Identification and characterization of heme-interacting proteins in the malaria parasite, Plasmodium falciparum.
The degradation of hemoglobin by the malaria parasite, Plasmodium falciparum, produces free ferriprotoporphyrin IX (FP) as a toxic by-product. In the presence of FP-binding drugs such as chloroquine, FP detoxification is inhibited, and the build-up of free FP is thought to be a key mechanism in parasite killing. In an effort to identify parasite proteins that might interact preferentially with FP, we have used a mass spectrometry approach. Proteins that bind to FP immobilized on agarose include P. falciparum glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH), P. falciparum glutathione reductase (PfGR), and P. falciparum protein disulfide isomerase. To examine the potential consequences of FP binding, we have examined the ability of FP to inhibit the activities of GAPDH and GR from P. falciparum and other sources. FP inhibits the enzymic activity of PfGAPDH with a Ki value of 0.2 microm, whereas red blood cell GAPDH is much less sensitive. By contrast, PfGR is more resistant to FP inhibition (Ki > 25 microm) than its human counterpart. We also examined the ability of FP to inhibit the activities of the additional antioxidant enzymes, P. falciparum thioredoxin reductase, which exhibits a Ki value of 1 microm, and P. falciparum glutaredoxin, which shows more moderate sensitivity to FP. The exquisite sensitivity of PfGAPDH to FP may indicate that the glycolytic pathway of the parasite is particularly susceptible to modulation by FP stress. Inhibition of this pathway may drive flux through the pentose phosphate pathway ensuring sufficient production of reducing equivalents to counteract the oxidative stress induced by FP build-up.
Department of Biochemistry and Co-operative Research Centre for Diagnostics, La Trobe University, Melbourne 3086, Victoria, Australia.
Naomi
N
Campanale
Christine
C
Nickel
Claudia A
CA
Daubenberger
Dean A
DA
Wehlan
Jeff J
JJ
Gorman
Nectarios
N
Klonis
Katja
K
Becker
Leann
L
Tilley
D000818:Animals; D004305:Dose-Response Relationship, Drug; D004912:Erythrocytes / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism; D054477:Glutaredoxins; D005980:Glutathione Reductase / Q000378:metabolism; D006418:Heme / Q000737:chemistry*; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D018384:Oxidative Stress; D010088:Oxidoreductases; D010427:Pentose Phosphate Pathway; D010455:Peptides / Q000737:chemistry; D010963:Plasmodium falciparum / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012685:Sepharose / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013880:Thioredoxin-Disulfide Reductase / Q000378:metabolism; D013879:Thioredoxins / Q000378:metabolism; D013997:Time Factors; D014357:Trypsin / Q000494:pharmacology
D005656:Fungal Proteins; D054477:Glutaredoxins; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; D006418:Heme; D013879:Thioredoxins; D012685:Sepharose; D010088:Oxidoreductases; D005980:Glutathione Reductase; D013880:Thioredoxin-Disulfide Reductase; D014357:Trypsin