RESUMO
Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals.
Assuntos
Blastocladiella/fisiologia , Proteínas Fúngicas/genética , Guanilato Ciclase/genética , Luz , Rodopsina/genética , Transdução de Sinais , Sequência de Aminoácidos , Sequência de Bases , Blastocladiella/genética , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas Fúngicas/metabolismo , Fusão Gênica , Guanilato Ciclase/metabolismo , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Rodopsina/metabolismo , Alinhamento de Sequência , Percepção VisualRESUMO
BACKGROUND: The α-proteobacterium Caulobacter crescentus inhabits low-nutrient environments and can tolerate certain levels of heavy metals in these sites. It has been reported that C. crescentus responds to exposure to various heavy metals by altering the expression of a large number of genes. RESULTS: In this work, we show that the ECF sigma factor σF is one of the regulatory proteins involved in the control of the transcriptional response to chromium and cadmium. Microarray experiments indicate that σF controls eight genes during chromium stress, most of which were previously described as induced by heavy metals. Surprisingly, σF itself is not strongly auto-regulated under metal stress conditions. Interestingly, σF-dependent genes are not induced in the presence of agents that generate reactive oxygen species. Promoter analyses revealed that a conserved σF-dependent sequence is located upstream of all genes of the σF regulon. In addition, we show that the second gene in the sigF operon acts as a negative regulator of σF function, and the encoded protein has been named NrsF (Negative regulator of sigma F). Substitution of two conserved cysteine residues (C131 and C181) in NrsF affects its ability to maintain the expression of σF-dependent genes at basal levels. Furthermore, we show that σF is released into the cytoplasm during chromium stress and in cells carrying point mutations in both conserved cysteines of the protein NrsF. CONCLUSION: A possible mechanism for induction of the σF-dependent genes by chromium and cadmium is the inactivation of the putative anti-sigma factor NrsF, leading to the release of σF to bind RNA polymerase core and drive transcription of its regulon.
Assuntos
Caulobacter crescentus/efeitos dos fármacos , Caulobacter crescentus/fisiologia , Regulação Bacteriana da Expressão Gênica , Metais Pesados/toxicidade , Fator sigma/metabolismo , Estresse Fisiológico , Cádmio/toxicidade , Caulobacter crescentus/genética , Caulobacter crescentus/metabolismo , Cromo/toxicidade , Perfilação da Expressão Gênica , Análise em Microsséries , ÓperonRESUMO
BACKGROUND: Cyclodextrins are suitable drug delivery systems because of their ability to subtly modify the physical, chemical, and biological properties of guest molecules through labile interactions by formation of inclusion and/or association complexes. Plant cysteine proteinases from Caricaceae and Bromeliaceae are the subject of therapeutic interest, because of their anti-inflammatory, antitumoral, immunogenic, and wound-healing properties. METHODS: In this study, we analyzed the association between beta-cyclodextrin (betaCD) and fraction P1G10 containing the bioactive proteinases from Carica candamarcensis, and described the physicochemical nature of the solid-state self-assembled complexes by Fourier transform infrared (FTIR) spectroscopy, thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray powder diffraction (XRD), and nuclear magnetic resonance (NMR), as well as in solution by circular dichroism (CD), isothermal titration calorimetry (ITC), and amidase activity. RESULTS AND DISCUSSION: The physicochemical analyses suggest the formation of a complex between P1G10 and betaCD. Higher secondary interactions, namely hydrophobic interactions, hydrogen bonding and van der Waals forces were observed at higher P1G10 : betaCD mass ratios. These results provide evidence of the occurrence of strong solid-state supramolecular non-covalent interactions between P1G10 and betaCD. Microcalorimetric analysis demonstrates that complexation results in a favorable enthalpic contribution, as has already been described during formation of similar betaCD inclusion compounds. The amidase activity of the complex shows that the enzyme activity is not readily available at 24 hours after dissolution of the complex in aqueous buffer; the proteinase becomes biologically active by the second day and remains stable until day 16, when a gradual decrease occurs, with basal activity attained by day 29. CONCLUSION: The reported results underscore the potential for betaCDs as candidates for complexing cysteine proteinases, resulting in supramolecular arrays with sustained proteolytic activity.