Gene Models, Expression Repertoire, and Immune Response of Plasmodium vivax Reticulocyte Binding Proteins.

Members of the Plasmodium vivax reticulocyte binding protein (PvRBP) family are believed to mediate specific invasion of reticulocytes by P. vivax. In this study, we performed molecular characterization of genes encoding members of this protein family. Through cDNA sequencing, we constructed full-length gene models and verified genes that are protein coding and those that are pseudogenes. We also used quantitative PCR to measure their in vivo transcript abundances in clinical P. vivax isolates. Like genes encoding related invasion ligands of P. falciparum, Pvrbp expression levels vary broadly across different parasite isolates. Through antibody measurements, we found that host immune pressure may be the driving force behind the distinctly high diversity of one of the family members, PvRBP2c. Mild yet significant negative correlation was found between parasitemia and the PvRBP2b antibody level, suggesting that antibodies to the protein may interfere with invasion.

Hsp72 Is an Intracellular Target of the α,ß-Unsaturated Sesquiterpene Lactone, Parthenolide.

The electrophilic natural product parthenolide has generated significant interest as a model for potential chemotherapeutics. Similar to other α,ß-unsaturated carbonyl electrophiles, parthenolide induces the heat shock response in leukemia cells, potentially through covalent adduction of heat shock proteins. Other thiol-reactive electrophiles have also been shown to induce the heat shock response as well as to covalently adduct members of the heat shock protein family, such as heat shock protein 72 (Hsp72). To identify sites of modification of Hsp72 by parthenolide, we used high-resolution tandem mass spectrometry to detect 10 lysine, histidine, and cysteine residues of recombinant Hsp72 as modified in vitro by 10 and 100 µM parthenolide. To further ascertain that modification of Hsp72 by parthenolide occurs inside cells and not simply as an in vitro artifact, an alkyne-labeled derivative of parthenolide was synthesized to enable enrichment and detection of protein targets of parthenolide using copper-catalyzed [3 + 2] azide-alkyne cycloaddition. The alkyne-labeled parthenolide derivative displays an half maximal inhibitory concentration (IC50) in undifferentiated acute monocytic leukemia cells (THP-1) of 13.1 ± 1.1 µM, whereas parthenolide has an IC50 of 4.7 ± 1.1 µM. Concentration dependence of protein modification by the alkyne-parthenolide derivative was demonstrated, as well as in vitro adduction of Hsp72. Following treatment of THP-1 cells in culture by the alkyne-parthenolide, adducted proteins were isolated with neutravidin resin and detected by immunoblotting in the enriched protein fraction. Hsp70 proteins were detected in the enriched proteins, indicating that Hsp70 proteins were adducted intracellularly by the alkyne-parthenolide derivative.