RESUMEN
It is well known that the co-chaperone p23 regulates Hsp90 chaperone activity in protein folding. In Plasmodium falciparum, a putative p23 (Pfp23) has been identified through genome analysis, but its authenticity has remained unconfirmed since co-immunoprecipitation experiments failed to show its interaction with P. falciparum Hsp90 (PfHsp90). Thus, recombinant Pfp23 and PfHsp90 proteins purified from expressed clones were used in this study. It was clear that Pfp23 exhibited chaperone activity by virtue of its ability to suppress citrate synthase aggregation at 45 degrees C. Pfp23 was also shown to interact with PfHsp90 and to suppress its ATPase activity. Analyses of modeled Pfp23-PfHsp90 protein complex and site-directed mutagenesis further revealed strategically placed amino acid residues, K91, H93, W94 and K96, in Pfp23 to be crucial for binding PfHsp90. Collectively, this study has provided experimental evidence for the inherent chaperone function of Pfp23 and its interaction with PfHsp90, a sequel widely required for client protein activation.
Asunto(s)
Proteínas HSP90 de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Plasmodium falciparum/metabolismo , Proteínas Protozoarias/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Adenosina Trifosfato/farmacología , Secuencia de Aminoácidos , Aminoácidos , Animales , Clonación Molecular , Biología Computacional , Electroforesis en Gel de Poliacrilamida , Cloruro de Magnesio/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Mutantes/metabolismo , Plasmodium falciparum/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Proteínas Protozoarias/química , Proteínas Recombinantes/metabolismo , Secuencias Repetitivas de Aminoácido , Eliminación de Secuencia , Relación Estructura-ActividadRESUMEN
BACKGROUND AND AIMS: Mutations on the a-determinant of hepatitis B virus surface antigen (HBsAg), capable of escaping detection and vaccination, are identified in HBsAg-positive/anti-HBs-positive vaccinated infants. We studied the prevalence of these mutants in HBsAg-negative/anti-HBc-positive chronic HBV carriers and patients with hepatocellular carcinoma (HCC). METHODS: DNA sequence coding for the antigenic a-determinant of HBsAg was amplified from either HCC genomic DNA or serum samples of the selected patients and sequenced. The replicative mutant genomes were reconstituted in vitro and their reactivity to commercial kits measured. RESULTS: Mutations within and/or outside the a-determinant were identified in patients seronegative for HBsAg. They were then reconstituted in vitro and transiently transfected into HepG2 cells. Culture medium containing secreted HBV viral particles was collected and assayed for their binding to commercial kits. Drastic decrease of reactivity to these kits was seen with most of the identified mutations, including those located outside the a-determinant. CONCLUSION: The existence of a more complex antigenic structure of HBsAg is indicated by the decreased reactivity to detection of mutations, some of which are outside the a-determinant, escape vaccination and may persist in seronegative patients. The high proportion of HBsAg mutants that are integrated in HCC genomes suggests a role of these mutants in hepatocarcinogenesis, possibly leading to mutant HBV-related HCC.