RESUMEN

The CED4/Apaf-1 family of proteins functions as critical regulators of apoptosis and NF-kappaB signaling pathways. A novel human member of this family, called CARD12, was identified that induces apoptosis when expressed in cells. CARD12 is most similar in structure to the CED4/Apaf-1 family member CARD4, and is comprised of an N-terminal caspase recruitment domain (CARD), a central nucleotide-binding site (NBS), and a C-terminal domain of leucine-rich repeats (LRR). The CARD domain of CARD12 interacts selectively with the CARD domain of ASC, a recently identified proapoptotic protein. In addition, CARD12 coprecipitates caspase-1, a caspase that participates in both apoptotic signaling and cytokine processing. CARD12 may assemble with proapoptotic CARD proteins to coordinate the activation of downstream apoptotic and inflammatory signaling pathways.

Asunto(s)

Apoptosis , Proteínas de Caenorhabditis elegans , Proteínas de Unión al Calcio/genética , Proteínas del Helminto/genética , Proteínas/genética , Animales , Especificidad de Anticuerpos , Factor Apoptótico 1 Activador de Proteasas , Caspasa 1/metabolismo , Línea Celular , Chlorocebus aethiops , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Bases de Datos Factuales , Expresión Génica , Genes Reporteros , Humanos , Immunoblotting , Riñón/citología , Riñón/metabolismo , Datos de Secuencia Molecular , Familia de Multigenes , Especificidad de Órganos , Estructura Terciaria de Proteína/fisiología , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transducción de Señal/fisiología , Transfección , Técnicas del Sistema de Dos Híbridos , Células Vero

RESUMEN

BCL10 belongs to the caspase recruitment domain (CARD) family of proteins that regulate apoptosis and NF-kappaB signaling pathways. Analysis of BCL10-deficient mice has revealed that BCL10 mediates NF-kappaB activation by antigen receptors in B and T cells. We recently identified a subclass of CARD proteins (CARD9, CARD11, and CARD14) that may function to connect BCL10 to multiple upstream signaling pathways. We report here that CARD10 is a novel BCL10 interactor that belongs to the membrane-associated guanylate kinase family, a class of proteins that function to organize signaling complexes at plasma membranes. When expressed in cells, CARD10 binds to BCL10 and signals the activation of NF-kappaB through its N-terminal effector CARD domain. We propose that CARD10 functions as a molecular scaffold for the assembly of a BCL10 signaling complex that activates NF-kappaB.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , FN-kappa B/metabolismo , Proteínas de Neoplasias/metabolismo , Nucleósido-Fosfato Quinasa/genética , Nucleósido-Fosfato Quinasa/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis , Proteína 10 de la LLC-Linfoma de Células B , Sitios de Unión , Proteínas Adaptadoras de Señalización CARD , Línea Celular , Membrana Celular/metabolismo , Genes Reporteros , Guanilato-Quinasas , Humanos , Mamíferos , Datos de Secuencia Molecular , Nucleósido-Fosfato Quinasa/química , Especificidad de Órganos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción Genética , Transfección , Dominios Homologos src

RESUMEN

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

Asunto(s)

Regulación hacia Abajo/genética , Neuronas/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/genética , Canales de Potasio/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Caribdotoxina/farmacología , Clonación Molecular , Electrofisiología , Epítopos/genética , Expresión Génica/fisiología , Haplorrinos , Humanos , Hibridación in Situ , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Riñón/citología , Cinética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades beta de los Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio de Gran Conductancia Activados por el Calcio , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , Neuronas/química , Péptidos/farmacología , Canales de Potasio/química , Estructura Cuaternaria de Proteína , ARN Mensajero/análisis , Análisis de Secuencia de ADN

RESUMEN

The nematode CED-4 protein and its human homolog Apaf-1 play a central role in apoptosis by functioning as direct activators of death-inducing caspases. A novel human CED-4/Apaf-1 family member called CARD4 was identified that has a domain structure strikingly similar to the cytoplasmic, receptor-like proteins that mediate disease resistance in plants. CARD4 interacted with the serine-threonine kinase RICK and potently induced NF-kappaB activity through TRAF-6 and NIK signaling molecules. In addition, coexpression of CARD4 augmented caspase-9-induced apoptosis. Thus, CARD4 coordinates downstream NF-kappaB and apoptotic signaling pathways and may be a component of the host innate immune response.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Apoptosis , Proteínas de Caenorhabditis elegans , Proteínas de Unión al Calcio/metabolismo , Proteínas Portadoras/metabolismo , Proteínas del Helminto/metabolismo , FN-kappa B/metabolismo , Proteínas/metabolismo , Secuencia de Aminoácidos , Factor Apoptótico 1 Activador de Proteasas , Secuencia de Bases , Proteínas Portadoras/genética , ADN Complementario , Humanos , Datos de Secuencia Molecular , Proteína Adaptadora de Señalización NOD1 , Homología de Secuencia de Aminoácido , Transducción de Señal

RESUMEN

Maturity-onset diabetes of the young 3 (MODY3) is a type of NIDDM caused by mutations in the transcription factor hepatocyte nuclear factor-1alpha (HNF-1alpha) located on chromosome 12q. We have identified four novel HNF-1alpha missense mutations in MODY3 families. In four additional and unrelated families, we observed an identical insertion mutation that had occurred in a polycytidine tract in exon 4. Among those families, one exhibited a de novo mutation at this location. We propose that instability of this sequence represents a general mutational mechanism in MODY3. We observed no HNF-1alpha mutations among 86 unrelated late-onset diabetic patients with relative insulin deficiency. Hence mutations in this gene appear to be most strongly associated with early-onset diabetes.

Asunto(s)

Cromosomas Humanos Par 12/genética , Proteínas de Unión al ADN , Diabetes Mellitus Tipo 2/genética , Mutación/genética , Proteínas Nucleares/genética , Factores de Transcripción/genética , Análisis Mutacional de ADN , Cartilla de ADN/química , Familia , Ligamiento Genético , Haplotipos , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Humanos , Linaje , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple

RESUMEN

Rhizobium meliloti produces an acidic exopolysaccharide, termed succinoglycan or EPS I, that is important for invasion of the nodules that it elicits on its host, Medicago sativa. Succinoglycan is a high-molecular-weight polymer composed of repeating octasaccharide subunits. These subunits are synthesized on membrane-bound isoprenoid lipid carriers, beginning with a galactose residue followed by seven glucose residues, and modified by the addition of acetate, succinate, and pyruvate. Biochemical characterizations of lipid-linked succinoglycan biosynthetic intermediates from previously identified exo mutant strains have been carried out in our laboratory (T. L. Reuber and G. C. Walker, Cell 74:269-280, 1993) to determine where each mutation blocks the biosynthetic pathway. We have carried out a fine structure genetic analysis of a portion of the cluster of exo genes present on the second symbiotic megaplasmid of R. meliloti and have identified several new genes. In addition, the DNA sequence of 16 kb of the exo cluster was determined and the genetic map was correlated with the DNA sequence. In this paper we present the sequence of a family of glycosyl transferases required for the synthesis of succinoglycan and discuss their functions.

Asunto(s)

Secuencia Conservada , Hexosiltransferasas/metabolismo , Polisacáridos Bacterianos/biosíntesis , Sinorhizobium meliloti/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Secuencia de Carbohidratos , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Genes Bacterianos , Genotipo , Hexosiltransferasas/biosíntesis , Hexosiltransferasas/genética , Datos de Secuencia Molecular , Familia de Multigenes , Sistemas de Lectura Abierta , Plásmidos , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/genética

RESUMEN

The major acidic exopolysaccharide of Rhizobium meliloti, termed succinoglycan, is required for nodule invasion and possibly nodule development. Succinoglycan is a polymer of octasaccharide subunits composed of one galactose residue, seven glucose residues, and acetyl, succinyl, and pyruvyl modifications, which is synthesized on an isoprenoid lipid carrier. A cluster of exo genes in R. meliloti are required for succinoglycan production, and the biosynthetic roles of their gene products have recently been determined (T.L. Reuber and G. C. Walker, Cell 74:269-280, 1993). Our sequencing of 16 kb of this cluster of exo genes and further genetic analysis of this region resulted in the discovery of several new exo genes and has allowed a correlation of the genetic map with the DNA sequence. In this paper we present the sequences of genes that are required for the addition of the succinyl and pyruvyl modifications to the lipid-linked intermediate and genes required for the polymerization of the octasaccharide subunits or the export of succinoglycan. In addition, on the basis of homologies to known proteins, we suggest that ExoN is a uridine diphosphoglucose pyrophosphorylase and that ExoK is a beta(1,3)-beta (1,4)-glucanase. We propose a model for succinoglycan biosynthesis and processing which assigns roles to the products of nineteen exo genes.

Asunto(s)

Genes Bacterianos , Familia de Multigenes , Polisacáridos Bacterianos/biosíntesis , Sinorhizobium meliloti/genética , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Carbohidratos , Mapeo Cromosómico , Secuencia Conservada , Glicósido Hidrolasas/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Sistemas de Lectura Abierta , Plásmidos , Homología de Secuencia de Aminoácido , Sinorhizobium meliloti/enzimología , Sinorhizobium meliloti/metabolismo , UTP-Glucosa-1-Fosfato Uridililtransferasa/genética

RESUMEN

Coliphage N4 virion-encapsidated, DNA-dependent RNA polymerase (vRNAP) is inactive on double-stranded N4 DNA; however, denatured promoter-containing templates are accurately transcribed. We report that all determinants of vRNAP promoter recognition exist in the template strand, indicating that this enzyme is a site-specific, single-stranded DNA-binding protein. We show that conserved sequences and the integrity of inverted repeats present at the promoters are essential for activity, suggesting the necessity for specific secondary structure. Evidence for such a structure is presented. We propose a model for in vivo utilization of vRNAP promoters in which template negative supercoiling yields single-strandedness at the promoter to reveal the determinants of vRNAP binding. This structure is stabilized by the binding of E. coli single-stranded DNA-binding protein to yield an "activated promoter."

Asunto(s)

ADN Viral/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Regiones Promotoras Genéticas , Virión/enzimología , Marcadores de Afinidad , Secuencia de Bases , ADN Viral/metabolismo , ARN Polimerasas Dirigidas por ADN/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación de Ácido Nucleico , Secuencias Repetitivas de Ácidos Nucleicos , Moldes Genéticos , Transcripción Genética

RESUMEN

Genetic experiments have indicated that succinoglycan (EPS I), the acidic Calcofluor-binding exopolysaccharide, of the nitrogen-fixing bacterium Rhizobium meliloti strain Rm1021 is required for nodule invasion and possibly for later events in nodule development on alfalfa and other hosts. Fourteen exo loci on the second megaplasmid have been identified that are required for, or affect, the synthesis of EPS I. Mutations in certain of these loci completely abolish the production of EPS I and result in mutants that form empty Fix- nodules. We have identified two loci, exoR and exoS, that are involved in the regulation of EPS I synthesis in the free-living state. Certain exo mutations which completely abolish EPS I production are lethal in an exoR95 or exoS96 background. Histochemical analyses of the expression of exo genes during nodulation using exo::TnphoA fusions have indicated that the exo genes are expressed most strongly in the invasion zone. In addition, we have discovered that R. meliloti has a latent capacity to synthesize a second exopolysaccharide (EPS II) that can substitute for the role(s) of EPS I in nodulation of alfalfa but not of other hosts. Possible roles for exopolysaccharides in symbiosis are discussed.

Asunto(s)

Polisacáridos Bacterianos/genética , Sinorhizobium meliloti/genética , Genes Bacterianos , Mutagénesis , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/fisiología , Sinorhizobium meliloti/fisiología , Simbiosis