Clinical Proteomics of the Neglected Human Malarial Parasite Plasmodium vivax

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

5‑Methylcytosine (5mC) and 5‑Hydroxymethylcytosine (5hmC) Enhance the DNA Binding of CREB1 to the C/EBP Half-Site Tetranucleotide GCAA

In human and mouse stem cells and brain, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) can occur outside of CG dinucleotides. Using protein binding microarrays (PBMs) containing 60-mer DNA probes, we evaluated the effect of 5mC and 5hmC on one DNA strand on the double-stranded DNA binding of the mouse B-ZIP transcription factors (TFs) CREB1, ATF1, and JUND. 5mC inhibited binding of CREB1 to the canonical CRE half-site |GTCA but enhanced binding to the C/EBP half-site |GCAA. 5hmC inhibited binding of CREB1 to all 8-mers except TGAT|GCAA, where binding is enhanced. We observed similar DNA binding patterns with ATF1, a closely related B-ZIP domain. In contrast, both 5mC and 5hmC inhibited binding of JUND. These results identify new DNA sequences that are well-bound by CREB1 and ATF1 only when they contain 5mC or 5hmC. Analysis of two X-ray structures examines the consequences of 5mC and 5hmC on DNA binding by CREB and FOS|JUN

The Epstein-Barr Virus B‑ZIP Protein Zta Recognizes Specific DNA Sequences Containing 5‑Methylcytosine and 5‑Hydroxymethylcytosine

The Epstein-Barr virus (EBV) B-ZIP transcription factor Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the sequence specific DNA binding of Zta to four kinds of double-stranded DNA (dsDNA): (1) DNA containing cytosine in both strands, (2) DNA with 5-methylcytosine (5mC) in one strand and cytosine in the second strand, (3) DNA with 5-hydroxymethylcytosine (5hmC) in one strand and cytosine in the second strand, and (4) DNA in which both cytosines in all CG dinucleotides contain 5mC. We compared these data to PBM data for three additional B-ZIP proteins (CREB1 and CEBPB homodimers and cJun|cFos heterodimers). With cytosine, Zta binds the TRE motif <b>TGA</b>^<b>C</b><b>/</b>_<b>G</b><b>TCA</b> as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as <b>MGAGTCA</b> and <b>TGAGMGA</b> (where <b>M</b> = 5mC), were preferentially bound. 5mC inhibits binding of Zta to both TRE motif half-sites <b>GTCA</b> and <b>CTCA</b>. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer more strongly bind the C/EBP half-site tetranucleotide <b>GCAA</b> when it contains 5mC. Zta also binds dsDNA sequences containing 5hmC in one strand, although the effect is less dramatic than that observed for 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, uncovering the potential for discovery of new viral and host regulatory programs controlled by EBV