RESUMEN

In fungi, heterotrimeric guanine-nucleotide binding proteins (G-proteins) are key elements of signal transduction pathways, which control growth, asexual and sexual development, as well as virulence. In this study, we have identified two genes encoding heterotrimeric G protein alpha subunits, named Gvm2 and Gvm3, from Valsa mali, the causal agent of apple Valsa canker. Characterization of Gvm2 and Gvm3 mutants indicates that Gvm3 may be a crucial regulator of vegetative growth. Deletion of the corresponding gene results in a 20% reduction in growth rate. Besides, Gvm2 and Gvm3 seem to be involved in asexual reproduction, and mutants are hypersensitive to oxidative and cell membrane stresses. Interestingly, both G protein alpha subunits were most probably involved in V. mali virulence. In infection assays using Malus domestica cv. 'Fuji' leaves and twigs, the size of lesions caused by deletion mutants △Gvm2, or △Gvm3 are significantly reduced. Furthermore, many genes encoding hydrolytic enzymes-important virulence factors in V. mali-are expressed at a lower level in these deletion mutants. Our results suggest that Gvm2 and Gvm3 play an important role in virulence probably by regulation of expression of cell wall degrading enzymes. △Gvm2, and △Gvm3 mutants were further analyzed with respect to their impact on the transcript levels of genes in the cAMP/PKA pathway. The expression of the genes encoding adenylate cyclase VmAC, protein kinase A (PKA) regulatory subunit VmPKR, and PKA catalytic subunit VmPKA1 are down-regulated in both mutants. Further analyses indicated that intracellular cAMP level and PKA activity are down-regulated in the △Gvm3 mutant, but are basically unchanged in the △Gvm2 mutant. Overall, our findings indicate that both Gvm2 and Gvm3 play diverse roles in the modulation of vegetative growth, asexual development, and virulence in V. mali.

Asunto(s)

Ascomicetos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Malus/microbiología , Malus/fisiología , Enfermedades de las Plantas/microbiología , Ascomicetos/genética , Ascomicetos/patogenicidad , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Subunidades alfa de la Proteína de Unión al GTP/genética , Eliminación de Gen , Prueba de Complementación Genética , Mutación , Fenotipo , Filogenia , Reproducción Asexuada/genética , Estrés Fisiológico , Virulencia/genética

RESUMEN

Heterotrimeric G-proteins form a major protein family, which participates in signal transduction. They are composed of three subunits, Gα, Gß and Gγ. The Gα subunit is further divided in four distinct families Gs, Gi/o, Gq/11 and G12/13. The goal of this work was to detect and classify members of the four distinct families, plus the Gß and the Gγ subunits of G-proteins from sequence alone. To achieve this purpose, six specific profile Hidden Markov Models (pHMMs) were built and checked for their credibility. These models were then applied to ten (10) proteomes and were able to identify all known G-protein and classify them into the distinct families. In a separate case study, the models were applied to twenty seven (27) arthropod proteomes and were able to give more credible classification in proteins with uncertain annotation and in some cases to detect novel proteins. An online tool, GprotPRED, was developed that uses these six pHMMs. The sensitivity and specificity for all pHMMs were equal to 100% with the exception of the Gß case, where sensitivity equals to 100%, while specificity is 99.993%. In contrast to Pfam's pHMM which detects Gα subunits in general, our method not only detects Gα subunits but also classifies them into the appropriate Gα-protein family and thus could become a useful tool for the annotation of G-proteins in newly discovered proteomes. GprotPRED online tool is publicly available for non-commercial use at http://bioinformatics.biol.uoa.gr/GprotPRED and, also, a standalone version of the tool at https://github.com/vkostiou/GprotPRED.

Asunto(s)

Secuencia de Aminoácidos/genética , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/genética , Anotación de Secuencia Molecular , Animales , Biología Computacional , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Proteínas de Unión al GTP Heterotriméricas , Mamíferos , Cadenas de Markov , Multimerización de Proteína/genética , Proteoma/genética , Transducción de Señal/genética , Programas Informáticos

RESUMEN

A comprehensive reevaluation of the G protein alpha subunit genes specifically expressed in taste buds in the tongue epithelium of rodents revealed that Gq and G14 of the Gq class and Gi2 and Ggust (Gt3, also known as gustducin) of the Gi class are expressed in mammalian taste buds. Meanwhile, a database search of fish genomes revealed the absence of a gene encoding an ortholog of the mammalian Ggust gene, which mediates sweet, umami, and bitter taste signals in mammalian taste receptor cells (TRCs). Histochemical screening identified two G protein alpha subunit genes, zfGia and zfG14, expressed in subsets of TRCs in zebrafish. The expression patterns of zfGia and zfG14 in taste buds were mutually exclusive, and the expression of known T1R and T2R genes in zebrafish was restricted to a subset of zfGia-expressing TRCs. These findings highlight the existence of a novel subset of TRCs in zebrafish that is absent in mammals and suggest that unidentified G protein-coupled receptors are expressed in zfG14-expressing TRCs and in zfGia-expressing TRCs where known T1R and T2R genes were not expressed in zebrafish. The existence of not only generalized but also specialized subsets of TRCs may imply a strong connection between the evolution of the peripheral gustatory system and the evolution of particular species.

Asunto(s)

Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Receptoras Sensoriales/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/anatomía & histología , Pez Cebra/metabolismo , Animales , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Subunidades alfa de la Proteína de Unión al GTP/genética , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Filogenia , Receptores Acoplados a Proteínas G/genética , Células Receptoras Sensoriales/citología , Transducción de Señal/fisiología , Proteínas de Pez Cebra/genética

RESUMEN

In many species, olfactory transduction is triggered by odorant molecules that interact with olfactory receptors coupled to heterotrimeric G-proteins. The role of G-protein-linked transduction in the olfaction of Drosophila is currently under study. Here, we supply a thorough description of the expression in the olfactory receptor organs (antennae and maxillary palps) of all known Drosophila melanogaster genes that encode for G-proteins. Using RT-polymerase chain reaction, we analyzed 6 Galpha (G(s), G(i), G(q), G(o), G(f), and concertina), 3 Gbeta (G(beta5), G(beta13F), and G(beta76C)), and 2 Ggamma genes (G(gamma1) and G(gamma30A)). We found that all Galpha protein-encoding genes showed expression in both olfactory organs, but G(f) mRNA was not detected in palps. Moreover, all the Gbeta and Ggamma genes are expressed in antennae and palps, except for G(beta76C). To gain insight into the hypothesis of different G-protein subunits mediating differential signaling in olfactory receptor neurons (ORNs), we performed immunohistochemical studies to observe the expression of several Galpha and Gbeta proteins. We found that Gs, Gi, Gq, and G(beta13F) subunits displayed generalized expression in the antennal tissue, including ORNs support cells and glial cells. Finally, complete coexpression was found between Gi and Gq, which are mediators of the cyclic adenosine monophosphate and IP3 transduction cascades, respectively.

Asunto(s)

Drosophila melanogaster/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Animales , Drosophila melanogaster/genética , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/clasificación , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/clasificación , Subunidades gamma de la Proteína de Unión al GTP/genética , Expresión Génica , Inmunohistoquímica , Neuronas Receptoras Olfatorias/metabolismo

RESUMEN

The opioid receptor-like 1 (NOP or ORL1) receptor is a G-protein-coupled receptor the endogenous ligand of which is the heptadecapeptide, nociceptin (Noc). NOP receptors are known to modulate pain processing at spinal, supraspinal, and peripheral levels. Previous work has demonstrated that NOP receptors inhibit N-type Ca2+ channel currents in rat sympathetic stellate ganglion (SG) neurons via pertussis toxin (PTX)-sensitive Galphai/o subunits. However, the identification of the specific Galpha subunit that mediates the Ca2+ current modulation is unknown. The purpose of the present study was to examine coupling specificity of Noc-activated NOP receptors to N-type Ca2+ channels in SG neurons. Small interference RNA (siRNA) transfection was employed to block the expression of PTX-sensitive Galpha subunits. RT-PCR results showed that siRNA specifically decreased the expression of the intended Galpha subunit. Evaluation of cell surface protein expression and Ca2+ channel modulation were assessed by immunofluorescence staining and electrophysiological recordings, respectively. Furthermore, the presence of mRNA of the intended siRNA target Galpha protein was examined by RT-PCR experiments. Fluorescence imaging showed that Galphai1, Galphai3, and Galphao were expressed in SG neurons. The transfection of Galphai1-specific siRNA resulted in a significant decrease in Noc-mediated Ca2+ current inhibition, while silencing of either Galphai3 or Galphao was without effect. Taken together, these results suggest that in SG neurons Galphai1 subunits selectively couple NOP receptors to N-type Ca2+ channels.

Asunto(s)

Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores Opioides/metabolismo , Ganglio Estrellado/citología , Animales , Anticuerpos/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Subunidades alfa de la Proteína de Unión al GTP/genética , Subunidades alfa de la Proteína de Unión al GTP/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Norepinefrina/farmacología , Péptidos Opioides/farmacología , Técnicas de Placa-Clamp , Toxina del Pertussis/farmacología , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Ratas , Transfección/métodos , omega-Conotoxina GVIA/farmacología , Receptor de Nociceptina , Nociceptina

RESUMEN

In insect olfactory receptor neurons, rapid and transient increases in inositol triphosphate (IP3) and Ca2+ are detected upon stimulation with pheromone or nonpheromonal odorants. This suggests that heterotrimeric guanine nucleotide binding proteins (G proteins) may transduce some odorant responses in insects. We obtained cDNA clones encoding three classes of G protein alpha subunits, Bm Go, Bm Gq, and Bm Gs, from the antennae of the adult male silkmoth (Bombyx mori). RT-PCR experiments showed that the mRNA of these G protein alpha subunits was also present in the various tissues of adult and larval insects. We used immunocytochemistry to localize these G protein alpha subunits in adult male and female antennae. In the adult male antennae, anti-Go antiserum stained the nerve bundles. In contrast, anti-Gq and anti-Gs antisera stained the inner and outer dendritic segments of the putative olfactory receptor neuron. The localizations of Bm Go, Bm Gq, and Bm Gs in the female antennae were the same as in the male antennae. The localizations of Bm Gq and Bm Gs suggest that each subunit mediates a subset of the odorant response.

Asunto(s)

Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Insectos/metabolismo , Neuronas Receptoras Olfatorias/metabolismo , Órganos de los Sentidos/citología , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting/métodos , Bombyx , Mapeo Encefálico , Clonación Molecular/métodos , Femenino , Subunidades alfa de la Proteína de Unión al GTP/clasificación , Subunidades alfa de la Proteína de Unión al GTP/genética , Expresión Génica/fisiología , Genes de Insecto , Inmunohistoquímica/métodos , Proteínas de Insectos/clasificación , Proteínas de Insectos/genética , Masculino , Ratones , Datos de Secuencia Molecular , Neuronas Receptoras Olfatorias/crecimiento & desarrollo , ARN Mensajero/biosíntesis , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Órganos de los Sentidos/crecimiento & desarrollo , Órganos de los Sentidos/metabolismo , Alineación de Secuencia/métodos , Factores Sexuales