Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3′-Kinase
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 ta...
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| Veröffentlicht in: | Biochemistry (Easton) 2017-08, Vol.56 (33), p.4335-4345 |
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| creator | Hassett, Matthew R Sternberg, Anna R Riegel, Bryce E Thomas, Craig J Roepe, Paul D |
| description | 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. |
| doi_str_mv | 10.1021/acs.biochem.7b00416 |
| format | Article |
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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. 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Sternberg, Anna R ; Riegel, Bryce E ; Thomas, Craig J ; Roepe, Paul D</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a411t-95161c5856595d3794bb60608e25ef39e6db35e1272a790815c2e6d3fa39a4103</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Class III Phosphatidylinositol 3-Kinases - biosynthesis</topic><topic>Class III Phosphatidylinositol 3-Kinases - chemistry</topic><topic>Class III Phosphatidylinositol 3-Kinases - genetics</topic><topic>Class III Phosphatidylinositol 3-Kinases - isolation & purification</topic><topic>Cloning, Molecular</topic><topic>Gene Expression</topic><topic>Plasmodium falciparum - enzymology</topic><topic>Plasmodium falciparum - genetics</topic><topic>Protein Domains</topic><topic>Protozoan Proteins - biosynthesis</topic><topic>Protozoan Proteins - chemistry</topic><topic>Protozoan Proteins - isolation & purification</topic><topic>Recombinant Proteins - biosynthesis</topic><topic>Recombinant Proteins - chemistry</topic><topic>Recombinant Proteins - genetics</topic><topic>Recombinant Proteins - isolation & purification</topic><topic>Saccharomyces cerevisiae - genetics</topic><topic>Saccharomyces cerevisiae - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Hassett, Matthew R</creatorcontrib><creatorcontrib>Sternberg, Anna R</creatorcontrib><creatorcontrib>Riegel, Bryce E</creatorcontrib><creatorcontrib>Thomas, Craig J</creatorcontrib><creatorcontrib>Roepe, Paul D</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Hassett, Matthew R</au><au>Sternberg, Anna R</au><au>Riegel, Bryce E</au><au>Thomas, Craig J</au><au>Roepe, Paul D</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3′-Kinase</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>2017-08-22</date><risdate>2017</risdate><volume>56</volume><issue>33</issue><spage>4335</spage><epage>4345</epage><pages>4335-4345</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><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.</abstract><cop>United States</cop><pub>American Chemical Society</pub><pmid>28719180</pmid><doi>10.1021/acs.biochem.7b00416</doi><tpages>11</tpages><orcidid>https://orcid.org/0000-0002-7079-3793</orcidid></addata></record> |
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| subjects | 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 |
| title | Heterologous Expression, Purification, and Functional Analysis of Plasmodium falciparum Phosphatidylinositol 3′-Kinase |
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