RESUMEN

K(+) efflux through K(+) channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel's selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K(+) channel. Either Mg(2+) or Ca(2+) can reduce K(+) efflux through MthK channels. However, Ca(2+), but not Mg(2+), can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca(2+) ions can partially dehydrate, enabling selective accessibility of Ca(2+) to a site at the entry to the selectivity filter. Ca(2+) binding at the site interacts with K(+) ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.

Asunto(s)

Activación del Canal Iónico , Canales de Potasio/metabolismo , Sitios de Unión , Calcio/metabolismo , Cationes Bivalentes/farmacología , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Iones/metabolismo , Modelos Biológicos , Simulación de Dinámica Molecular , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Fuerza Protón-Motriz , Termodinámica

RESUMEN

Ligand binding sites within proteins can interact by allosteric mechanisms to modulate binding affinities and control protein function. Here we present crystal structures of the regulator of K+ conductance (RCK) domain from a K+ channel, MthK, which reveal the structural basis of allosteric coupling between two Ca2+ regulatory sites within the domain. Comparison of RCK domain crystal structures in a range of conformations and with different numbers of regulatory Ca2+ ions bound, combined with complementary electrophysiological analysis of channel gating, suggests chemical interactions that are important for modulation of ligand binding and subsequent channel opening.

Asunto(s)

Calcio/metabolismo , Canales de Potasio Calcio-Activados/química , Potasio/metabolismo , Regulación Alostérica , Sitios de Unión , Calcio/química , Cationes Bivalentes , Cationes Monovalentes , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Humanos , Activación del Canal Iónico , Transporte Iónico , Membrana Dobles de Lípidos/química , Potenciales de la Membrana , Modelos Moleculares , Mutación , Técnicas de Placa-Clamp , Potasio/química , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo

RESUMEN

Regulator of K(+) conductance (RCK) domains form a conserved class of ligand-binding domains that control the activity of a variety of prokaryotic and eukaryotic K(+) channels. Structural analysis of these domains by X-ray crystallography has provided insight toward mechanisms underlying ligand binding and channel gating, and thus the experimental strategies aimed at determining structures of liganded and unliganded forms of the domains may be useful in analysis of other ligand-binding domains. Here, we describe a basic strategy for crystallographic analysis of the RCK domain from the MthK channel, for determination of its Ca(2+)-bound structure.

Asunto(s)

Calcio/metabolismo , Canales de Potasio Calcio-Activados/química , Canales de Potasio Calcio-Activados/metabolismo , Sitios de Unión , Cromatografía de Afinidad , Cromatografía en Gel , Cristalografía por Rayos X , Polietilenglicoles/química , Canales de Potasio Calcio-Activados/aislamiento & purificación , Estructura Terciaria de Proteína , Sonicación

RESUMEN

RCK domains control activity of a variety of K(+) channels and transporters through binding of cytoplasmic ligands. To gain insight toward mechanisms of RCK domain activation, we solved the structure of the RCK domain from the Ca(2+)-gated K(+) channel, MthK, bound with Ba(2+), at 3.1 Å resolution. The Ba(2+)-bound RCK domain was assembled as an octameric gating ring, as observed in structures of the full-length MthK channel, and shows Ba(2+) bound at several positions. One of the Ba(2+) sites, termed C1, overlaps with a known Ca(2+)-activation site, determined by residues D184 and E210. Functionally, Ba(2+) can activate reconstituted MthK channels as observed in electrophysiological recordings, whereas Mg(2+) (up to 100 mM) was ineffective. Ba(2+) activation was abolished by the mutation D184N, suggesting that Ba(2+) activates primarily through the C1 site. Our results suggest a working hypothesis for a sequence of ligand-dependent conformational changes that may underlie RCK domain activation and channel gating.

Asunto(s)

Proteínas Arqueales/química , Bario/química , Methanobacteriaceae , Canales de Potasio Calcio-Activados/química , Secuencias de Aminoácidos , Sitios de Unión , Calcio/química , Complejos de Coordinación/química , Cristalografía por Rayos X , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Potenciales de la Membrana , Modelos Moleculares , Fosfatidiletanolaminas/química , Fosfatidilgliceroles/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína

RESUMEN

Regulator of K(+) conductance (RCK) domains control the activity of a variety of K(+) transporters and channels, including the human large conductance Ca(2+)-activated K(+) channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca(2+)-gated K(+) channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca(2+). Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca(2+)-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca(2+)-binding sites, resulting in a stoichiometry of 24 Ca(2+) ions per channel, is consistent with the steep relation between [Ca(2+)] and MthK channel activity. Comparison of Ca(2+)-bound and unliganded RCK domains suggests a physical mechanism for Ca(2+)-dependent conformational changes that underlie gating in this class of channels.

Asunto(s)

Sitios de Unión/genética , Calcio/metabolismo , Activación del Canal Iónico/genética , Modelos Moleculares , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Estructura Terciaria de Proteína , Cristalografía por Rayos X , Electrofisiología , Membrana Dobles de Lípidos/metabolismo

RESUMEN

Necroptosis represents a form of alternative programmed cell death that is dependent on the kinase RIP1. RIP1-dependent necroptotic death manifests as increased reactive oxygen species (ROS) production in mitochondria and is accompanied by loss of ATP biogenesis and eventual dissipation of mitochondrial membrane potential. Here, we show that tumor necrosis factor alpha (TNF-α)-induced necroptosis requires the adaptor proteins FADD and NEMO. FADD was found to mediate formation of the TNF-α-induced pronecrotic RIP1-RIP3 kinase complex, whereas the IκB Kinase (IKK) subunit NEMO appears to function downstream of RIP1-RIP3. Interestingly, loss of RelA potentiated TNF-α-dependent necroptosis, indicating that NEMO regulates necroptosis independently of NF-κB. Using both pharmacologic and genetic approaches, we demonstrate that the overexpression of antioxidants alleviates ROS elevation and necroptosis. Finally, elimination of BAX and BAK or overexpression of Bcl-x(L) protects cells from necroptosis at a later step. These findings provide evidence that mitochondria play an amplifying role in inflammation-induced necroptosis.

Asunto(s)

Apoptosis/fisiología , Proteína de Dominio de Muerte Asociada a Fas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Necrosis/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Animales , Western Blotting , Citometría de Flujo , Proteínas Activadoras de GTPasa/metabolismo , Técnicas de Inactivación de Genes , Inmunoprecipitación , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , FN-kappa B/metabolismo , Especies Reactivas de Oxígeno , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Factor de Transcripción ReIA/antagonistas & inhibidores , Factor de Transcripción ReIA/genética , Proteína Destructora del Antagonista Homólogo bcl-2/deficiencia , Proteína X Asociada a bcl-2/deficiencia

RESUMEN

The aim of this study was to develop a chiral gas chromatographic method for the separation of compounds likely to be found in the EMDE synthesis of methylamphetamine, a heavily abused stimulant drug. Here we describe the separation of the enantiomers of ephedrine, pseudoephedrine, chlorinated intermediates and methylamphetamine using fluorinated acid anhydrides as chemical derivatization reagents prior to gas chromatographic analysis on a 2,3-di-O-methyl-6-t-butyl silyl-ß-cyclodextrin stationary phase (CHIRALDEX™ B-DM). Separation of the enantiomers of pseudoephedrine, methylamphetamine and chloro-intermediates was achieved using PFPA derivatization, and enantiomers of ephedrine using TFAA derivatization, in run times of less than 40 minutes. The use of HFBA as a derivatization reagent for this set of analytes is also discussed.

Asunto(s)

Cromatografía de Gases/métodos , Drogas Ilícitas/síntesis química , Drogas Ilícitas/aislamiento & purificación , Metanfetamina/síntesis química , Metanfetamina/aislamiento & purificación , Cromatografía de Gases/normas , Fluorocarburos/química , Drogas Ilícitas/química , Metanfetamina/química , Estándares de Referencia , Estereoisomerismo

RESUMEN

The environmental niche of each fungus places distinct functional demands on the cell wall. Hence cell wall regulatory pathways may be highly divergent. We have pursued this hypothesis through analysis of Candida albicans transcription factor mutants that are hypersensitive to caspofungin, an inhibitor of beta-1,3-glucan synthase. We report here that mutations in SKO1 cause this phenotype. C. albicans Sko1 undergoes Hog1-dependent phosphorylation after osmotic stress, like its Saccharomyces cerevisiae orthologues, thus arguing that this Hog1-Sko1 relationship is conserved. However, Sko1 has a distinct role in the response to cell wall inhibition because 1) sko1 mutants are much more sensitive to caspofungin than hog1 mutants; 2) Sko1 does not undergo detectable phosphorylation in response to caspofungin; 3) SKO1 transcript levels are induced by caspofungin in both wild-type and hog1 mutant strains; and 4) sko1 mutants are defective in expression of caspofungin-inducible genes that are not induced by osmotic stress. Upstream Sko1 regulators were identified from a panel of caspofungin-hypersensitive protein kinase-defective mutants. Our results show that protein kinase Psk1 is required for expression of SKO1 and of Sko1-dependent genes in response to caspofungin. Thus Psk1 and Sko1 lie in a newly described signal transduction pathway.

Asunto(s)

Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/fisiología , Candida albicans/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Represoras/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Factores de Transcripción/fisiología , Antifúngicos/farmacología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Caspofungina , Pared Celular , Equinocandinas/farmacología , Lipopéptidos , Modelos Biológicos , Mutación , Ósmosis , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Represoras/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

The cell wall of Candida albicans lies at the crossroads of pathogenicity and therapeutics. It contributes to pathogenicity through adherence and invasion; it is the target of both chemical and immunological antifungal strategies. We have initiated a dissection of cell wall function through targeted insertional mutagenesis of cell wall-related genes. Among 25 such genes, we were unable to generate homozygous mutations in 4, and they may be essential for viability. We created homozygous mutations in the remaining 21 genes. Insertion mutations in SUN41, Orf19.5412, Orf19.1277, MSB2, Orf19.3869, and WSC1 caused hypersensitivity to the cell wall inhibitor caspofungin, while two different ecm33 insertions caused mild caspofungin resistance. Insertion mutations in SUN41 and Orf19.5412 caused biofilm defects. Through analysis of homozygous sun41Delta/sun41Delta deletion mutants and sun41Delta/sun41Delta+pSUN41-complemented strains, we verified that Sun41 is required for biofilm formation and normal caspofungin tolerance. The sun41Delta/sun41Delta mutant had altered expression of four cell wall damage response genes, thus suggesting that it suffers a cell wall structural defect. Sun41 is required for inducing disease, because the mutant was severely attenuated in mouse models of disseminated and oropharyngeal candidiasis. Although the mutant produced aberrant hyphae, it had no defect in damaging endothelial or epithelial cells, unlike many other hypha-defective mutants. We suggest that the sun41Delta/sun41Delta cell wall defect is the primary cause of its attenuated virulence. As a small fungal surface protein with predicted glucosidase activity, Sun41 represents a promising therapeutic target.

Asunto(s)

Biopelículas , Candida albicans/fisiología , Candida albicans/patogenicidad , Proteínas Fúngicas/metabolismo , Animales , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candidiasis/microbiología , Candidiasis/patología , Caspofungina , Equinocandinas/farmacología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Riñón/efectos de los fármacos , Riñón/microbiología , Riñón/patología , Lipopéptidos , Masculino , Ratones , Ratones Endogámicos BALB C , Pruebas de Sensibilidad Microbiana , Mutación/genética , Virulencia/efectos de los fármacos

RESUMEN

The fungal pathogen Candida albicans is frequently associated with catheter-based infections because of its ability to form resilient biofilms. Prior studies have shown that the transcription factor Bcr1 governs biofilm formation in an in vitro catheter model. However, the mechanistic role of the Bcr1 pathway and its relationship to biofilm formation in vivo are unknown. Our studies of biofilm formation in vitro indicate that the surface protein Als3, a known adhesin, is a key target under Bcr1 control. We show that an als3/als3 mutant is biofilm-defective in vitro, and that ALS3 overexpression rescues the biofilm defect of the bcr1/bcr1 mutant. We extend these findings with an in vivo venous catheter model. The bcr1/bcr1 mutant is unable to populate the catheter surface, though its virulence suggests that it has no growth defect in vivo. ALS3 overexpression rescues the bcr1/bcr1 biofilm defect in vivo, thus arguing that Als3 is a pivotal Bcr1 target in this setting. Surprisingly, the als3/als3 mutant forms a biofilm in vivo, and we suggest that additional Bcr1 targets compensate for the Als3 defect in vivo. Indeed, overexpression of Bcr1 targets ALS1, ECE1, and HWP1 partially restores biofilm formation in a bcr1/bcr1 mutant background in vitro, though these genes are not required for biofilm formation in vitro. Our findings demonstrate that the Bcr1 pathway functions in vivo to promote biofilm formation, and that Als3-mediated adherence is a fundamental property under Bcr1 control. Known adhesins Als1 and Hwp1 also contribute to biofilm formation, as does the novel protein Ece1.

Asunto(s)

Biopelículas/crecimiento & desarrollo , Candida albicans/fisiología , Candida albicans/patogenicidad , Moléculas de Adhesión Celular/fisiología , Proteínas Fúngicas/fisiología , Proteínas Proto-Oncogénicas c-bcr/fisiología , Animales , Candida albicans/genética , Candidiasis/etiología , Candidiasis/genética , Candidiasis/fisiopatología , Cateterismo/efectos adversos , Adhesión Celular/genética , Adhesión Celular/fisiología , Proteínas Fúngicas/genética , Regulación Fúngica de la Expresión Génica , Genes Fúngicos/fisiología , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Ratones , Ratones Endogámicos BALB C , Mutación , Proteínas Proto-Oncogénicas c-bcr/genética , Ratas , Ratas Sprague-Dawley

RESUMEN

The ESCRT-I, -II, and -III protein complexes function to create multivesicular bodies (MVBs) for sorting of proteins destined for the lysosome or vacuole. Prior studies with Saccharomyces cerevisiae have shown that the ESCRT-III protein Snf7p interacts with the MVB pathway protein Bro1p as well as its homolog Rim20p. Rim20p has no role in MVB formation, but functions in the Rim101p pH-response pathway; Rim20p interacts with transcription factor Rim101p and is required for the activation of Rim101p by C-terminal proteolytic cleavage. We report here that ESCRT-III proteins Snf7p and Vps20p as well as all ESCRT-I and -II proteins are required for Rim101p proteolytic activation in S. cerevisiae. Mutational analysis indicates that the Rim20p N-terminal region interacts with Snf7p, and an insertion in the Rim20p "Bro1 domain" abolishes this interaction, as determined with two-hybrid assays. Disruption of the MVB pathway through mutations affecting non-ESCRT proteins does not impair Rim101p processing. The relationship between the MVB pathway and Rim101p pathway is conserved in Candida albicans, because mutations in four ESCRT subunit genes abolish alkaline pH-induced filamentation, a phenotype previously seen for rim101 and rim20 mutants. The defect is suppressed by expression of C-terminally truncated Rim101-405p, as expected for mutations that block Rim101p proteolytic activation. These results indicate that the ESCRT complexes govern a specific signal transduction pathway and suggest that the MVB pathway may provide a signal that regulates pH-responsive transcription.

Asunto(s)

Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Complejos Multiproteicos/metabolismo , Saccharomyces cerevisiae/metabolismo , Candida albicans/genética , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Proteínas Fúngicas/genética , Concentración de Iones de Hidrógeno , Mutación/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Proteínas Represoras , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo

RESUMEN

Mineral and short-chain carboxylic acid vapours and NO(x) gases were reacted with cyclohexene oxide (1,2-epoxycyclohexane) to quantitatively produce specific, thermally stable cyclohexyl derivatives. Subsequent analysis of these derivatives by gas chromatography with mass spectroscopy and flame ionisation detection afforded a multi analyte method for the assay of these gaseous acidic atmospheric species. Derivatisation was found to be quantitative for the derivatives tested and the method highly sensitive (to 0.3 mg/m3 for a 30 l sample), accurate, precise and free from apparent interferences. The technique has been applied to "acid stack gases" and a number of other acid rich atmospheres and the results obtained show good agreement with the single analyte wet chemical determinations indicating that the approach has considerable potential as a routine analytical method for measuring such atmospheric pollutants. The high specificity of the reaction mechanism and its potential for the analysis of analyte mixtures is illustrated in the assay of nitric acid and its acid anhydride, dinitrogen pentoxide.

Asunto(s)

Ácidos/química , Contaminantes Atmosféricos/análisis , Monitoreo del Ambiente/métodos , Compuestos Epoxi/química , Gases/análisis , Cromatografía de Gases y Espectrometría de Masas/métodos

RESUMEN

Salud organizacional es un volumen que forma parte de la serie Teoría y Práctica Organizacional, en el cual proporciona las técnicas necesarias para recoger la opinión del personal o detectar alguna anomalía. A través de él se delínean los procedimientos para desarrollar, administrar e interpretar encuestas organizacionales que capacitarán al gerente de empresas para aplicar en forma apropiada la técnica de la encuestra y analizar sus resultados en forma efectiva

Asunto(s)

Diagnóstico , Salud , Organizaciones