Naturally-acquired humoral immune responses against the N- and C-termini of the Plasmodium vivax MSP1 protein in endemic regions of Brazil and Papua New Guinea using a multiplex assay.

BACKGROUND: Progress towards the development of a malaria vaccine against Plasmodium vivax, the most widely distributed human malaria parasite, will require a better understanding of the immune responses that confer clinical protection to patients in regions where malaria is endemic. METHODS: Glutathione S-transferase (GST) and GST-fusion proteins representing the N- terminus of the merozoite surface protein 1 of P. vivax, PvMSP1-N, and the C-terminus, PvMSP1-C, were covalently coupled to BioPlex carboxylated beads. Recombinant proteins and coupled beads were used, respectively, in ELISA and Bioplex assays using immune sera of P. vivax patients from Brazil and PNG to determine IgG and subclass responses. Concordances between the two methods in the seropositivity responses were evaluated using the Kappa statistic and the Spearman's rank correlation. RESULTS: The results using this methodology were compared with the classical microtitre enzyme-linked immnosorbent assay (ELISA), showing that the assay was sensitive, reproducible and had good concordance with ELISA; yet, further research into different statistical analyses seems desirable before claiming conclusive results exclusively based on multiplex assays. As expected, results demonstrated that PvMSP1 was immunogenic in natural infections of patients from different endemic regions of Brazil and Papua New Guinea (PNG), and that age correlated only with antibodies against the C-terminus part of the molecule. Furthermore, the IgG subclass profiles were different in these endemic regions having IgG3 predominantly recognizing PvMSP1 in Brazil and IgG1 predominantly recognizing PvMSP1 in PNG. CONCLUSIONS: This study validates the use of the multiplex assay to measure naturally-acquired IgG antibodies against the merozoite surface protein 1 of P. vivax.

Structural and biochemical studies of TREX1 inhibition by metals. Identification of a new active histidine conserved in DEDDh exonucleases.

TREX1 is the major exonuclease in mammalian cells, exhibiting the highest level of activity with a 3'-->5' activity. This exonuclease is responsible in humans for Aicardi-Goutières syndrome and for an autosomal dominant retinal vasculopathy with cerebral leukodystrophy. In addition, this enzyme is associated with systemic lupus erythematosus. TREX1 belongs to the exonuclease DEDDh family, whose members display low levels of sequence identity, while possessing a common fold and active site organization. For these exonucleases, a catalytic mechanism has been proposed that involves two divalent metal ions bound to the DEDD motif. Here we studied the interaction of TREX1 with the monovalent cations lithium and sodium. We demonstrate that these metals inhibit the exonucleolytic activity of TREX1, as measured by the classical gel method, as well as by a new technique developed for monitoring the real-time exonuclease reaction. The X-ray structures of the enzyme in complex with these two cations and with a nucleotide, a product of the exonuclease reaction, were determined at 2.1 A and 2.3 A, respectively. A comparison with the structures of the active complexes (in the presence of magnesium or manganese) explains that the inhibition mechanism is caused by the noncatalytic metals competing with distinct affinities for the two metal-binding sites and inducing subtle rearrangements in active centers. Our analysis also reveals that a histidine residue (His124), highly conserved in the DEDDh family, is involved in the activity of TREX1, as confirmed by mutational studies. Our results shed further light on the mechanism of activity of the DEDEh family of exonucleases.

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

STAT1 regulates lipopolysaccharide- and TNF-alpha-dependent expression of transporter associated with antigen processing 1 and low molecular mass polypeptide 2 genes in macrophages by distinct mechanisms.

Transporter associated with Ag processing 1 and low molecular mass polypeptide 2 (LMP2) are essential for class I MHC function and share a common bidirectional promoter. In murine bone marrow-derived macrophages, LPS and TNF-alpha induced Tap1 and up-regulated Lmp2, which is constitutively expressed at low levels. These two genes are induced by LPS and TNF-alpha with distinct kinetics, at 6 and 12-24 h, respectively. Using macrophages derived from the TNF-alpha receptors of knockout mice, we found that induction by LPS is not due to the autocrine production of TNF-alpha. In macrophages from STAT-1 knockout mice, neither LPS nor TNF-alpha induced the expression of Tap1 or Lmp2. The shared promoter contains several areas that can be controlled by STAT-1, such as the proximal and distal IFN-gamma activation site (GAS) boxes in the direction of the Tap1 gene. By making deletions of the promoter, we determined that only the proximal GAS box is required for LPS induction of Tap1 and Lmp2. In contrast, TNF-alpha induction of these two genes is dependent on the IFN regulatory factor-1 and NF-kappaB boxes, and not on the GAS box. Our experiments using gel shift analysis and Abs indicated that STAT1 binds to the GAS box in nuclear extracts from LPS-treated macrophages. The nuclear extracts obtained from macrophages treated with TNF-alpha bound to the IFN regulatory factor-1 and NF-kappaB boxes. These results show that LPS and TNF-alpha regulate the induction of Tap1 and Lmp2 through STAT1, but use distinct areas of the promoter.