Protein complex directs hemoglobin-to-hemozoin formation in Plasmodium falciparum

Abstract

Malaria parasites use hemoglobin (Hb) as a major nutrient source in the intraerythrocytic stage, during which heme is converted to hemozoin (Hz). The formation of Hz is essential for parasite survival, but to date, the underlying mechanisms of Hb degradation and Hz formation are poorly understood. We report the presence of a ∼200-kDa protein complex in the food vacuole that is required for Hb degradation and Hz formation. This complex contains several parasite proteins, including falcipain 2/2′, plasmepsin II, plasmepsin IV, histo aspartic protease, and heme detoxification protein. The association of these proteins is evident from coimmunoprecipitation followed by mass spectrometry, coelution from a gel filtration column, cosedimentation on a glycerol gradient, and in vitro protein interaction analyses. To functionally characterize this complex, we developed an in vitro assay using two of the proteins present in the complex. Our results show that falcipain 2 and heme detoxification protein associate with each other to efficiently convert Hb to Hz. We also used this in vitro assay to elucidate the modes of action of chloroquine and artemisinin. Our results reveal that both chloroquine and artemisinin act during the heme polymerization step, and chloroquine also acts at the Hb degradation step. These results may have important implications in the development of previously undefined antimalarials.

Complex nature of malaria parasite hemoglobin degradation

“…A number of other questions remain and some important ones are raised by Chugh et al's intriguing report (8):…”

Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

“…S3A in the supplemental material) (34). In order to evaluate if compounds from the 1,2,4-oxadiazole series might also interfere with the hemoglobin degradation pathway, we measured the activity of compounds 1 and 2 in an in vitro hemozoin formation assay based on enzymatic or autocatalytic reactions (see Fig.…”

“…S3B to D and Table S4 in the supplemental material). In vitro, heme can be released from hemoglobin by falcipain 2 and subsequently converted to hemozoin by the heme detoxification protein (HDP) or via an autocatalytic mechanism (34,41). The autocatalytic formation of hemozoin appears to be slightly inhibited by compounds 1 and 2 (IC 50 s, 452 M and 587 M, respectively, compared to an IC 50 of 43.5 M for amodiaquine; see Table S4 in the supplemental material).…”

Antimalarials inhibit hematin crystallization by unique drug–surface site interactions

“…A group of authors has argued that several neutral lipids are present in the DV as mesoscopic phases that accumulate hematin and serve as crystallization medium (41)(42)(43); this scenario has been supported by numerical modeling (44). High-resolution electron microscopy and X-ray tomography, however, have failed to detect lipid phases larger than 25 nm, interpreted in favor of growth from the aqueous environment (10)(11)(12), where crystallization may be assisted by a protein complex that catalyzes hematin dimerization (45). In a previous paper, we reconciled these seemingly opposite viewpoints by suggesting that β-hematin crystals grow from a thin shroud of lipid that coats their surface (46), a mechanism that is qualitatively consistent with experimental observations (47).…”