An iron regulatory-like protein expressed in Plasmodium falciparum displays aconitase activity

https://doi.org/10.1016/j.molbiopara.2005.05.004Get rights and content

Abstract

Plasmodium falciparum iron regulatory-like protein (PfIRPa) has homology to both mammalian iron regulatory proteins and aconitases and is capable of binding RNA iron response elements. We examined the subcellular localization of PfIRPa and its enzymatic properties at low oxygen tension. Differential digitonin permeabilization of isolated trophozoites with subsequent Western blot analysis suggests that the localization of PfIRPa is predominantly in the membranous compartments of the parasite, such as the mitochondrion. Immunofluorescence analysis showed that PfIRPa colocalizes with heat shock protein 60 (Hsp60), a mitochondrial marker, and is also present in the parasitic cytosol/food vacuole. Under conditions favoring the formation of an iron–sulfur cluster, recombinant PfIRPa (rPfIRPa) had aconitase activity as detected by a colorimetric NADPH-MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) assay. As assessed by the hydration of cis-aconitate spectrophotometrically at 240 nm, rPfIRPa had high affinity for cis-aconitate (Km = 3.5 μM) but a low turnover number (Kcat =3.3 s−1). The overall catalytic efficiency (Kcat/Km) of rPfIRPa was similar in magnitude to human cytosolic IRP1/aconitase and human mitochondrial aconitase. PfIRPa immunoprecipitated from parasite lysates also had aconitase activity, as assessed by an MTT-based assay. Our results provide evidence that PfIRPa localizes in the mitochondrion and in the cytosol/food vacuole and is able to demonstrate aconitase activity. Further understanding of the role of PfIRPa/aconitase in the regulation of P. falciparum homeostasis may contribute towards the development of novel antimalarial strategies against plasmodial species.

Introduction

The major energy supply for the asexual erythrocytic stage of Plasmodium falciparum is the process of glycolysis [1], but the classical association between glycolysis and the tricarboxylic acid (TCA) cycle is not apparent [2]. The sequencing of the plasmodial genome has revealed that all of the major TCA cycle enzymes are present in P. falciparum [3], and gene expression profiles have shown that the mRNA levels of the genes encoding these enzymes are almost synchronously increased during the middle and late trophozoite stages (20–36 h post-invasion of red blood cells) [2], [3], [4]. The mRNA levels of the trigger enzyme of the TCA cycle, citrate synthase, are maximal 45 h post-invasion, i.e. 9–15 h after maximum mRNA levels of all other TCA cycle enzymes [2]. However, it has not been determined if the TCA cycle has a vital role in providing energy for the asexual erythrocytic parasites [5]. Progress in sequencing the plasmodial genome and the search for novel antimalarial targets stimulated the cloning and partial characterization of five P. falciparum genes that may be involved in the TCA cycle: malate dehydrogenase [6], the flavoprotein and iron–sulfur subunits of succinate dehydrogenase [7], [8], lactate-dehydrogenase-like malate dehydrogenase [9], isocitrate dehydrogenase [5] and a gene with homology for iron regulatory protein/aconitase genes of other species [10], [11]. The intracellular localization of these proteins in P. falciparum remains to be completed.

The connection of the TCA cycle with glycolysis in the asexual stages of P. falciparum is not completely evident, as expression has been recently detected for the α and β chains of pyruvate dehydrogenase [12], but not for dihydrolipoamide S-acetyl transferase, the typical links between glycolysis and the TCA cycle [2]. In addition, the expression of pyruvate dehydrogenase, which is typically cytosolic in eukaryotic systems, has been mapped to the apicoplast of plasmodial parasites [12]. The expression of TCA cycle genes is synchronized with the expression of a large number of genes in the mitochondrial genome, including those involved in electron transport [2]. These data are consistent with the possibility that electron transport occurs in the mitochondria of intraerythrocytic P. falciparum parasites, which is supported by the detection of enzymatic activities of dihydroorotate dehydrogenase, cytochrome c reductase, and cytochrome c oxidase in asexual stage parasites [13]. Additionally, mitochondrial respiration has been reported in asexual parasites of P. falciparum [13], [14] and P. berghei [15]. The production of NADH and FADH2 in the citric acid cycle is critical for oxidative phosphorylation, as the oxidation of NADH and FADH2 is coupled to the formation of ATP. Although earlier studies suggested that purified P. falciparum and P. yoelii mitochondria do not oxidize NADH-linked substrates such as α-ketoglutarate, malate, or pyruvate [16], it has been recently demonstrated that NADH-quinone oxidoreductase and a malate-quinone oxidoreductase (FAD-dependent) are present in P. yoelii yoelii asexual stage parasites. The addition of ADP to the digitonin-permeabilized isolated P. yoelii mitochondria containing malate induced an increase in respiration, indicating the transition from resting to phosphorylating state respiration. Oligomycin inhibited respiration in agreement with the operation of oxidative phosphorylation in the P. yoelii mitochondria [17].

It has been proposed that the TCA cycle enzymes in asexual malaria parasites may serve primarily to generate reducing equivalents for maintaining the redox status of the mitochondrion [5], to produce ATP [17] or to provide other metabolic intermediates such as succinyl-CoA [1] or glutamate [18]. Aconitase, which facilitates the conversion of citrate to isocitrate through the intermediate cis-aconitate, is one of the essential enzymes associated with this cycle. In higher eukaryotes, the catalytically inactive form of aconitase has the capacity to function as an iron regulatory protein (IRP-1, also known as iron response element-binding protein 1) [19]. Previous studies [10], [11] have demonstrated that a protein expressed in P. falciparum has homology to mammalian iron regulatory proteins and has the capacity to bind specifically to putative RNA iron response elements present in the plasmodial system. The purpose of the present study was to determine the subcellular localization of P. falciparum iron regulatory-like protein (PfIRPa) and to investigate its possible aconitase function.

Section snippets

Maintenance of P. falciparum growth in culture

P. falciparum (strain 3D7) was grown in culture flasks containing RPMI-1640 supplemented with 25 mM HEPES, 23 mM sodium bicarbonate, 10 mM glucose, 10% (v/v) heat-inactivated human plasma (0+ or A+), and washed human erythrocytes (A+) at 2–2.5% hematocrit. The growth medium was replaced daily and the cultures were gassed with a mixture of 90% N2, 5% CO2 and 5% O2 [20]. Ring stage synchronization was achieved by the lysis of cells containing mature parasites with alanine or sorbitol [21]. The

Results

To ascertain the subcellular localization of PfIRPa, we permeabilized infected cells with digitonin, which solubilizes the cholesterol-rich plasma membrane at low concentrations, whereas higher concentrations are required to solubilize organellar membranes [26], [27]. Enolase [28] and the mitochondrial heat shock protein Hsp60 [29] were used as markers for the cytosolic and mitochondrial fractions, respectively, as described before [29]. Saponin-isolated trophozoites were incubated with

Discussion

Our previous cloning and expression of plasmodial IRP (PfIRPa) in a bacterial expression system facilitated its characterization as an RNA-binding protein [11]. Our present results indicate that PfIRPa is localized partially in the plasmodial mitochondrion and partially in the cytosol/food vacuole, and that under appropriate conditions this molecule undergoes the necessary changes to display aconitase activity. The reduction of intracellular citrate concentration upon maturation of malaria

Acknowledgements

The authors acknowledge the grant support provided in part by grants RO1-A144857-05 from the National Institute of Allergy and Infectious Diseases, UH1-HL03679-07 from The National Heart, Lung and Blood Institute and the Office of Research on Minority Health, and Howard University GCRC grant MO1-RR10284-06. The authors are grateful to Drs. John R. Guest and John C. Wootton for fruitful discussions and insightful help. The technical assistance of Ms. Tiffany N. Johnson is appreciated.

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