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Artemisinin Binds and Inhibits the Activity of Plasmodium falciparum Ddi1, a Retroviral Aspartyl Protease.

ABSTRACT: Reduced sensitivity of the human malaria parasite, Plasmodium falciparum, to Artemisinin and its derivatives (ARTs) threatens the global efforts towards eliminating malaria. ARTs have been shown to cause ubiquitous cellular and genetic insults, which results in the activation of the unfolded protein response (UPR) pathways. The UPR restores protein homeostasis, which otherwise would be toxic to cellular survival. Here, we interrogated the role of DNA-damage inducible protein 1 (PfDdi1), a unique proteasome-interacting retropepsin in mediating the actions of the ARTs. We demonstrate that PfDdi1 is an active A₂ family protease that hydrolyzes ubiquitinated proteasome substrates. Treatment of P. falciparum parasites with ARTs leads to the accumulation of ubiquitinated proteins in the parasites and blocks the destruction of ubiquitinated proteins by inhibiting the PfDdi1 protease activity. Besides, whereas the PfDdi1 is predominantly localized in the cytoplasm, exposure of the parasites to ARTs leads to DNA fragmentation and increased recruitment of the PfDdi1 into the nucleus. Furthermore, we show that Ddi1 knock-out Saccharomycescerevisiae cells are more susceptible to ARTs and the PfDdI1 protein robustly restores the corresponding functions in the knock-out cells. Together, these results show that ARTs act in multiple ways; by inducing DNA and protein damage and might be impairing the damage recovery by inhibiting the activity of PfDdi1, an essential ubiquitin-proteasome retropepsin.

SUBMITTER: Onchieku NM

PROVIDER: S-EPMC8621276 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDuring erythrocytic schizogony, Plasmodium falciparum interacts with the human erythrocyte membrane when it enters into, grows within and escapes from the erythrocyte. An interaction between the P. falciparum M18 aspartyl aminopeptidase (PfM18AAP) and the human erythrocyte membrane protein spectrin was recently identified using phage display technology. In this study, recombinant (r) PfM18AAP was characterized and the interaction between the enzyme and spectrin, as well as other erythrocyte membrane proteins, analyzed.MethodsrPfM18AAP was produced as a hexahistidine-fusion protein in Escherichia coli and purified using magnetic bead technology. The pI of the enzyme was determined by two-dimensional gel electrophoresis and the number of subunits in the native enzyme was estimated from Ferguson plots. The enzymatic activity over a pH and temperature range was tested by a coupled enzyme assay. Blot overlays were performed to validate the spectrin-PfM18AAP interaction, as well as identify additional interactions between the enzyme and other erythrocyte membrane proteins. Sequence analysis identified conserved amino acids that are expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization.ResultsrPfM18AAP has a molecular weight of ~67 kDa and the enzyme separated as three entities with pI 6.6, 6.7 and 6.9. Non-denaturing gel electrophoresis indicated that rPfM18AAP aggregated into oligomers. An in vitro coupled enzyme assay showed that rPfM18AAP cleaved an N-terminal aspartate from a tripeptide substrate with maximum enzymatic activity at pH 7.5 and 37 degrees C. The spectrin-binding region of PfM18AAP is not found in Homo sapiens, Saccharomyces cerevisiae and otherPlasmodium species homologues. Amino acids expected to be involved in cofactor binding, substrate cleavage and quaternary structure stabilization, are conserved. Blot overlays with rPfM18AAP against spectrin and erythrocyte membrane proteins indicated that rPfM18AAP binds to spectrin, as well as to protein 4.1, protein 4.2, actin and glyceraldehyde 3-phosphate dehydrogenase.ConclusionStudies characterizing rPfM18AAP showed that this enzyme interacts with erythrocyte spectrin and other membrane proteins. This suggests that, in addition to its proposed role in hemoglobin digestion, PfM18AAP performs other functions in the erythrocyte host and can utilize several substrates, which highlights the multifunctional role of malaria enzymes.

Project description:BackgroundArtemisinin-based combination therapies are the recommended first-line treatments of falciparum malaria in all countries with endemic disease. There are recent concerns that the efficacy of such therapies has declined on the Thai-Cambodian border, historically a site of emerging antimalarial-drug resistance.MethodsIn two open-label, randomized trials, we compared the efficacies of two treatments for uncomplicated falciparum malaria in Pailin, western Cambodia, and Wang Pha, northwestern Thailand: oral artesunate given at a dose of 2 mg per kilogram of body weight per day, for 7 days, and artesunate given at a dose of 4 mg per kilogram per day, for 3 days, followed by mefloquine at two doses totaling 25 mg per kilogram. We assessed in vitro and in vivo Plasmodium falciparum susceptibility, artesunate pharmacokinetics, and molecular markers of resistance.ResultsWe studied 40 patients in each of the two locations. The overall median parasite clearance times were 84 hours (interquartile range, 60 to 96) in Pailin and 48 hours (interquartile range, 36 to 66) in Wang Pha (P<0.001). Recrudescence confirmed by means of polymerase-chain-reaction assay occurred in 6 of 20 patients (30%) receiving artesunate monotherapy and 1 of 20 (5%) receiving artesunate-mefloquine therapy in Pailin, as compared with 2 of 20 (10%) and 1 of 20 (5%), respectively, in Wang Pha (P=0.31). These markedly different parasitologic responses were not explained by differences in age, artesunate or dihydroartemisinin pharmacokinetics, results of isotopic in vitro sensitivity tests, or putative molecular correlates of P. falciparum drug resistance (mutations or amplifications of the gene encoding a multidrug resistance protein [PfMDR1] or mutations in the gene encoding sarco-endoplasmic reticulum calcium ATPase6 [PfSERCA]). Adverse events were mild and did not differ significantly between the two treatment groups.ConclusionsP. falciparum has reduced in vivo susceptibility to artesunate in western Cambodia as compared with northwestern Thailand. Resistance is characterized by slow parasite clearance in vivo without corresponding reductions on conventional in vitro susceptibility testing. Containment measures are urgently needed. (ClinicalTrials.gov number, NCT00493363, and Current Controlled Trials number, ISRCTN64835265.)

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Artemisinin Binds and Inhibits the Activity of Plasmodium falciparum Ddi1, a Retroviral Aspartyl Protease.

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