Yeast Model Uncovers Dual Roles of Mitochondria in the Action of Artemisinin

Artemisinins, derived from the wormwood herb Artemisia annua, are the most potent antimalarial drugs currently available. Despite extensive research, the exact mode of action of artemisinins has not been established. Here we use yeast, Saccharamyces cerevisiae, to probe the core working mechanism of this class of antimalarial agents. We demonstrate that artemisinin's inhibitory effect is mediated by disrupting the normal function of mitochondria through depolarizing their membrane potential. Moreover, in a genetic study, we identify the electron transport chain as an important player in artemisinin's action: Deletion of NDE1 or NDI1, which encode mitochondrial NADH dehydrogenases, confers resistance to artemisinin, whereas overexpression of NDE1 or NDI1 dramatically increases sensitivity to artemisinin. Mutations or environmental conditions that affect electron transport also alter host's sensitivity to artemisinin. Sensitivity is partially restored when the Plasmodium falciparum NDI1 ortholog is expressed in yeast ndi1 strain. Finally, we showed that artemisinin's inhibitory effect is mediated by reactive oxygen species. Our results demonstrate that artemisinin's effect is primarily mediated through disruption of membrane potential by its interaction with the electron transport chain, resulting in dysfunctional mitochondria. We propose a dual role of mitochondria played during the action of artemisinin: the electron transport chain stimulates artemisinin's effect, most likely by activating it, and the mitochondria are subsequently damaged by the locally generated free radicals

Geographical Affinities of the HapMap Samples

The HapMap samples were collected for medical-genetic studies, but are also widely used in population-genetic and evolutionary investigations. Yet the ascertainment of the samples differs from most population-genetic studies which collect individuals who live in the same local region as their ancestors. What effects could this non-standard ascertainment have on the interpretation of HapMap results?We compared the HapMap samples with more conventionally-ascertained samples used in population- and forensic-genetic studies, including the HGDP-CEPH panel, making use of published genome-wide autosomal SNP data and Y-STR haplotypes, as well as producing new Y-STR data. We found that the HapMap samples were representative of their broad geographical regions of ancestry according to all tests applied. The YRI and JPT were indistinguishable from independent samples of Yoruba and Japanese in all ways investigated. However, both the CHB and the CEU were distinguishable from all other HGDP-CEPH populations with autosomal markers, and both showed Y-STR similarities to unusually large numbers of populations, perhaps reflecting their admixed origins.The CHB and JPT are readily distinguished from one another with both autosomal and Y-chromosomal markers, and results obtained after combining them into a single sample should be interpreted with caution. The CEU are better described as being of Western European ancestry than of Northern European ancestry as often reported. Both the CHB and CEU show subtle but detectable signs of admixture. Thus the YRI and JPT samples are well-suited to standard population-genetic studies, but the CHB and CEU less so

Homozygosity Mapping Places the Acrodermatitis Enteropathica Gene on Chromosomal Region 8q24.3

Acrodermatitis enteropathica (AE) is a rare autosomal recessive pediatric disease characterized by dermatitis, diarrhea, alopecia, and growth failure. The disease results from insufficient uptake of zinc by the intestine and can be fatal unless the diet is supplemented with zinc. To map the gene responsible for AE, a genomewide screen was performed on 17 individuals, including 4 affected individuals, in a consanguineous Jordanian family. Three markers—D8S373, D10S212, and D6S1021—had a pattern consistent with tight linkage to a recessive disease: one allele in the affected sibs and multiple alleles in unaffected sibs and parents. Two-point parametric linkage analysis using FASTLINK identified one region, D8S373, with a maximum LOD score >1.5 (1.94 at D8S373: recombination fraction .001). Twelve additional markers flanking D8S373 were used to genotype the extended family, to fine-map the AE gene. All five affected individuals—including one who was not genotyped in the genomewide screen—were found to be homozygous for a common haplotype, spanning ∼3.5 cM, defined by markers D8S1713 and D8S2334 on chromosomal region 8q24.3. To support these mapping data, seven consanguineous Egyptian families with eight patients with AE were genotyped using these markers, and six patients from five families were found to be homozygous in this region. Multipoint analysis with all consanguineous families, by Mapmaker/Homoz, resulted in a maximum LOD score of 3.89 between D8S1713 and D8S373. Sliding three-point analysis resulted in a maximum LOD score of 5.16 between markers D8S1727 and D8S1744

The Genetic Screen for Artemisinin-Resistant Mutations Identified Genes in the Electron Transport Chain or in the Pathway of Respiratory Control

<div><p>(A) The three mutants isolated display increased resistance to artemisinin. YPGE plates with or without 4 μM artemisinin were used. <i>nde1Δ ndi1Δ</i> exhibited severe growth defect in nonfermentable media.</p><p>(B) Increased activities of NADH dehydrogenases exacerbate artemisinin sensitivity, and Sip5 may be positioned upstream of NADH dehydrogenases. Plates are all SG-Ura (with or without 4 μM artemisinin) to prevent plasmid loss. <i>ADH1-NDE1</i> and <i>ADH1-SIP5</i> here denote constructs that express <i>NDE1</i> and <i>SIP5</i> under the control of <i>ADH1</i> promoter. The results of <i>ADH1-NDI1</i> are similar to that of <i>ADH1-NDE1</i> and are not shown on the two plates<i>.</i></p><p>(C) Expression of PfNDI1 in <i>ndi1</i>Δ restores yeast sensitivity to artemisinin. Plates used here are SG-Ura (with or without 8 μM artemisinin).</p><p>Art, artemisinin; SG, synthetic yeast media with glycerol as the carbon source; WT, wild type.</p></div

Artemisinin Inhibits Yeast Respiratory Growth by Depolarizing the Mitochondrial Membrane

<div><p>(A) Artemisinin (Art) inhibits yeast growth in nonfermentable media. In YPD the effect of artemisinin is minimal, whereas in YPG, artemisinin is highly effective.</p><p>(B) Yeast growth is inhibited by artemisinin in YPG with an IC₅₀ that is comparable to that required to kill cultured malaria parasites. Relative growth in the presence of artemisinin was measured against to that of the yeast grown in the absence of artemisinin. Experiments shown were performed three times in liquid YPG media. Error bars represent standard errors of the mean for each assay.</p><p>(C) Artemisinin depolarizes mitochondrial membrane. The peak shift toward the left represents a decrease of fluorescence signal indicating the loss of membrane potential. Cells were grown in YPG with or without artemisinin (Art) for 2 h.</p></div

Artemisinin Generates ROS in Yeast

<div><p>When applicable, 8 μM artemisinin was used.</p><p>(A) Artemisinin-resistant strains generate fewer ROS. Yeast untreated with artemisinin was used as the control. The experiment was performed three times with similar results. NDE1 denotes the overexpressor strain of <i>NDE1</i> driven by <i>ADH1</i> promoter.</p><p>(B) Isolated artemisinin-resistant strains are not cross-resistant to paraquat or peroxide. Shown here are the wild-type (WT) parental strain (BY4742), <i>nde1</i>Δ and <i>ndi1</i>Δ on YPD plates without or with 0.02% paraquat.</p><p>(C) Iron is possibly involved in artemisinin (Art) activation. Addition of BPS to the medium reduces yeast's sensitivity to artemisinin, whereas BPS alone does not enhance general yeast survival on drug-free plates. We did not use a higher amount of BPS to further reduce the iron level because a severe reduction in iron dramatically affects yeast growth on YPG.</p></div

Hephaestin, a ceruloplasmin homologue implicated in intestinal iron transport, is defective in the sla mouse

Iron is essential for many cellular functions; consequently, disturbances of iron homeostasis, leading to either iron deficiency or iron overload, can have significant clinical consequences. Despite the clinical prevalence of these disorders, the mechanism by which dietary iron is absorbed into the body is poorly understood. We have identified a key component in intestinal iron transport by study of the sex-linked anaemia (sla) mouse, which has a block in intestinal iron transport. Mice carrying the sla mutation develop moderate to severe microcytic hypoChromic anaemia. Although these mice take up iron from the intestinal lumen into mature epithelial cells normally, the subsequent exit of iron into the circulation is diminished. As a result, iron accumulates in enterocytes and is lost during turnover of the intestinal epithelium. Biochemical studies have failed to identify the underlying difference between sla and normal mice, therefore, we used a genetic approach to identify the gene mutant in sla mice. We describe here a novel gene, Heph, encoding a transmembrane-bound ceruloplasmin homologue that is mutant in the sla mouse and highly expressed in intestine. We suggest that the hephaestin protein is a multicopper ferroxidase necessary for iron egress from intestinal enterocytes into the circulation and that it is an important link between copper and iron metabolism in mammals