RESUMEN
BACKGROUND: HNF1A gene regulates liver-specific genes, and genes that have a role in glucose metabolism, transport, and secretion of insulin. HNF1A gene mutations are frequently associated with type 2 diabetes. HNF1A protein has three domains: the dimerization domain, the DNA-binding domain, and the trans-activation domain. Some mutations in the dimerization or DNA-binding domains have no influence on the normal allele, while others have dominant negative effects. The I27L, A98V, and S487N polymorphisms are common variants of the HNF1A gene; they have been found in T2D and non-diabetic subjects. METHODS AND RESULTS: We searched for mutations in the first three exons of the HNF1A gen in an Amerindian population of 71 diabetic patients. DNA sequencing revealed the previously reported I27L polymorphism (c.79A>C) in 53% of diabetic patients and in 67% of the control group. Thus, the I27L/L27L polymorphism might be a marker of Amerindians. In addition, we found the c.422_423InsT mutation in the HNF1A gene of one patient, which had not been previously reported. This mutation resulted in a frame shift of the open reading frame and a new translation stop in codon 187, leading to a truncated polypeptide of 186 amino acids (Q141Hfs*47). This novel mutation affects the DNA-binding capacity of the mutant HNF1A protein by EMSA; its intracellular localization by fluorescence and confocal microscopy, and a dominant-negative effect affecting the DNA-binding capacity of the normal HNF1A by EMSA. We also studied the homology modeling structure to understand the effect of this mutation on its DNA-binding capacity and its dominant negative effect. CONCLUSION: The HNF1A Q141Hfs*47 mutant polypeptide has no DNA-binding capacity and exerts a dominant negative effect on the HNF1A protein. Therefore, it might produce severe phenotypic effects on the expression levels of a set of ß-cell genes. Consequently, its screening should be included in the genetic analysis of diabetic patients after more functional studies are performed.
RESUMEN
We previously demonstrated that, in the early stages of tau processing in Alzheimer's disease, the N-terminal part of the molecule undergoes a characteristic cascade of phosphorylation and progressive misfolding of the proteins resulting in a structural conformation detected by Alz-50. In this immunohistochemical study of AD brain tissue, we have found that C-terminal truncation of tau at Asp-421 was an early event in tau aggregation and analyzed the relationship between phospho-dependent tau epitopes located at the C-terminus with truncation at Glu-391. The aim of this study was to determine whether C-terminal truncation may trigger events leading to the assembly of insoluble PHFs from soluble tau aggregates present in pre-tangle cells. Our findings suggest that there is a complex interaction between phosphorylated and truncated tau species. A model is presented here in which truncated tau protein represents an early neurotoxic species while phosphorylated tau species may provide a neuroprotective role in Alzheimer's disease.
RESUMEN
The cell surface of Candida albicans is enriched with highly glycosylated mannoproteins that are involved in the interaction with host tissues. N- and O-glycosylation are post-translational modifications that initiate in the endoplasmic reticulum, and finalize in the Golgi. The KRE2/MNT1 family encode a set of multifunctional mannosyltransferases that participate in O-, N- and phosphomannosylation. In order to gain insights into the substrate specificities of these enzymes, recombinant forms of Mnt1, Mnt2, and Mnt5 were expressed in Pichia pastoris and the enzyme activities characterized. Mnt1 and Mnt2 showed a high specificity for α-methylmannoside and α1,2-mannobiose as acceptor substrates. Notably, they also used Saccharomyces cerevisiaeO-mannans as acceptors and generated products with more than three mannose residues, suggesting than Mnt1 and Mnt2 could be the mannosyltransferases adding the fourth and fifth mannose residue to the O-mannans in C. albicans. Mnt5 only recognized α-methylmannoside as acceptor, suggesting that participates in the addition of the second mannose residues to the N-glycan outer chain.
Asunto(s)
Candida albicans/enzimología , Proteínas Fúngicas/química , Mananos/biosíntesis , Manosiltransferasas/química , Proteínas Fúngicas/genética , Manosiltransferasas/genética , Modelos Químicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
RNA helicases are widely conserved key enzymes that perform multiple functions in RNA metabolism. Here, we present the cloning, expression and functional characterization of the EhDEAD1 RNA helicase in the protozoan parasite Entamoeba histolytica. According to its primary structure, EhDEAD1 is evolutionary related to yeast DED1 and human DDX3X RNA helicases, both involved in translation and cell cycle regulation. The EhDEAD1 predicted amino acid sequence exhibits the nine conserved motifs described for the DEAD-box SFII superfamily members reported in other organisms and it is evolutionary close to protozoan homologues. Purified recombinant EhDEAD1 protein presented ATPase activity and it was able to bind and unwind RNA in an ATPase-dependent manner in vitro. RT-PCR assays showed that EhDead1 gene is overtranscribed in the cell cycle S phase. Moreover, inhibition of EhDead1 gene expression by antisense RNA seemed to facilitate transition from S to G2/M phase. Intriguingly, our results showed that EhDEAD1 was unable to rescue two yeast Ded1 RNA helicase mutants affected in translation, in spite of the high sequence homology with yeast DED1.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , ARN Helicasas DEAD-box/metabolismo , Entamoeba histolytica/enzimología , ARN Protozoario/genética , Secuencia de Aminoácidos , Animales , Secuencia Conservada , ARN Helicasas DEAD-box/genética , Ensayo de Cambio de Movilidad Electroforética , Entamoeba histolytica/genética , Entamoeba histolytica/crecimiento & desarrollo , Prueba de Complementación Genética , Rayos Láser , Datos de Secuencia Molecular , Filogenia , ARN Mensajero , ARN Protozoario/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase S/fisiología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de AminoácidoAsunto(s)
Antiprotozoarios/farmacología , Resistencia a Múltiples Medicamentos , Entamoeba histolytica/efectos de los fármacos , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Animales , Clonación Molecular , Emetina/farmacología , Entamoeba histolytica/genética , Amplificación de Genes , Genes MDR , Genes Protozoarios , Fenotipo , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Transcripción GenéticaRESUMEN
Ornithine decarboxylase, a rate-limiting enzyme in polyamine biosynthesis in eukaryotes, was stabilized and purified from trophozoites of the parasite protozoan E. histolytica. Analytical electrophoresis revealed the presence in the purified preparations of a major polypeptide of 45 kDa and barely detectable amounts of two other proteins of 70 and 120 kDa. Both the 45 and 70 kDa polypeptides were recognized by a mouse anti-ODC monoclonal antibody. The major polypeptide exhibited amino terminal sequence homology in the range of 40-73% with ODCs from other organisms. The immunoreactive polypeptide of 70 kDa was not identified. The molecular masses of 216 and 45 kDa determined for the native enzyme by gel filtration and for the major polypeptide by SDS-PAGE, respectively, suggest that the amoeba ODC is a homopentamer. Dialysis against hydroxylamine rendered the enzyme activity fully dependent on pyridoxal 5'-phosphate (PLP). As expected for an oligomeric enzyme, ODC activity exhibited sigmoidal kinetics when it was measured as a function of increasing concentrations of L-ornithine and PLP yielding S(0.5) values of 0.45 and 0.18 mM, respectively. Purified ODC was inhibited by 1,3-diaminopropane and 2,4-diamino-2-butanone but was largely insensitive to inhibition by alpha-difluoromethylornithine (DFMO), indicating that the enzyme may not be a suitable target for this anti-parasitic drug. Other features of the amoeba ODC were common with the enzyme from prokaryotes and eucaryotes.
Asunto(s)
Entamoeba histolytica/enzimología , Ornitina Descarboxilasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Cromatografía de Afinidad , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Immunoblotting , Cinética , Datos de Secuencia Molecular , Peso Molecular , Ornitina/farmacología , Ornitina Descarboxilasa/química , Ornitina Descarboxilasa/metabolismo , Inhibidores de la Ornitina Descarboxilasa , Fosfato de Piridoxal/farmacologíaRESUMEN
A 309 bp DNA fragment from Entamoeba histolytica was amplified by PCR using primers derived from the Acanthamoeba castellanii consensus TATA-box binding protein amino acid sequence. The amplified fragment was used to isolate cDNA and genomic DNA clones containing an ORF encoding the complete E. histolytica TATA-box binding protein (Ehtbp, 702 bp, 234 aa, molecular mass 26 kDa). The EhTBP functional domain showed 55% sequence identity to that of Homo sapiens, 54% to A. castellanii and 37% to Plasmodium falciparum TBPs. In Southern blot experiments we detected a single Ehtbp band, which was transcribed as a 1.3 kb mRNA containing a 420 nt 5' untranslated region. However, the probe hybridized with the 0.8 and 1.5 Mb chromosomes, suggesting that this sequence is diploid. In situ PCR assays showed two signals in 95% of trophozoites, one located in the nucleus and another in EhkO, the novel DNA-containing organelle recently reported. The recombinant E. histolytica TATA-box binding protein was expressed in Escherichia coli. Antibodies against it recognized two proteins of 26 and 29 kDa in E. histolytica nuclear extracts. Confocal microscopy immunofluorescence analysis located the protein in both the nucleus and EhkO.