Allosteric regulation of the Plasmodium falciparum cysteine protease falcipain-2 by heme

https://doi.org/10.1016/j.abb.2015.03.007Get rights and content

Highlights

  • The malaria parasite depends upon aminoacids from hemoglobin during its erythrocytic cycle.

  • Hemoglobin digestion releases toxic, pro-oxidant heme.

  • Heme perform bound to falcipain-2 with a 1:1 stoichiometry.

  • Heme induces non-competitive inhibition of falcipain-2.

  • Falcipain-2 inhibition by heme might be a control-point allowing its crystallization into hemozoin.

Abstract

During the erythrocytic cycle of Plasmodium falciparum malaria parasites break down host hemoglobin, resulting in the release of free heme (ferriprotoporphyrin IX). Heme is a generator of free radicals that cause oxidative stress, but it is detoxified by crystallization into hemozoin inside the food vacuole. We evaluated the interaction of heme and heme analogues with falcipain-2, a P. falciparum food vacuole cysteine protease that plays a key role in hemoglobin digestion. Heme bound to falcipain-2 with a 1:1 stoichiometry, and heme inhibited falcipain-2 activity against both human hemoglobin and chromogenic peptide substrates through a noncompetitive-like mechanism. A series of porphyrin analogues was screened for inhibition of falcipain-2, demonstrating a minor contribution of iron to heme–falcipain-2 interaction, and revealing dependence on both propionic and vinyl groups for inhibition of falcipain-2 by heme. Docking and molecular dynamics simulation unveiled a novel, inducible heme-binding moiety in falcipain-2 adjacent to the catalytic site. Kinetic data suggested that the noncompetitive-like inhibition was substrate inhibition induced by heme. Collectively these data suggest that binding of heme to falcipain-2 may limit the accumulation of free heme in the parasite food vacuole, providing a means of heme detoxification in addition to crystallization into hemozoin.

Introduction

Malaria is one of the most severe and widespread tropical diseases in the world [1]. According to estimates from the World Health Organization, there were up to 207 million episodes of malaria and about 627,000 deaths in 2012 [2], with more than 90% of the deaths in sub-Saharan Africa [3]. Almost half the world’s population lives in malaria-risk areas, and 95% of cases are caused by Plasmodium falciparum and Plasmodium vivax [3]. P. falciparum, the most virulent parasite, is responsible for nearly all serious illnesses and deaths from malaria, and has developed resistance to most available drugs [4]. Therefore, new antimalarial therapies, ideally directed against new targets, are greatly needed [4], [5], [6], [7], [8].

The portion of the life cycle of malaria parasites that causes human disease involves infection of erythrocytes. During this cycle, P. falciparum digests up to 80% of erythrocyte hemoglobin to obtain amino acids for protein synthesis, maintain osmotic balance, and provide space for the growing parasite [9], [10], [11], [12], [13]. Hemoglobin is hydrolyzed inside the food vacuole, a lysosome-like organelle, by the action of several types of proteases, including plasmepsin aspartic proteases and falcipain cysteine proteases. Treatment with cysteine protease inhibitors leads to the accumulation of undigested hemoglobin in the food vacuole, blocking parasite development and leading to cures of murine malaria [14], [15], [16]. The targets of antimalarial cysteine protease inhibitors appear to be principally falcipain-2 and falcipain-3, two similar papain family cysteine proteases [17]. Disruption of the falcipain-2 gene inhibited hemoglobin degradation, with accumulation of intact hemoglobin inside the parasite food vacuole, but parasites survived, presumably due to rescue after expression of falcipain-3 late in the erythrocytic cycle [18]. Disruption of the falcipain-3 gene was not possible, suggesting that this protease is essential for erythrocytic parasites [19].

High resolution crystallographic data confirmed that falcipain-2 is structurally similar to homologous papain family proteases, with a hydrophobic catalytic site composed of the Cys42, His174, and Asn204 residues (Cys25, His159, and Asn175 accordingly to papain convention) [12]. Mature falcipain-2 has two unusual motifs for a papain family protease, an N-terminal “nose” domain and a C-terminal “arm” domain. The nose motif is required for mature protease folding [20] and the arm-like β-hairpin is required for interaction with hemoglobin [21], [22].

Due to its Fe ion, heme exerts toxicity via oxidative stress [23], and thus heme crystallization [24], [25], aggregation [26] and metabolism [27], [28] are evolutionary mechanisms for heme detoxification. Impairment of any of these processes might result in the accumulation of free heme species, and consequently in increased oxidative stress. Hemoglobin iron is almost entirely in the ferrous state. During hemoglobin digestion in the acidic food vacuole, free heme is generated, (Fig. 1A) and iron is oxidized to the ferric state. The electrons released in this oxidation can combine with molecular oxygen to produce reactive oxygen species, leading to parasite oxidative stress [9]. Malaria parasites address this situation by converting heme into hemozoin, an inert heme moiety which is a cyclic ferriprotoporphyrin IX dimer in which the propionate group of a heme molecule coordinates the Fe(III) in the center of its partner [29], [30], [31]. Approximately 90% of the iron found in infected erythrocytes is in the form of hemozoin and restricted to the food vacuole [32], [32], leaving 10% available for interaction with other molecules. The exact mechanism of hemozoin formation is not fully understood, and identification of a protein chaperone involved in the modulation of this conversion has been intently pursued. In vitro studies reported lipid-driven conversion [33], [34], and spontaneous conversion between heme and hemozoin has also been proposed [35]. A heme detoxification protein (HDP) involved in the conversion of heme into hemozoin has also been reported [36].

In this work, we have explored the possibility that the interaction of free heme with falcipains provides a means of detoxification in addition to heme crystallization into hemozoin. We describe the interaction between recombinant falcipain-2, heme, and a series of heme analogs (Fig. 1). Heme and heme analogues inhibited falcipain-2 at nanomolar concentrations, with a complex allosteric regulation of falcipain-2 by both substrate and product, supporting a key role for falcipains in the control of Plasmodium metabolism.

Section snippets

Chemicals

All reagents were of analytical grade. All buffers were freshly prepared. Heme and protoporphyrin IX were purchased from Sigma–Aldrich (Brazil). Bilirubin, deuteroporphyrin, mesoporphyrin IX dimethyl ester, deuteroporphyrin IX 2-vinyl, 4-hydroxymethyl, coproporphyrin III, N-methyl mesoporphyrin, deuteroporphyrin IX 2,4 (4,2) hydroxyethyl vinyl, N-methyl protoporphyrin IX and isohematoporphyrin were purchased from Frontier Scientific. Before use in the experiments described below the compounds

Heme inhibits falcipain-2 activity against both synthetic and natural substrates

We evaluated the effect of heme on the activity of falcipain-2. Human hemoglobin was incubated with 20 nM falcipain-2 in the presence of varying concentrations of heme, and cleavage products were resolved by SDS–PAGE. Incubation with 2 μM or 5 μM free heme inhibited the cleavage of hemoglobin (Fig. 2A). Free heme also inhibited the action of falcipain-2 against the fluorogenic substrate Z-Phe-Arg-AMC, with an IC50 of 515 ± 14 nM (Fig. 2B).

pH dependence of the inhibitory effect of heme

Falcipain activity showed a strict dependence on pH, with

Discussion

We evaluated the interaction between heme, a breakdown product of hemoglobin, and falcipain-2, a principal hemoglobinase of P. falciparum. We found that heme interacts with falcipain-2 and inhibits its hemoglobinase activity. Inhibition of falcipain-2 by heme was optimal at the pH of the P. falciparum food vacuole, the site of hemoglobin digestion. Heme binding to falcipain-2 appears to occur in a specific pattern, with a single binding site on the enzyme, and moderate affinity, with apparent

Acknowledgments

The authors thank Jennifer Legac and Jiri Gut for assistance with enzyme expression and purification. This research was supported by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), INCT-CNPq; Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro Carlos Chagas Filho (FAPERJ), and Instituto Nacional de Metrologia, Normalização e Qualidade Industrial (INMETRO). Funding agencies had no involvement in

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