Protein translation in Plasmodium parasites

Review

Protein translation in Plasmodium parasites Katherine E. Jackson1*, Saman Habib2*, Magali Frugier3, Rob Hoen4, Sameena Khan5, James S. Pham1, Lluı´s Ribas de Pouplana6,7, Miriam Royo4, Manuel A.S. Santos8, Amit Sharma5 and Stuart A. Ralph1 1

Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010 Australia 2 Division of Molecular and Structural Biology, Central Drug Research Institute–Council of Scientific and Industrial Research, PO Box 173, Chattar Manzil, Mahatma Gandhi Marg, Lucknow 226001, India 3 Architecture et Re´activite´ de l’ARN, Universite´ de Strasbourg, Centre National de la Recherche Scientifique, Institut de Biologie Mole´culaire et Cellulaire, 15 rue Rene´ Descartes, 67084 Strasbourg CEDEX, France 4 Combinatorial Chemistry Unit, Barcelona Science Park, University of Barcelona, carrer Baldiri Reixac 10, 08028 Barcelona, Spain 5 Structural and Computational Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Road, New Delhi 110 067, India 6 Institucio´ Catalana de Recerca i Estudis Avanc¸ats, Passeig Lluı´s Companys 1, Barcelona 08010, Catalonia, Spain 7 Institute for Research in Biomedicine, Carrer Baldiri Reixac 10, Barcelona 08028, Catalonia, Spain 8 RNA Biology Laboratory, Department of Biology and CESAM, University of Aveiro, 3810-193 Aveiro, Portugal

The protein translation machinery of the parasite Plasmodium is the target of important anti-malarial drugs, and encompasses many promising targets for future drugs. Plasmodium parasites have three subcellular compartments that house genomes; the nucleus, mitochondrion and apicoplast, and each requires its own compartmentalized transcription and translation apparatus for survival. Despite the availability of the complete genome sequence that should reveal the requisite elements for all three compartments, our understanding of the translation machineries is patchy. We review what is known about cytosolic and organellar translation in Plasmodium and discuss the molecules that have been identified through genome sequencing and post-genomic analysis. Some translation components are yet to be found in Plasmodium, whereas others appear to be shared between translationally active organelles. Plasmodium translation in the post-genomic era Since the sequencing of the P. falciparum genome, and the complete or near-complete genome sequencing of a handful of other Plasmodium species, there have been tremendous advances in the characterization of the molecules governing host–pathogen interactions. Less striking has been the pursuit in Plasmodium species of one of the basic processes of life – the translation of RNA into protein. Perhaps the very centrality of this process to biology has made it a less attractive research topic in a parasite whose most captivating behaviors involve interplay with its host. Nevertheless, protein translation is an important field of Plasmodium research. Although translation is more easily studied in model eukaryotes and prokaryotes, departures from these

*

Corresponding author: Ralph, S.A. ([email protected]) These authors contributed equally to this work.

models observed in Plasmodium and other apicomplexan parasites reveal important aspects of diversity among translation systems. Most phylogenetic neighbors of Plasmodium have poorly characterized protein translation and, where mechanisms have been studied (in ciliates for example), they appear to differ in important aspects from what is

Glossary Apicoplast: non-photosynthetic plastid with its own circular genome, found in most Apicomplexa. Arose via secondary endosymbiosis. Cytosol: the intracellular fluid found within cells, excluding that within membrane-bound organelles. Isoacceptors: tRNAs that have different anticodons but are charged with the same amino acid. Mitochondrion: endosymbiotic organelle with its own small mitochondrial chromosome. Generally plays a major role in energy conversion. Peptide exit tunnel: the site, formed by proteins and rRNA, where the nascent polypeptide chain leaves the ribosome. P-granules: translationally silent storage particles containing high levels of non-translated mRNA. Related to P-bodies, but differ in their inability to degrade mRNA. Plastid: endosymbiotic organelle found in plants, algae and some protists. Houses photosynthetic machinery in some eukaryotes as well as several biosynthetic pathways. RNA polymerase III: the enzyme responsible for transcribing many non-coding RNA molecules. These include the tRNA and 5S rRNA molecules essential for translation, and also other structural, catalytic and regulatory RNA molecules. Translation elongation factors: proteins that facilitate the steps involved in the protein translation elongation. EF-Tu allows the correct aminoacyl-tRNA to enter the ribosome, EF-Ts is the nucleotide exchange factor for EF-Tu, and EF-G is the translocase that allows the mRNA and tRNA to move one codon along the ribosome after each round of elongation. Translation initiation factors: a large group of proteins responsible for effective initiation of protein synthesis. In eukaryotes, eIF-4A (eukaryotic initiation factor) is a helicase that prepares the mRNA for ribosome binding eIF-4B is responsible for the recognition of the mRNA cap, eIF-4E and eIF-4G direct ribosomes to the mRNA cap, eIF-5A stimulates formation of the first peptide bond in translation, and eiF1 prevents premature association of the large and small ribosomal subunits to increase specificity in initiation codon recognition. tRNA synthetase: one of a family of enzymes that catalyze the ligation of an amino acid to its cognate tRNA to form an aminoacyl-tRNA (also referred to as charged tRNA) ready for use in protein translation. In most cases a distinct tRNA synthetase is required for each amino acid.

1471-4922/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.pt.2011.05.005 Trends in Parasitology, October 2011, Vol. 27, No. 10

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Review known for Plasmodium. A more pressing reason to study Plasmodium translation is its role as the target for several of the very few successful antimalarial drugs and a suite of promising inhibitors. This precedent for successful drugs urges more research in this field to improve our understanding of the mechanisms of action and resistance to existing drugs and to explore rationally the potential for new translation-blocking drugs. Three compartments, three translation machines? Plasmodium, as do plants, algae, and the majority of other Apicomplexans, possesses three active compartments of translation: the cytosol, mitochondrion and a relic plastid termed the apicoplast (see Glossary) or apicomplexan plastid. Apicomplexans contain, therefore, a mixture of translation machinery, with eukaryotic components for cytosolic mRNA translation and prokaryotic-like components for mRNA translation in the mitochondria and apicoplast. The apicoplast is non-photosynthetic and appears to supply the parasite with precursors of heme, isoprenes, fatty acids, and iron–sulfur clusters [1] whereas the mitochondria is involved in energy conversion and pyrimidine synthesis [2]. The cytosolic protein translation machinery is responsible for translation of an estimated 5385 genes encoded by the nuclear genome, whereas the apicoplast machinery translates less than 50 genes encoded by the 35 kb circular apicoplast genome [3]. The plastid encodes genes relevant for its own translation, including rRNAs, tRNAs, ribosomal proteins, and the translation elongation factor Tu (EF-Tu), and also a handful of other poorly characterized proteins unrelated to translation (Figure 1). Most proteins resident in the apicoplast are encoded by the nuclear genome, translated in the cytoplasm, and transported into the apicoplast using specific targeting signals [4]. Detection of plastid-encoded EF-Tu by western blot and immunofluorescence has provided evidence for translation in P. falciparum plastids [5]. Indirect evidence of apicoplast translation also comes from the identification of ribosome-like particles carrying plastid-specific rRNA and mRNA [6]. The efficacy of inhibitors targeting apicoplast translation components also confirms the importance of this machinery (reviewed in [7,8]). Plasmodium spp. have the smallest mitochondrial genome known to date (6 kb), and this encodes only three proteins, namely cytochrome c oxidase subunits I and III (Cox1 and Cox3) and cytochrome b (Cytb) [2,9]. The mitochondrial genome does not encode tRNA genes but encodes small fragmented rRNAs [10]. Despite the apparent incompleteness of the mitochondrial translational machinery and lack of direct evidence for translation in mitochondria, indirect evidence suggests that mitochondrial translation is active and is essential. A major antimalarial drug (atovaquone) targets the mitochondrial cytochrome bc1 complex, and point mutations in the mitochondrial cytb gene correlate with resistance to this drug [11]. Cytochrome c oxidase activity, the requirement of the complex for mitochondrial electron transport and its inhibition by cyanide also suggest that the mitochondrial cox genes are translated in the mitochondrion [12,13]. 468

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Amino acids P. falciparum parasites have multiple means of obtaining amino acids for protein synthesis, but the most important for growth in vivo remain unclear. Of the 20 canonical amino acids, Plasmodium possesses biosynthetic pathways for Asn, Gln, Gly, Pro, Asp and Glu [14]. However, very low amounts of these amino acids are incorporated into Plasmodium proteins [15]. This limited capacity for de novo amino acid synthesis means that, during in vitro culture at least, to charge its own tRNAs Plasmodium relies largely on amino acids released from the digestion of host hemoglobin in the parasite food vacuole and on amino acid uptake from the extracellular space [16]. Human hemoglobin is rich in most amino acids, but is low in Cys, Glu, Gln and Met, and lacks Ile; these could be rate-limiting for protein synthesis. Supplementation of Plasmodium with these five essential amino acids is sufficient for short-term parasite development [17], and recent evidence has shown that Ile alone is sufficient to support parasite growth [16]. However, for some Plasmodium strains Met supplementation is also required for optimal growth [16]. The dependence on such exogenous amino acids means that parasite protein synthesis relies on amino acid transport across the erythrocyte plasma membrane, food-vacuole membrane, and its own plasma membrane [18,19]. In addition to the canonical 20 amino acids, P. falciparum incorporates two others. The first is the redoxregulating selenocysteine, which is incorporated into very few proteins (only four have been identified so far) [20,21]. The second is formyl-methionine (fMet), that is probably used as the initiator amino acid in the apicoplast. The transferase responsible for adding the formyl group to the charged fMet tRNA is apicoplast-targeted in the related parasite Toxoplasma [22]. The peptide deformylase required to process such amino acids is also trafficked to the apicoplast in Plasmodium [23,24], and inhibition of this enzyme results in parasite death [25]. No corresponding enzymes appear to be targeted to the mitochondrion [22]. Components of the translation machinery tRNA Of the three Plasmodium genomes, nuclear, apicoplast and mitochondrial, only the first two encode tRNAs [3,26,27] and correspond to compartments in which translation has been demonstrated [5]. A total of 46 tRNA genes, coding for 45 tRNA isoacceptors (initiator and elongator tRNAMet are encoded by two different genes), are found in the nuclear genome whereas the apicoplast genome contains 35 genes encoding 26 tRNA isoacceptors. With the exception of the apicoplast initiator tRNAMet, characterized by a unique variable region (11 nucleotides), they all resemble other eukaryotic tRNAs [28]. Indeed, they have the potential to adopt a canonical tertiary fold because they contain all conserved and semi-conserved nucleotides necessary to form the well-known L-shaped structure [28,29]. Some but not all Plasmodium species have very low G/C contents in their genomes (less than 20% G/C for P. falciparum [3]). Cytosolic tRNAs have a G/C content of 56% whereas apicoplast-encoded tRNAs have 26% G/C [28]. Higher G/C content in tRNA and rRNA species has been associated

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Amino acids sourced by: • Hemoglobin breakdown • De novo synthesis • Import from circulation

35 kb apicoplast genome encodes: • All rRNAs (save 5s rRNA) • 35 tRNAs (a full set) • 17-18 ribosomal proteins (number varies between apicomplexans) • EF-Tu • No aaRSs

23 Mb nuclear genome encodes: • Asexual and sexual stage rRNA molecules • 46 tRNAs (a full set) • Ribosomal proteins • 36 aaRSs

Apicoplast targeted molecules: • Some ribosomal proteins • 8 initiation, elongation and release factors? • 20 aaRSs?

Mitochondrially targeted molecules: • Some rRNAS? • All tRNAs (aminoacylated) • All ribosomal proteins? • 5 initiation, elongation and release factors?

3 kb Mitochondrial genome encodes: • Fragmented rRNA molecules • No tRNA • No ribosomal proteins • No translation initiation, elongation or release factors • No aaRSs TRENDS in Parasitology

Figure 1. Translation in Plasmodium: a tale of many compartments. Translation takes place in the Plasmodium apicoplast, mitochondrion and cytosol (including at the rough ER). The apicoplast genome, like that of most plastids, encodes its own tRNA and rRNA molecules, and also many ribosomal proteins, but imports many other nuclear-encoded proteins needed for its translation. The mitochondrion appears to import all proteins and tRNAs needed for translation of mitochondrial-encoded transcripts, and it is unclear if its fragmented rRNA is functional. Some of the amino acids necessary for translation in these three compartments can be synthesized by the parasite, but others are imported from the extracellular milieu, or can be sourced from hemoglobin degraded in the food vacuole.

with higher optimum growth temperatures or increased structural stability, although it is hard to see why these should vary between the apicoplast and cytosol. The consequences for translational efficiency of the unusual G/C content of the genomes and tRNAs of P. falciparum are completely unknown. Finally, only two introns were identified in the anticodon domain of these tRNAs [30]; one

intron is located in the nuclear tRNATyrGTA and another in the apicoplast tRNALeuTAA. The most striking observation is that there is only one gene copy per tRNA isoacceptor in the nuclear genome, and therefore Plasmodium has the fewest known tRNA genes of any eukaryote. Whether this limits translation efficiency in the parasite or whether its RNA polymerase III machinery 469

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is regulated by as yet unknown factors is unclear. If the different tRNAs are equally abundant, the most-used codons in the genome will have relatively fewer tRNA molecules available per codon. Codons for Asn, in particular, are highly used in low-complexity regions of Plasmodium proteins, and it has been proposed that a relative limitation of tRNAAsn might slow down local translation of these regions, with implications for translation accuracy and efficiency and for proper cotranslational folding of the relevant proteins [31]. Nuclear, plastid and mitochondrial genomes show differences in codon usage, but every amino acid is used at least once in each of the three compartments, and therefore a full complement of tRNAs and aminoacyltRNA synthetases should be necessary for translation of each genome. tRNA synthetases Aminoacyl-tRNA synthetases (aaRSs) are the enzymes responsible for the esterification of a specific amino acid, or its precursor, to its compatible cognate tRNA to form aminoacyl-tRNAs. These enzymes belong to two structurally distinct classes, apparently evolving from two distinct ancestors via convergent evolution [32]. The increase in complexity of the aaRSs matches that of their tRNA substrates, whose structure can also be divided into two distinct structural domains, namely the acceptor-arm domain and the anticodon-arm domain [33]. Plasmodium has genes for only 37 aaRSs, and these are apparently sufficient to translate the nuclear, apicoplast, and mitochondrial genomes [34]. The reduction from

a theoretical maximum of more than 60 to 37 aaRS genes implies that several Plasmodium aaRSs aminoacylate tRNAs that are active in more than one subcellular compartment. Studies in Toxoplasma gondii suggest that in this species all mitochondrial tRNAs are in fact aminoacylated externally and transported into the mitochondria for use in protein synthesis [22,35]. Such a mechanism presumably suffers from the deficiency that imported tRNAs would be unable to be recharged readily, and it is currently unknown if these tRNAs are in some way recycled or are degraded after a single use. To our knowledge this is the first example of a translation machinery that depends fully on transport of charged tRNAs from another cellular compartment. The mitochondria of unrelated trypanosomatid parasites also import all tRNAs [36], but in that case are charged by mitochondrial-targeted tRNA aaRS enzymes. In P. falciparum the subcellular distribution of most aaRSs remains unclear, although computational studies suggest that most of these proteins are either targeted to the apicoplast or the cytosol, but not to the mitochondria [34]. The two isoleucyl-tRNA synthetases have been shown to be targeted to the apicoplast and cytosol, respectively [37]. Several aaRS enzymes are present only once in the genome, despite their apparent requirement in the apicoplast and the cytosol. In those cases we predict that these will be dually targeted to both compartments (Table 1). In each case the gene models start with a predicted apicoplast targeting sequence [4], but whether all isoforms contain this is unknown. Some aaRSs in Toxoplasma are dual-targeted to the

Table 1. Analysis of the full complement of Plasmodium aaRSs and related enzymesa Enzyme Alanine tRNA synthetase Arginine tRNA synthetase Asparagine tRNA synthetase Asparagine tRNA synthetase Cysteine tRNA synthetase Glutamate tRNA synthetase Glutamine tRNA synthetase Glutamate tRNA amidotransferase subunit A Glutamate-tRNA amidotransferase subunit B Glycine tRNA synthetase Histidine tRNA synthetase Isoleucine tRNA synthetase Leucine tRNA synthetase Lysine tRNA synthetase Methionine tRNA synthetase Methionine tRNA formyltransferase Phenylalanine tRNA synthetase subunits

Proline tRNA synthetase Serine tRNA synthetase Threonine tRNA synthetase Tryptophan tRNA synthetase Tyrosine tRNA synthetase Valine tRNA synthetase a

Apicoplast

Cytosol

PFI0680c b PFE0475w PFE0715w

PFL0900c b PFB0525w PFA0145c

MAL13P1.281 not used PFD0780w PFF1395c

PF13_0257 PF13_0170 not used not used

PFI1645c PFL1210w PF08_0011 PF14_0166 PF10_0053 MAL13P1.67 PFL1540c

PFI1240c PFL0770w

PF14_0428 PF13_0179 PFF1095w PF13_0262 PF10_0340 not used PFF0180w PF11_0051 PFA0480w PFL0670c PF07_0073

PFL2485c PF11_0181 PFC0470w

PF13_0205 MAL8P1.125 PF14_0589

Single copy; dual targeted? PF13_0354

PF10_0149

PF14_0198

PF11_0270

The number of enzymes predicted in Plasmodium, and their supposed cellular localization, are shown. Subcellular localization is predicted on the basis of possession of trafficking sequences for the apicoplast or mitochondrion, or from experimental localization in the case of the isoleucyl tRNA synthetases [37]. We postulate that the aaRS enzymes that are found only once in the genome are dual-targeted to the cytosol and the apicoplast by mechanisms yet to be characterized.

b

Systematic gene designation.

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Review cytosol and apicoplast, but the underlying mechanisms are unclear [22]. Dual-targeted enzymes of apicoplast origin are particularly attractive drug targets because these enzymes are often similar to bacterial versions and significantly different from the human enzymes. Molecules that block such enzymes should effectively arrest translation of the main genomes of the parasite without significantly affecting orthologous human aaRSs. L–D amino acid discrimination In many cases, aaRSs do not exclude D-amino acids when charging tRNA. The stereospecificity of these enzymes is therefore not absolute, and this charging error can be toxic to cells [38]. Early studies on bacterial tyrosyl-tRNA synthetases, and later on S. cerevisiae tyrosyl-, tryptophanyl- and aspartyl-tRNA synthetases, demonstrated transfer of the D-isomer of their cognate amino acid to the corresponding tRNA species. This activity can be compensated for by D-tyrosyl-tRNATyr deacylase (DTD), which hydrolyzes the misacylated tRNA back into D-tyrosine and free tRNA [38,39]. DTD can also recognize other tRNAs charged with corresponding D-amino acids [39] and could function as a general proofreading mechanism against D-amino acid incorporation. Disruptions of dtd in bacteria and yeast are viable, but lead to increased toxicity of D-amino acids [38]. Similarly, inhibitors of Plasmodium DTD enhance the toxicity of D-amino acids, suggesting that D-amino acids from the host cell would indeed present a potential challenge for the parasites [40]. Recently, the structure of the P. falciparum DTD enzyme (PfDTD) was solved, shedding light on the mechanism for its deacylation reaction [40,41]. The availability of a PfDTD structure and the inhibitory nature of drugs modeled on PfDTD suggest that hydrolysis of misacylated tRNA should be explored further as a novel and unique drug target. Ribosomes The ribosome is a large complex of macromolecules that manufactures proteins in all eukaryotic and prokaryotic cells. Substantial information on this molecular giant has been generated to atomic resolution, and its intricate mechanisms have been unraveled using X-ray crystallography and electron microscopy in model organisms [42]. Very little has been published on the protein members of the Plasmodium ribosomes, but the rRNA has received considerable attention, and has several features not found in other eukaryotes. As with other plastid-bearing eukaryotes, Plasmodium possesses three sets of rRNAs: cytoplasmic rRNAs, encoded in the nucleus and responsible for the majority of protein synthesis, as well as mitochondrial and plastid rRNAs that are encoded by the corresponding genomes and are responsible for protein synthesis in these organelles. Apicomplexan parasites contain rRNA gene units that are comprised of 18S, 28S, and 5.8S genes separated by the internal transcribed spacer regions and flanked by external transcribed spacer regions [43–48]. Many apicomplexan parasites contain a small number of structurally distinct rRNA gene units that are dispersed in their genomes. These rRNA gene units are transcribed in a stage-specific manner, thereby reducing the total number of simultaneously active gene units [43–48]. Based on

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differences in expression patterns and differences in the rRNA sequences, the existence of three structurally different ribosome types has been proposed. These are referred to as A-type (asexual liver and blood stages), O-type (ookinete) and S-type (mosquito sporozoite stages). Controlling expression of different sets of rRNA genes could help the parasite to regulate ribosomes as a function of the host environment in which the parasite finds itself [49]. In addition to the nuclear genome, both the apicoplast and mitochondrial genomes contain rDNA sequences [9,50,51]. The latter appear to be polycistronically transcribed and, as with the cytosolic forms, appear to be developmentally regulated [52]. Initiation, elongation and release factors All translation factors required for initiation, elongation and release of the polypeptide chain in the cytosol, apicoplast and mitochondrion are encoded by the parasite nucleus, with the exception of the apicoplast EF-Tu that is encoded by the apicoplast genome. Most of the translation factors required for cytosolic translation are currently annotated in the P. falciparum genome (Table 2), however, the majority of their functional interactions await confirmation. Among these cytosolic factors, mRNA cap-binding by eIF-4E has been reported in P. falciparum, and also interactions of eIF-4G with eIF-4E, eIF-4A (PfH45), and poly(A)-binding protein, PABP [53–55]. Identification of native protein interactions in the parasite have indicated contacts between the phosphoprotein eEF-1b and the G protein eEF-1a (the functional homologs of prokaryotic EF-Ts and EF-Tu, respectively) with two additional proteins, eEF-1d and eEF-1g, also associated with eEF-1b [56,57]. We have been unable to find an eIF4B coding sequence in the P. falciparum genome; however, the sequence of this molecule is poorly conserved between distant eukaryotes. The translation initiation factor, eIF5A from P. falciparum, contains the unique amino acid hypusine and has been shown to be post-translationally modified by parasite deoxyhypusine synthase and hydroxylase [58–60]. The presence of an active translation machinery is established in the P. falciparum apicoplast [5], and a case for mitochondrial translation in apicomplexans has been made [12,22]. Putative prokaryotic-like initiation factors for both of these organelles are apparent in the genome, in some cases with clear targeting presequences (Table 2). Recent data show that peptide chain elongation in the apicoplast is facilitated by plastid-encoded EF-Tu and nuclear-encoded EF-Ts, presumably in conjunction with an apicoplast-targeted EF-G [61]. The nuclear-encoded EF-Tu and an EF-G are predicted to contain mitochondrial transit peptides, but mitochondrial EF-Ts is lacking in all Plasmodium species. In prokaryotes, two release factors RF-1 and RF-2 decode the stop codons UAA/UAG and UAA/UGA, respectively [62]. Apicoplast open reading frames (ORFs) terminate with either UAA or UGA. The apicoplast RF-2 candidate (PFD0480w) has a strong targeting prediction but lacks a conserved stop-codon-specificity recognition domain. Thus, the plastid RF-1 candidate (PF14_0265), with a PXN (Pro-x-Asn) motif replacing the conserved bacterial PXT (Pro-x-Thr) codon recognition motif, could 471

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Table 2. P. falciparum apicoplast and mitochondrion translation factorsa Translation process Initiation

Elongation

Termination

Ribosome recycling

Factor IF-1 IF-2

Apicoplast PF14_0658 b PFE0830c

IF-3 EF-Tu EF-Ts EF-G RF-1 RF-2 RF-3 RRF

MAL8P1.27 Apicoplast-encoded (X95276) PFC0225c PFF0115c PF14_0265 PFD0480w Not found PFB0390w

Mitochondrion Not found d PF08_0018 PF13_0069 (only cytb has an AUG start codon and can require tRNAfMet for initiation) Not found MAL13P1.164 Not found PFL1590c MAL7P1.20 Not found Not found

a

Organellar assignment is in nearly all cases predicted from possession of appropriate trafficking signals. The apicoplast EF-Tu is encoded on the apicoplast genome, and the localization of the nuclear-encoded apicoplast EF-Ts has been verified [61].

b

Systematic gene designation.cSome mitochondria lack an IF-1

function with an altered decoding specificity for UAA/UGA. Each of the three mitochondrial ORFs terminates at a UAA stop codon, and a single RF (MAL7P1.20) could therefore suffice for peptide chain release in this organelle. A prokaryotic RF-3, which would normally help RF1 and RF2 to dissociate from the ribosome, is lacking in many bacteria [63], and is not apparent in the P. falciparum genome. Apicoplast genes, transcribed as mono- or polycistrons, have short intergenic spaces and lack the purine-rich prokaryotic ribosome-binding Shine–Dalgarno (SD) sequence upstream of their AUG codons, with the corresponding apicoplast sequences being generally A/T-rich. This is similar to the green alga Euglena gracilis where a group of chloroplast mRNAs lacking the SD sequence are characterized by a 50 -UTR with a high A/T content [64]. The complementary pyrimidine-rich anti-SD sequence at the 30 end of the bacterial small ribosomal subunit, 16S rRNA, is also replaced by an A/T-rich sequence in the apicoplastencoded ssu16S rRNA. Similar to mammalian mitochondrial genes that have very short 50 -UTRs, the three P. falciparum mitochondrial mRNAs do not use the SD interaction to recognize the start codon and presumably have other mechanisms for interaction with the ribosome and fragmented rRNA of the organelle for selection of the correct AUG. Control and regulation of translation A large number of proteomic studies have examined individual life stages of Plasmodium, or compared transitions from one stage to another (reviewed in [65]). Although these studies show shifts in protein composition between stages or treatments, the lack of corresponding transcriptome analyses for the same parasites makes it unclear whether expression regulation takes place at the transcriptional, translational or post-translational levels. The earliest mRNA microarrays indicate that the timing of mRNA production is generally well correlated with known timing of protein function [66]. This suggests that Plasmodium transcripts are produced as needed, or just before the time they are required to be translated. Despite a strong correlation between transcriptome and proteome, several studies 472

have found a significant delay between maximum mRNA abundance and peak protein expression, indicating that post-transcriptional regulation is involved [67–71]. During asexual replication in the erythrocyte, consensus motifs in the 5’- and 3’-UTRs are proposed to be involved in posttranscriptional regulation of gene expression [67,69], however, very few transcriptional regulators have been identified so far. This absence has lead to theories that Plasmodium transcription is ‘hard-wired’ [72] and regulation of expression depends on post-transcriptional and post-translational mechanisms such as proteolytic cleavage, glycosylation, phosphorylation, myristoylation, acetylation and ubiquitination [73]. Recent studies on Plasmodium eIF2a and its interacting kinases have shed some light on specific post-transcriptional mechanisms regulating the initiation of translation. The Plasmodium eIF2a kinase, IK1, was demonstrated to mediate the response to amino acid starvation by phosphorylating eIF2a [74]. Intriguingly, the Plasmodium salivary gland sporozoites have been successful at exploiting the eIF2a kinase, IK2, classically associated with stress responses in other eukaryotes, to repress translation. This IK2-mediated repression of translation maintains the parasites in a latent state in preparation for differentiation into liver stages [75]. In recent years there have been major developments in our understanding of translation regulators during zygote development. Translation repression in P-granules has been implicated in the regulation of over 370 proteins during the sexual development of Plasmodium. Mair and colleagues [68,71] have demonstrated that transcripts produced in the gametocyte stage are silenced and stabilized in P-granules with the aid of the conserved DEAD-box RNA helicase, DOZI, and the Sm-like factor, CITH, in preparation for translation following gamete fertilization. It is noteworthy that the cores of Plasmodium P-granules do not contain any of the RNA degradation factors annotated in the genome (e.g. XRN1, Lsm1–7, UPF1–3, DCP1 and 2) [71]. This suggests Plasmodium P-granules are not mRNA degradation depots, but are predominantly involved in stable storage of translationally repressed transcripts to be co-coordinated for transcription following fertilization.

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Drug targets Translation is a longstanding and major focus for drug development in Plasmodium. The relevant enzymes in the apicoplast and mitochondria in particular are attractive targets for malaria chemotherapy due to their prokaryotic origin. The promise of anti-translation inhibitors as antimalarials is highlighted by the number of inhibitors of protein translation already used for the clinical treatment of malaria (Table 3), of which doxycycline is perhaps the most widely known. Despite its potency and efficacy, doxycycline and other tetracyclines, and also clindamycin, exhibit an inhibition behavior known as the ‘delayed-death’ phenomenon [76]. In delayed death, parasites show no growth inhibition in the first asexual cycle during treatment, but die in the second asexual cycle, even if drug is washed out in the first cycle. This effect is observed for some but not all translation-blocking inhibitors [77,78]. Although the cause of delayed death is unknown, indirect evidence suggests that delayed-death inhibitors target the apicoplast. For example, tetracyclines block expression of the apicoplast genome, producing defects in apicoplast genome replication, apicoplast segregation and protein trafficking into the apicoplast [76]. Delayed death raises concerns about using apicoplast inhibitors as antimalarials; however, in combination these compounds are effective against malaria. Two combination therapies that each include one inhibitor of apicoplast translation, fosmidomycin/clindamycin [79] and azithromycin/chloroquine [80], are currently in Phase III clinical trials. Clindamycin and azithromycin inhibit by binding at

the peptide exit tunnel formed by the interaction of the 23s rRNA with the 50s ribosomal subunit. Some antibiotics that attack ribosomal RNA rather than ribosomal proteins themselves [42,81] have antimalarial activity. Specifically, the ribosome-blocking antibiotics telithromycin and quinupristin–dalfopristin target the parasite apicoplast and result in delayed death, suggesting inhibition of apicoplast translation [82]. Recent P. berghei studies show that probable apicoplast translation inhibitors (azithromycin, clindamycin and doxycycline) also block development of the apicoplast during exo-erythrocytic schizogony in the liver, leading to impaired parasite maturation [83]. Azithromycin also inhibits of apicoplast replication in different stages of Plasmodium berghei (blood, liver and mosquito) [84]. These data strengthen the rationale for prophylactic use of such drugs. The Plasmodium translation systems have a number of other potentially interesting drug targets, namely translation elongation factors, tRNA synthetases and S-adenosylmethionine-decarboxylase. For example, amythiamicin-A and kirromycin are effective against EF-Tu and demonstrate anti-malarial activity [61,85] and the EF-G inhibitor, fusidic acid, also inhibits P. falciparum growth [86]. In addition to organellar translation factors, proteins involved in post-translational modification of cytosolic translation initiation and elongation factors have been proposed as targets for antimalarials. Plasmodial S-adenosyl-methionine-decarboxylase, required for biosynthesis of spermidine and a precursor for hypusine synthesis on eIF-5A, and deoxyhypusine hydroxylase have been

Table 3. Molecular targets of some known inhibitors of Plasmodium protein translation Target Apicoplast LSU 23s rRNA

Compounds Thiostrepton Clindamycin

Comments Thiostrepton has been shown to bind 23s rRNA [89,90] Clindamycin-resistant Toxoplasma and Plasmodium have mutations in the small subunit of the apicoplast rRNA [91], although it has not been established that these mutations are causal for Plasmodium [92]. Clindamycin has been used in humans in combination with fosmidomycin [79] Azithromycin The polypeptide exit tunnel formed by the 23s rRNA, ribosomal proteins L4 and L22 is thought to be the site of action of azithromycin. Mutations in apicoplast 23s rRNA confer azithromycin resistance [93] Chloramphenicol Chloramphenicol has some delayed death effect [94] but has not been specifically shown to block apicoplast translation Telithromycin Telithromycin has some delayed death effect [82] but has not been specifically shown to block apicoplast translation Quinupristin–dalfopristin The quinupristin–dalfopristin combination has some delayed death effect [82] but has not been specifically shown to block apicoplast translation The polypeptide exit tunnel of the ribosome formed by the 23s rRNA, ribosomal Azithromycin Ribosomal protein L4 (PfRpl4) proteins L4 and L22 is thought to be the site of action of azithromycin. Mutations in apicoplast targeted L4 confer azithromycin resistance [93] The polypeptide chain formed by the 23s rRNA, ribosomal proteins L4 and L22 is Ribosomal protein L22 (PfRpl22) Azithromycin thought to be the site of action of Azithromycin [93] Tetracyclines The tetracycline antibiotic doxycycline is used as an antimalarial prophylactic and Apicoplast 16s rRNA inhibits translation in the apicoplast [76,78,95]. To our knowledge tetracyclines have not formally been demonstrated to inhibit via interaction with 16s rRNA, but this is the probable site of action Mutations in EF-Tu bestow resistance to amythiamicin (EF-Tu is encoded by the Amythiamicin Apicoplast EF-Tu apicoplast genome) [85] Kirromycin Kirromycin-EF-Tu complex observed [85] Actinonin Actinonin is assumed to inhibit apicoplast translation via inhibition of deformylation Peptide deformylase of the initiator fMet [25] Treatment with tetracycline was found to lead to decreased transcription of Tetracyclines Mitochondrial SSU rRNA apicoplast-encoded genes as well as mitochondrion-encoded transcripts [95]. Tetracycline also modifies mitochondrial morphology [78]

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Review identified as potential targets [60,87]. A serine/threonine protein phosphatase 2C that regulates nucleotide exchange activity of EF-1b in P. falciparum has also been suggested as a candidate chemotherapeutic target [88]. Unanswered questions Plasmodium has three translationally active compartments, the nucleus, apicoplast and mitochondrion, each with distinct evolutionary origins. These origins are reflected in differing mechanisms controlling translation, presenting three separate biological stories, and three potential sets of targets for inhibition. Several aspects of these stories remain to be revealed: ribosomal proteins for each compartment are generally poorly annotated, and the recent structural elucidation of ribosomes in model organisms provides a template to improve the organization and understanding of Plasmodium ribosomal proteins. The Plasmodium translation initiation, elongation and release factors are also incompletely understood. Several components appear to be missing, and more work will be required to clarify whether or how translation proceeds in their absence. Mitochondrial translation in particular remains a black box. We have no understanding of how tRNAs used in the mitochondria are charged, of how charged tRNAs are imported, nor what happens to these tRNAs after use. Indeed, direct evidence for translation in mitochondria is absent. The mitochondrion of Plasmodium could be the target of important inhibitors of bacterial-type translation, but the lack of a direct mitochondrial translation marker complicates the assessment of the effects of inhibitors on this organelle, and thus impedes the development of future inhibitors. Future work on these topics should provide not only a better understanding of how protein translation proceeds in a eukaryote very distant from well-characterized models, but also opportunities for drug development on the most solidly established of anti-infective drug targets. Acknowledgments The authors are funded by an European Union FP7 Collaborative Project Grant HEALTH-F3-2009-223024 – Mephitis. S.A.R. is funded by Australian Research Council Future fellowship FT0990350. The authors acknowledge and apologize to the authors of many important studies in this field whose findings had to be omitted or referred to only via review papers owing to space constraints.

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