RESUMEN
During mammalian development, some methylated cytosines (5mC) in CG dinucleotides are iteratively oxidized by TET dioxygenases to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). The effect of these cytosine oxidative products on the sequence-specific DNA binding of transcription factors is being actively investigated. Here, we used the electrophoretic mobility shift assay (EMSA) to examine C/EBPα and C/EBPß homodimers binding to all 25 chemical forms of a CG dinucleotide for two DNA sequences: the canonical C/EBP 8-mer TTGC|GCAA and the chimeric C/EBP|CRE 8-mer TTGC|GTCA. 5hmC in the CG dinucleotide in the C/EBP|CRE motif 8-mer TGAC|GCAA inhibits binding of C/EBPß but not C/EBPα. Binding was increased by 5mC, 5fC and 5caC. Circular dichroism monitored thermal denaturations for C/EBPß bound to the C/EBP|CRE motif confirmed the EMSA. The structural differences between C/EBPα and C/EBPß that may account for the difference in binding 5hmC in the 8-mer TGAC|GCAA are explored.
Asunto(s)
Proteína beta Potenciadora de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/genética , Metilación de ADN/genética , Factores de Transcripción/genética , 5-Metilcitosina/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/química , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/química , Cristalografía por Rayos X , Citosina/análogos & derivados , Citosina/metabolismo , Nucleótidos de Citosina/genética , ADN/química , ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Motivos de Nucleótidos/genética , Factores de Transcripción/metabolismoRESUMEN
Recent reports highlight the severity and the morbidity of disease caused by the long neglected malaria parasite Plasmodium vivax. Due to inherent difficulties in the laboratory-propagation of P. vivax, the biology of this parasite has not been adequately explored. While the proteome of P. falciparum, the causative agent of cerebral malaria, has been extensively explored from several sources, there is limited information on the proteome of P. vivax. We have, for the first time, examined the proteome of P. vivax isolated directly from patients without adaptation to laboratory conditions. We have identified 153 proteins from clinical P. vivax, majority of which do not show homology to any previously known gene products. We also report 29 new proteins that were found to be expressed in P. vivax for the first time. In addition, several proteins previously implicated as anti-malarial targets, were also found in our analysis. Most importantly, we found several unique proteins expressed by P. vivax.This study is an important step in providing insight into physiology of the parasite under clinical settings.