Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3'-Kinase.

Therapeutic Approaches

Abstract

The Plasmodium falciparum malarial parasite genome appears to encode one and only one phosphatidylinositol 3'-kinase (PI3K), and sequence analysis suggests that the enzyme is a "class III"- or "Vps34"-type PI3K. PfVps34 has generated excitement as a possible druggable target and potentially a key target of artemisinin-based antimalarials. In this study, we optimize the PfVps34 gene for heterologous expression in yeast, purify the protein to homogeneity, use a recently validated quantitative assay for phosphatidylinositol 3'-phosphate production from phosphatidylinositol ( Hassett et al., companion paper; DOI 10.1021/acs.biochem.7b00416 ) to quantify activity and drug inhibition of that activity, and investigate the importance of key residues in the enzyme's catalytic and "N-lobe" domains. Data suggest that PfVps34 is indeed inhibited by artemisinin and related drugs but only under conditions that cleave the drugs' endoperoxide bridge to generate reactive alkylating agents.

Authors

Hassett, Matthew R; Sternberg, Anna R; Riegel, Bryce E; Thomas, Craig; Roepe, Paul D;

Keywords

  • Class III Phosphatidylinositol 3-Kinases/ biosynthesis
  • Class III Phosphatidylinositol 3-Kinases/ chemistry
  • Class III Phosphatidylinositol 3-Kinases/ genetics
  • Class III Phosphatidylinositol 3-Kinases/ isolation & purification
  • Cloning, Molecular
  • Gene Expression
  • Plasmodium falciparum/ enzymology
  • Plasmodium falciparum/ genetics
  • Protein Domains
  • Protozoan Proteins/ biosynthesis
  • Protozoan Proteins/ chemistry
  • Protozoan Proteins/ isolation & purification
  • Recombinant Proteins/ biosynthesis
  • Recombinant Proteins/ chemistry
  • Recombinant Proteins/ genetics
  • Recombinant Proteins/ isolation & purification
  • Saccharomyces cerevisiae/ genetics
  • Saccharomyces cerevisiae/ metabolism

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