RESUMEN

Salicylic acid (SA) is an important phytohormone that is involved in the regulation of plant defence, growth and development. A large number of proteins have been shown to have the ability to interact with SA, and NPR4 has been demonstrated to be a receptor of SA that plays significant roles in the innate immune response of plants. In this study, Spodoptera frugiperda (Sf9) cells were used to express full-length AtNPR4 from Arabidopsis thaliana. To facilitate crystallization, T4 lysozyme (T4L) was added to the N-terminus of the AtNPR4 protein. The recombinant T4L-AtNPR4 protein was expressed, purified and crystallized using the sitting-drop and hanging-drop vapour-diffusion methods. The T4L-AtNPR4 crystals have symmetry consistent with space group C2, with unit-cell parameters a = 93.7, b = 85.8, c = 88.2 Å, ß = 90° and one molecule per asymmetric unit. The best crystal diffracted to a resolution of 2.75 Å. Structure determination is in progress.

Asunto(s)

Proteínas de Arabidopsis/química , Arabidopsis/química , Proteínas Recombinantes de Fusión/química , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Bacteriófago T4/química , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Baculoviridae/genética , Baculoviridae/metabolismo , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Muramidasa/química , Muramidasa/genética , Muramidasa/metabolismo , Inmunidad de la Planta/genética , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ácido Salicílico/química , Ácido Salicílico/inmunología , Ácido Salicílico/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Sf9 , Spodoptera , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/metabolismo

RESUMEN

Nucleotide binding proteins are involved in many important cellular processes and form one of the largest protein families. Traditionally, the identification of nucleotide binding motif, such as the ATP binding P-loop, has relied on the comparison of protein sequences, consideration of the function of each of the proteins and the identification of signature motifs within the sequence. Sometimes, it is difficult to identify nucleotide binding proteins based on sequence alignment because of increased evolutionary distances. In such cases, structural alignments can provide a better guide for comparing specific features of sequences because the overall structures of these motifs are conserved despite low sequence identity. In the present study, on the basis of bioinformatics and structural comparison of three representative protein structures of Ham1 superfamily, YjjX, YggV, and YhdE, previously identified as nucleotide binding proteins, we have identified a novel nucleotide binding motif (T/SXXXXK/R). The importance of this signature motif in binding of nucleotides was validated using site directed mutagenesis. Mutations of conserved residues of the loop either decreased or completely abolished the nucleotide binding activity of the protein. We used the conserved motif identified in the study to search for other proteins having a similar motif. Two proteins, GTP cyclohydrolase II and dephospho-CoA pyrophosphorylase showed presence of the loop, suggesting that this nucleotide binding motif is not unique in the Ham1 superfamily, but represents a novel NTP recognition motif.

Asunto(s)

Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Nucleótidos/metabolismo , Secuencia de Aminoácidos , Bacterias/química , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Hidrólisis , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Unión Proteica , Alineación de Secuencia

RESUMEN

MICU2 has been reported to interact with MICU1 and participate in the regulation of mitochondrial Ca(2+) uptake, although the molecular determinants underlying the function of MICU2 is unknown. In order to characterize MICU2 we screened a series of N-terminal and C-terminal truncations and obtained constructs which can be expressed in abundance, giving rise to soluble samples to enable subsequent characterizations. Size exclusion chromatography (SEC) and multi-angle laser light scattering (MALLS) revealed that MICU2 exists as a monomer in Ca(2+)-free conditions but forms a dimer in Ca(2+)-bound conditions. Unlike MICU1, the C-helix domain of MICU2 exhibits no influence on protein conformation in both Ca(2+)-free and Ca(2+)-bound forms. Furthermore, mutation of the first EF-hand abolishes the ability of MICU2 to switch to a dimer in the presence of Ca(2+), indicating that the first EF-hand is not only involved in Ca(2+) binding but also in conformational change. Our pull-down and co-immunoprecipitation assays suggest that, in addition to disulfide bonds, salt bridges also contribute to MICU1-MICU2 heterodimer formation.

RESUMEN

In recent years, hand-foot-and-mouth disease (HFMD), which is caused by Enteroviruses, has emerged as a serious illness. It affects mainly children under the age of five and results in high fatality rates. Enterovirus 71 (EV71) is the main causative agent of HFMD in China and currently there are no effective anti-viral drugs available to treat HFMD. In the present study, we screened compounds for inhibition of proliferation of EV71. Compound YZ-LY-0 stalled the life cycle of EV71. The inhibitor exhibited EC50 value of 0.29 µm against SK-EV006 strain of EV71. Notably, YZ-LY-0 had low cytotoxicity (CC50 > 100 µM) and a high selectivity index (over 300) in Vero and RD cells. YZ-LY-0 in combination with an EV71 RdRp inhibitor or an entry inhibitor showed an antagonistic effect at very low concentrations. However, at higher concentrations the inhibitors exhibited a synergistic effect in inhibiting viral replication. Preliminary results on investigation of the mechanism of inhibition indicate that YZ-LY-0 does not block the entry of the virus in the host cell, but instead inhibits an early stage of EV71 replication. Our studies provide a potential clinical therapeutic option against EV71 infections and suggest that a combined application of YZ-LY-0 with other inhibitors could be more effective in the treatment of HFMD.

Asunto(s)

Antivirales/farmacología , Enterovirus/efectos de los fármacos , Enfermedad de Boca, Mano y Pie/virología , Replicación Viral/efectos de los fármacos , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Enterovirus/fisiología , Humanos , Células Vero

RESUMEN

The Arabidopsis thaliana glutathione peroxidase 3 (GPX3) gene encodes a glutathione peroxidase with roles in H2O2 homeostasis and signalling. The GPX3 gene sequence was cloned into pGEX-6P1 and overexpressed in Escherichia coli. The GPX3 protein was purified to homogeneity in two chromatographic steps. Various lengths of the GPX3 sequence were used to obtain proteins that yielded crystals using vapour-diffusion techniques, but only GPX3ΔN36 (lacking 36 amino acids from the N-terminus) showed a good diffraction pattern. Its crystals diffracted to 2.8 Šresolution and belonged to space group P65, with unit-cell parameters a = b = 98.241, c = 42.057 Å.

Asunto(s)

Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Glutatión Peroxidasa/química , Glutatión Peroxidasa/genética , Proteínas de Arabidopsis/aislamiento & purificación , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Glutatión Peroxidasa/aislamiento & purificación , Sincrotrones