Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3'-Kinase.
Matthew R HassettAnna R SternbergBryce E RiegelCraig J ThomasPaul D RoepePublished in: Biochemistry (2017)
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.
Keyphrases
- plasmodium falciparum
- protein kinase
- poor prognosis
- saccharomyces cerevisiae
- binding protein
- genome wide
- acute coronary syndrome
- drug induced
- high throughput
- long non coding rna
- tyrosine kinase
- amino acid
- emergency department
- electronic health record
- dna methylation
- transcription factor
- resting state
- machine learning
- genome wide identification