RESUMEN
The orphan nuclear receptor HNF4alpha and the LIM homeodomain factor Isl1 are co-expressed in pancreatic beta-cells and are required for the differentiation and function of these endocrine cells. HNF4alpha activates numerous genes and mutations in its gene are associated with maturity onset diabetes of the young. Cofactors and transcription factors that interact with HNF4alpha are crucial to modulate its transcriptional activity, since the latter is not regulated by conventional ligands. These transcriptional partners interact mainly through the HNF4alpha AF-1 module and the ligand binding domain, which contains the AF-2 module. Here, we showed that Isl1 could enhance the HNF4alpha-mediated activation of transcription of the HNF1alpha, PPARalpha and insulin I promoters. Isl1 interacted with the HNF4alpha AF-2 but also required the HNF4alpha carboxy-terminal F domain for optimal interaction and transcriptional synergy. More specifically, we found that naturally occurring HNF4alpha isoforms, differing only in their F domain, exhibited different abilities to interact and synergize with Isl1, extending the crucial transcriptional modulatory role of the HNF4alpha F domain. HNF4alpha interacted with both the homeodomain and the first LIM domain of Isl1. We found that the transcriptional synergy between HNF4alpha and Isl1 involved an increase in HNF4alpha loading on promoter. The effect was more pronounced on the rat insulin I promoter containing binding sites for both HNF4alpha and Isl1 than on the human HNF1alpha promoter lacking an Isl1 binding site. Moreover, Isl1 could mediate the recruitment of the cofactor CLIM2 resulting in a further transcriptional enhancement of the HNF1alpha promoter activity.
Asunto(s)
Factor Nuclear 4 del Hepatocito/fisiología , Proteínas de Homeodominio/fisiología , Proteínas del Tejido Nervioso/fisiología , Factores de Transcripción/fisiología , Transcripción Genética , Animales , Proteínas de Unión al ADN/metabolismo , Factor Nuclear 1 del Hepatocito/genética , Humanos , Insulina/genética , Proteínas con Dominio LIM , Proteínas con Homeodominio LIM , Ligandos , PPAR alfa/genética , Regiones Promotoras Genéticas , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Activación TranscripcionalRESUMEN
Mutations in the hepatocyte nuclear factor 4alpha (HNF-4alpha) gene are associated with one form of maturity-onset diabetes of the young (MODY1). The R154X mutation generates a protein lacking the E-domain which is required for normal HNF-4alpha functions. Since pancreatic beta-cell dysfunction is a feature of MODY1 patients, we compared the functional properties of the R154X mutant in insulin-secreting pancreatic beta-cells and non-beta-cells. The R154X mutation did not affect nuclear localisation in beta-cells and non-beta-cells. However, it did lead to a greater impairment of HNF-4a function in beta-cells compared to non-beta-cells, including a complete loss of transactivation activity and a dominant-negative behaviour. .
Asunto(s)
Proteínas de Unión al ADN , Diabetes Mellitus Tipo 1/genética , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Mutación Puntual , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Células COS , Línea Celular , Núcleo Celular/metabolismo , Cartilla de ADN/genética , Diabetes Mellitus Tipo 1/metabolismo , Factor Nuclear 4 del Hepatocito , Humanos , Islotes Pancreáticos/metabolismo , Datos de Secuencia Molecular , Fosfoproteínas/química , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/química , Activación TranscripcionalRESUMEN
A genomic region of 12 kb encompassing the gene encoding the superoxide dismutase (SOD) of Toxoplasma gondii has been cloned. The gene contains four exons of 121, 42, 381 and 59 bp which are separated by three introns of 321, 202, and 577 bp, respectively. The open reading frame can be translated into a protein of 201 amino acids with a molecular mass of 22.6 kDa. Alignment indicated that it is a FeSOD, a type only found in bacteria, protozoa and chloroplast of higher plants. Recombinant SOD was expressed in a Escherichia coli double mutant lacking both MnFeSOD and FeSODs. The presence of iron as metal cofactor was confirmed by measurements of iron by absorption mass spectrometry and electron paramagnetic resonance studies. Semi-quantitative reverse transcribed polymerase chain reaction experiments showed a similar amount of SOD transcripts in two developmental stages of T. gondii. Antibodies raised against the purified recombinant protein detected SOD protein in both bradyzoite and tachyzoite forms suggesting this SOD might be essential for the intracellular growth of both developmental stages. Southern blot analysis indicated that SOD occured as a single copy gene in T. gondii genome.
Asunto(s)
Proteínas Protozoarias/genética , Superóxido Dismutasa/genética , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Western Blotting , Clonación Molecular , ADN Complementario/análisis , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli/enzimología , Genes Protozoarios , Hierro/análisis , Ratones , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Alineación de Secuencia , Espectrofotometría Atómica , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Toxoplasma/enzimología , Toxoplasma/metabolismoRESUMEN
The cDNA encoding the Toxoplasma gondii microneme protein MIC1 and the corresponding gene have been cloned and sequenced. The MIC1 gene contains three introns. The cDNA encodes a 456 amino acid (aa) sequence, with a typical signal sequence and no other trans-membrane domain. The protein contains a tandemly duplicated domain with conservation of cysteines and presents distant homology with the Plasmodium sp. microneme protein TRAP-SSP2. The MIC1 protein from tachyzoite lysates and a PMAL recombinant expressing the N-terminal duplicated domain of the protein bound to the surface of putative host cells, suggesting a possible involvement of MIC1 in host cell binding/recognition.
Asunto(s)
Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas Protozoarias , Toxoplasma/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular , Moléculas de Adhesión Celular/química , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , Genes Protozoarios/genética , Intrones/genética , Datos de Secuencia Molecular , Peso Molecular , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Toxoplasma/metabolismo , Células VeroRESUMEN
In non fatal and synchronous P. chabaudi rodent malaria, we observed at the stage of parasitaemia peak, an alteration (50 % decrease) in LCAT activity. This decrease could be related partly to hepatic dysfunction, and mainly to circulating inhibitors released into blood from parasitized red blood cells at each end of a schizogonic cycle. This decrease in LCAT activity, at this step of the infection, accounts for part of the dyslipoproteinemia previously observed (i.e., increase in cholesterol and phospholipids into VLDL-LDL and decrease in the EC series and delayed conversion of Tg-rich lipoproteins into LDL-HDL. At a prepatent step of infection and after the parasitaemia peak, the alterations observed in LCAT activity, (respectively, increase and then decrease), would be related to similar changes in levels of cholesterol of HDL associated to complex changes in triacylglyceride transport and metabolism.
