDA en medio NA en relación a la cepa silvestre. Sin embargo el mutante de deleción ΔabrC2 y el doble mutante ΔabrC1/C2 mostraron niveles de producción de antibióticos similares a los de esta cepa parental. Los estudios de expresión indican que ambas quinasas se expresan y lo hacen a niveles similares y que son cotranscritas compartiendo un mismo promotor. En cuanto a las capacidades fosforilativas de las quinasas, su capacidad para autofosforilarse ha sido confirmada utilizando fósforo marcado radiactivamente. Además, se observó (indirectamente) que el regulador se encuentra más fosforilado en el mutante simple ΔabrC1. [Conclusiones]: Se ha observado que ambas quinasas son funcionales a nivel transcripcional y capaces de fosforilar al regulador. La eficiencia de la quinasa AbrC2 para fosforilar el regulador es mayor que la de la quinasa AbrC1 (inferida a partir de la producción de antibióticos de los mutantes individuales de las quinasas y la toxicidad derivada de la sobreexpresión del regulador) y ambas son capaces de autofosforilarse, habiéndose identificado en ambas el residuo fosforilable.Peer Reviewe

A new calmodulin-binding motif for inositol 1,4,5-trisphosphate 3-kinase regulation

© The Authors Journal compilation © 2014 Biochemical Society. IP3-3K [Ins(1,4,5)P33-kinase] is a key enzyme that catalyses the synthesis of Ins(1,3,4,5)P4, using Ins(1,4,5)P3and ATP as substrates. Both inositides, substrate and product, present crucial roles in the cell. Ins(1,4,5)P3is a key point in Ca2+metabolism that promotes Ca2+release from intracellular stores and together with Ins(1,3,4,5)P4regulates Ca2+homoeostasis. In addition, Ins(1,3,4,5)P4is involved in immune cell development. It has been proved that Ca2+/CaM (calmodulin) regulates the activity of IP3-3K, via direct interaction between both enzymes. Although we have extensive structural knowledge of the kinase domains of the three IP3-3K isoforms, no structural information is available about the interaction between IP3-3K and Ca2+/CaM. In the present paper we describe the crystal structure of the complex between human Ca2+/CaM and the CaM-binding region of human IP3-3K isoform A (residues 158-183) and propose a model for a complex including the kinase domain. The structure obtained allowed us to identify all of the key residues involved in the interaction, which have been evaluated by site-directed mutagenesis, pull-down and fluorescence anisotropy experiments. The results allowed the identification of a new CaM-binding motif, expanding our knowledge about how CaM interacts with its partners.Peer Reviewe

Noncatalytic aspartate at the exonuclease domain of proofreading DNA polymerases regulates both degradative and synthetic activities

Most replicative DNA polymerases (DNAPs) are endowed with a 3′-5′ exonuclease activity to proofread the polymerization errors, governed by four universally conserved aspartate residues belonging to the Exo I, Exo II, and Exo III motifs. These residues coordinate the two metal ions responsible for the hydrolysis of the last phosphodiester bond of the primer strand. Structural alignment of the conserved exonuclease domain of DNAPs from families A, B, and C has allowed us to identify an additional and invariant aspartate, located between motifs Exo II and Exo III. The importance of this aspartate has been assessed by site-directed mutagenesis at the corresponding Asp of the family B ϕ29 DNAP. Substitution of this residue by either glutamate or alanine severely impaired the catalytic efficiency of the 3′-5′ exonuclease activity, both on ssDNA and dsDNA. The polymerization activity of these mutants was also affected due to a defective translocation following nucleotide incorporation. Alanine substitution for the homologous Asp in family A T7 DNAP showed essentially the same phenotype as ϕ29 DNAP mutant D121A. This functional conservation, together with a close inspection of ϕ29 DNAP/DNA complexes, led us to conclude a pivotal role for this aspartate in orchestrating the network of interactions required during internal proofreading of misinserted nucleotides.Peer Reviewe

The new InsP<inf>3</inf>Kinase inhibitor BIP-4 is competitive to InsP<inf>3</inf> and blocks proliferation and adhesion of lung cancer cells

© 2015 Elsevier Inc. All rights reserved. As ectopic expression of the neuronal inositol-1,4,5-trisphosphate-3-kinase A (InsP3Kinase) in tumor cells increases the metastatic potential, InsP3Kinase is an interesting target for tumor therapy. Recently, we have identified a membrane-permeable InsP3Kinase inhibitor (BAMB-4) exhibiting an IC50-value of 20 μM. Here we characterized a new InsP3Kinase inhibitor which shows a 130-fold lower IC50 value (157 ± 57 nM) as compared to BAMB-4. We demonstrate that this nitrophenolic compound, BIP-4, is non-competitive to ATP but competitive to InsP3, thus exhibits a high selectivity for inhibition of InsP3Kinase activity. Docking analysis suggested a putative binding mode of this molecule into the InsP3Kinase active site. Determination of cellular uptake in lung cancer cells (H1299) revealed that 6% of extracellular BIP-4 is internalized by non-endosomal uptake, showing that BIP-4 is not trapped inside endo/lysosomes but is available to inhibit cellular InsP3Kinase activity. Interestingly, we found that BIP-4 mediated inhibition of InsP3Kinase activity in the two lung cancer cell lines H1299 and LN4323 inhibited proliferation and adhesion at IC50 values of 3 μM or 2 μM, respectively. InsP3Kinase inhibition did not alter ATP-induced calcium signals but significantly reduced the level of Ins(1,3,4,5,6)P5. From these data we conclude that the inhibitory effect of BIP-4 on proliferation and adhesion of lung cancer cells does not result from alterations of calcium but from alterations of inositol phosphate signals. In summary, we reveal that inhibition of cellular InsP3Kinase by BIP-4 impairs proliferation and adhesion and therefore BIP-4 might be a promising compound to reduce the metastatic potential of lung carcinoma cells.Peer Reviewe