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Molecular & Biochemical Parasitology 187 (2013) 21– 31

Contents lists available at SciVerse ScienceDirect

Molecular & Biochemical Parasitology

eview

nique posttranslational modifications in eukaryotic translation factors and theiroles in protozoan parasite viability and pathogenesis

imisha Mittala, Gowri Subramaniana, Peter Bütikoferb, Rentala Madhubalaa,∗

School of Life Sciences, Jawaharlal Nehru University, New Delhi, IndiaInstitute for Biochemistry and Molecular Medicine, University of Bern, Switzerland

r t i c l e i n f o

rticle history:eceived 5 July 2012eceived in revised form 4 November 2012ccepted 5 November 2012vailable online 28 November 2012

eywords:ukaryotic elongation factor 1Aukaryotic initiation factor 5Aukaryotic elongation factor 2

a b s t r a c t

Protozoan parasites are one of the major causes of diseases worldwide. The vector transmitted parasitesexhibit complex life cycles involving interactions between humans, protozoa, and arthropods. In orderto adapt themselves to the changing microenvironments, they have to undergo complex morphologicaland metabolic changes. These changes can be brought about by expressing a new pool of proteins in thecell or by modifying the existing repertoire of proteins via posttranslational modifications (PTMs). PTMsinvolve covalent modification and processing of proteins thereby modulating their functions. Some ofthese changes may involve PTMs of parasite proteins to help the parasite survive within the host and thevector. Out of many PTMs known, three are unique since they occur only on single proteins: ethanolaminephosphoglycerol (EPG) glutamate, hypusine and diphthamide. These modifications occur on eukaryotic

thanolamine phosphoglycerolypusineiphthamiderotozoan parasites

elongation factor 1A (eEF1A), eukaryotic initiation factor 5A (eIF5A) and eukaryotic elongation factor 2(eEF2), respectively. Interestingly, the proteins carrying these unique modifications are all involved inthe elongation steps of translation. Here we review these unique PTMs, which are well conserved inprotozoan parasites, and discuss their roles in viability and pathogenesis of parasites. Characterizationof these modifications and studying their roles in physiology as well as pathogenesis will provide newinsights in parasite biology, which may also help in developing new therapeutic interventions.

© 2012 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222. Hypusine modification of eIF5A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2.1. Functional significance of eIF5A and hypusine modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242.2. Hypusine modification of eIF5A in protozoan parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

3. Diphthamide modification of eEF2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.1. Functional significance of eEF2 and diphthamide modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.2. Diphthamide modification of eEF2 in protozoan parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

4. EPG modification of eEF1A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 284.1. Functional significance of eEF1A and EPG modification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294.2. EPG modification of eEF1A in protozoan parasites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

5. Concluding remarks and future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Appendix A. Supplementary data. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: eEF1A, eukaryotic elongation factor 1A; eIF5A, eukaryotic initiation facDHS, deoxyhypusine synthase; DOHH, deoxyhypusine hydroxylase.∗ Corresponding author. Tel.: +91 11 26742630; fax: +91 11 26742630.

E-mail address: madhubala@mail.jnu.ac.in (R. Madhubala).

166-6851/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.molbiopara.2012.11.001

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

tor 5A; eEF2, eukaryotic elongation factor 2; EPG, ethanolamine phosphoglycerol;

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. Introduction

The genomic content of every organism is limited, yet the cod-ng capacity, i.e. the corresponding proteome, is very diverse. A cellas two basic ways to diversify its proteome: first, at the trans-riptional level via alternate splicing and trans-splicing of mRNAsnd second, through posttranslational modifications (PTMs) of pro-eins. The regulation of gene expression in kinetoplastids takeslace at multiple levels, such as trans-splicing, polyadenylation,RNA stability, transcript elongation, RNA translation and pro-

ein stability. Transcription in trypanosomatids generates largeolycistronic transcripts, which are processed to monocistronicRNAs by polyadenylation and trans-splicing of a mini-exon, or

plice-leader, to primary transcripts. As a result, regulation ofene expression in kinetoplastids takes place mainly at the post-ranscriptional level.

PTMs refer to a broad array of covalent modifications androcessing of proteins. These modifications can be reversible or

rreversible. Reversible modifications include the specific attach-ent of small molecules, such as phosphate, acetate, ADP-ribose,MP, methyl and hydroxyl groups to specific amino acid residuesf a given protein. These modifications may not only affect the bio-hemical properties of proteins but also modulate their functions,ocalization, turnover and interactions with other macromolecules.hus, PTMs increase the functional diversity of a given protein,hereby adding a layer of complexity to the proteome in eukaryotes

nd to a limited extent in prokaryotes [1,2].

Among many protein modifications known till date, threef them are exceptional as they occur only on single proteins:thanolamine phosphoglycerol (EPG) glutamate, hypusine and

ig. 1. Translation elongation cycle. (1) eEF1A along with GTP escorts specific aminoacubunits and a peptidyl tRNA at the P site. Following GTP hydrolysis, the aa-tRNA is tranhe growing polypeptide from peptidyl tRNA to aa-tRNA. (3) eEF2A bound to GTP assists

ssist eEF2A in this process. The peptidyl tRNA moves from A to P site. (4) Uncharged tRhree post-translational modifications of elongation factors are: (A) EPG modification ofiphthamide is involved in maintaining the reading frame during translocation. The role

al Parasitology 187 (2013) 21– 31

diphthamide modifications of eukaryotic elongation factor 1A(eEF1A), eukaryotic initiation factor 5A (eIF5A) and eukaryotic elon-gation factor 2 (eEF2), respectively. Interestingly, all three proteinsare involved in the elongation step of translation in eukaryotes[3]. Translation is a basic event required by the cell to make pro-teins. Therefore, homologs of translation factors involved in thisprocess can be found in eukaryotes, archaea and bacteria. Trans-lation involves three main steps: (i) initiation, (ii) elongation, and(iii) termination (Fig. 1). During initiation, a 43S pre-initiation com-plex is formed comprising of the 40S subunit, eIF2-GTP, Met-tRNAi,and eIF3. This is joined by cap binding complex of eIF4F and thefactors eIF4A and eIF4B which further assist the complex in bind-ing mRNA, thus forming the 48S complex. This complex associateswith the 60S large ribosomal subunit to form the 80S ribosome [4],which has a mettRNA bound at its P site. The process starts with acognate aminoacyl tRNA brought to the A site of the ribosome inform of a ternary complex with eEF1A and GTP. Following hydrol-ysis of the bound GTP to GDP, eEF1A is released, while the growingpolypeptide is transferred from the P site tRNA to the aminoacyltRNA at the A site. The peptidyl tRNA is then translocated from theA to the P site by GTP-bound eEF2, whereby eIF5A has been sug-gested to assist eEF2 in translocation. This cycle repeats until theribosome reaches the stop codon where the process stalls, resultingin termination of elongation (Fig. 1).

The three translation factors eEF1A, eEF2 and eIF5A are phyloge-netically well conserved [3]. The eukaryotic translation elongation

factor complex consists of three or four subunits: eEF1A, eEF1B�, eEF1 �, eEF1B � and eEF1B �. It is a GTP-binding complex,where GTP acts as a positive allosteric regulator of eEF1A [5]. Theintrinsic GTPase activity of eEF1A is very low but is enhanced by

yl tRNA (aa-tRNA) to the ribosome bound with mRNA at the interface of the twosferred to the A site of the large subunit. (2) Peptidyl transferase reaction transfersin translocation of the ribosome, followed by GTP hydrolysis. eIF5A is supposed toNA exits the ribosome from the E site. (5) Ribosome ready to start another cycle.

eEF1A. (B) Hypusine modification of eIF5A. (C) Diphthamide modification of eEF2.s of hypusine and EPG in translation are not known.

N. Mittal et al. / Molecular & Biochemical Parasitology 187 (2013) 21– 31 23

Fig. 2. Post-translational modification of eIF5A showing hypusine formation (a) Schematic representation of hypusine biosynthesis on eIF5A. (b) Predicted structure of yeaste nation

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IF5A (PDB:3ER0) showing the conserved lysine residue (Lys51) involved in hypusi

factor of 105 after binding to the ribosome [6]. eEF1A is mod-fied at several amino acid side chains by phosphorylation [7],ysine methylation [8] and EPG addition. eEF2 is also a GTPase,

hich in its GTP bound form catalyses the co-ordinated move-ent of the two tRNA molecules, the mRNA and conformational

hanges in the ribosome, following which its GTPase activity is acti-ated. The diphthamide modification located at the tip of domainV of eEF2 helps in the maintenance of the reading frame [9]. Inddition, eEF2 is phosphorylated by calcium-dependent kinases,eading to down-regulation of the protein [10]. eIF5A, initiallynown as eIF4D, is well conserved from yeasts to humans andontains a lysine residue that is modified to hypusine. In addition,nother lysine residue can be acetylated, resulting in inactivation ofIF5A [11].

A diverse group of protozoan parasites represents a major causef diseases in humans. Usually, these parasites have a digenetic lifeycle, where one form of the parasite resides in an insect vector andhe other infective form within the human host. Due to their com-lex life cycles, parasites have to undergo dramatic morphologicalnd metabolic changes to adapt to the changing extracellular envi-onments in the hosts and vectors. At least some of these changesay involve PTMs of parasitic proteins to help the parasite sur-

ive within the host and the vector [12]. The above-mentionedodifications of eEF1A, eEF2 and eIF5A are well conserved in pro-

ozoan parasites. This review describes the biosynthetic pathwaysnd physiological significance of these unique modifications inrotozoan parasites and discusses their possible roles in parasiteiology.

. Hypusine modification of eIF5A

eIF5A is a small, acidic protein that is highly conserved inrchaea and eukaryotes [13]. It is the only protein carrying thepecific polyamine-derived amino acid hypusine [N�-(4-amino-2-ydroxybutyl) lysine]. Hypusine was first identified in bovine brainxtracts [14] and, subsequently, as modified amino acid in eIF4D15,16]. Hypusine is synthesized post-translationally in a two step

rocess (Fig. 2a): first, deoxyhypusine synthase (DHS) catalyses theransfer of a 4-aminobutyl moiety from spermidine to the � aminoroup of a specific lysine residue of eIF5A, in a NAD+-dependenteaction, thus forming the intermediate deoxyhypusine. In a second

in ball and stick model (white color).

step, deoxyhypusine is hydroxylated to hypusine by deoxyhypu-sine hydroxylase (DOHH) [13].

eIF5A as well as the two enzymes, DHS and DOHH, are wellconserved among eukaryotes. In addition, a significant amino acidsequence identity is observed between eukaryotic eIF5A and itsorthologs, aIF5A and EF-P, in archaebacteria and eubacteria, respec-tively [13]. Crystal structures have been determined and structuralmodeling has been done for yeast and human eIF5A (PDB 3er0,3cpf), four aIF5A proteins (PDB 1eif, 2eif, 1iz6, 1bkb), and twoLeishmania proteins (PDB 1x6o, 1xtd), showing only minor differ-ences between organisms [17]. eIF5A has two domains, a basicN-terminal and an acidic C-terminal domain. Hypusine modifica-tion takes place on a conserved lysine residue (Lys50 in humans)in the N-terminal domain. Structure of yeast eIF5A (PDB: 3ER0)with the conserved lysine residue (Lys51) involved in hypusinationis shown in ball and stick model in Fig. 2b. Amino acid substi-tution and site directed mutagenesis studies have demonstratedthat the lysine residue is a strict requirement for hypusination totake place [18]. Multiple alignments of eIF5A sequences from var-ious organisms reveal that there is a high degree of amino acidsequence conservation, especially among the residues forming thehypusination loop [19]. The fact that hypusine is restricted to eIF5Aand that the two enzymes required for hypusine modification areconserved, implies a vital role of eIF5A and hypusine in the cell[20].

Early studies on rabbit reticulocytes identified eIF5A as trans-lation initiation factor [21,22]. More recently, the role of eIF5Ahas been implicated directly in translation elongation. eIF5A wasshown to interact with structural components of 80S ribosomes andeEF2 [23]. Polysome profiles from temperature sensitive mutantsof yeast for eIF5A revealed an increase in polysome/monosomeratio at the restrictive temperature, suggesting a block in trans-lation elongation. The profile was similar to that in wild type cellstreated with sordarin, an inhibitor of eEF2, suggesting that eIF5Amight function together with eEF2 to promote ribosomal translo-cation [24]. Moreover, a strain harboring both an eIF5A mutantand a translation initiation (eIF4E) mutant showed an interme-diate polysome profile phenotype, further supporting the role of

eIF5A at the elongation step of translation [25]. Furthermore, inde-pendent evidence for a role of eIF5A in translation elongation wasobtained by measuring the ribosome transit time (the time duringwhich a growing nascent polypeptide chain remains attached to

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he translating ribosome, i.e. the time of elongation plus the timef termination). The ribosome transit was significantly delayed forifferent eIF5A temperature-sensitive mutants at the restrictiveemperature [24], supporting the notion that eIF5A is involved inhe elongation step of translation.

.1. Functional significance of eIF5A and hypusine modification

In general, eukaryotes contain two or more isoforms of eIF5A26]. S. cerevisiae encodes two isoforms, TIF51A and TIF51B, whichhare ∼90% amino acid sequence identity. Both of them are of simi-ar size, but are regulated by heme and oxygen in opposite manner,hus showing differential expression under aerobic and anaerobiconditions [27]. Contrary to this, in mammals eIF5A1 is constitu-ively expressed in all tissues, while the expression of eIF5A2 isimited to testis and some parts of brain [26].

Hypusination affects the subcellular localization of eIF5A. Whileon-hypusinated eIF5A is equally distributed between the nucleusnd cytoplasm, the hypusinated form is exclusively present in theytoplasm. Furthermore, mutant eIF5A having various amino acidubstitutions at Lys50 shows a subcellular distribution profile sim-lar to non-hypusinated form [28].

eIF5A and its hypusine modification are essential for growthf yeast. Null mutants of eIF5A in yeast were not able to sur-ive [18]. Similarly, null mutants of eIF5A carrying a plasmid with

TIF51A gene having a Lys51Arg point mutation were not ableo grow since they lacked hypusinated eIF5A [18]. In mammals,IF5A2 is up-regulated in many cancers [29], hence considered asn oncogene whereas eIF5A1 is highly expressed in proliferatingells. Expression pattern of eIF5A1 shows that it is ubiquitouslyxpressed in mice during all post-implantation days [30]. In addi-ion, when CHO cells were treated with mono guanyl diamine GC7, aHS inhibitor, it curtailed the growth of cells without depleting the

permidine or eIF5A precursor levels, demonstrating an essentialole of hypusine in cellular proliferation [31]. Nishimura et al. [79]urther investigated the role of eIF5A in mammalian development,here they showed its essentiality in embryogenesis. eIF5A has

able 1andidate proteins involved in hypusine biosynthesis in L. infantum, T. brucei gambiense a

al Parasitology 187 (2013) 21– 31

also been shown to have pro-apoptotic function. Over-expressionof eIF5A1 induced apoptosis in colon carcinoma cells [32] via mito-chondrial apoptotic pathway. Overproduction and accumulation ofunmodified eIF5A precursor (but not the hypusinated form) upontransduction with eIF5A adeno-viral vectors has been implicated inapoptosis [33].

2.2. Hypusine modification of eIF5A in protozoan parasites

Like in all other eukaryotes, the hypusine pathway is well con-served in kinetoplastids and apicomplexans. Synthesis of hypusineis one way by which polyamines are covalently incorporated inproteins. Polyamines (spermidine, putrescine and spermine) arepresent at high concentrations in many protozoan parasites, wherethey are required for essential functions and viability. There areseveral reports highlighting the importance of polyamines in para-sites and their potential roles as drug targets (reviewed in [34]).These studies have been done by manipulating polyamine levelsusing various inhibitors of the enzymes involved in the polyaminepathway. In addition, treatment of neoplastic cells in culture with�-difluoromethylornithine, an inhibitor of ornithine decarboxylase(ODC), the first enzyme of this pathway, leads to cessation of cellgrowth due to depletion of putrescine and spermidine [35]. Thiscessation was due to depletion of hypusine synthesis resulting inaccumulation of immature eIF5A [36]. Together, these studies high-light the essential roles of polyamines and hypusine in parasitesurvival, development and pathogenesis.

Kinetoplastids are flagellated protozoans and include the genusTrypanosoma and Leishmania. Leishmania has two genes codingfor DHS, one on chromosome 34 (LinJ.34.0350) and one on chro-mosome 20 (LinJ.20.0270) (http://tritrypdb.org/tritrypdb/), withboth of them showing little sequence homology to human DHS(P49366). Pfam domain assignments of DHSL34 and DHSL20

show the presence of the deoxyhypusine synthase domain(http://pfam.sanger.ac.uk) (Table 1). Interestingly, only DHSL34exhibits deoxyhypusine synthase activity [37,38]. In Leishmania,chromosomal null mutants of DHSL34 could only be obtained in

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he presence of a rescuing episome that contained the DHSL34 gene,uggesting that DHSL34 is essential for L. donovani. These results aren line with a previous study showing that the hypusination path-

ay plays a vital role in L. donovani survival and proliferation [37].he second enzyme of this pathway, DOHH, has been character-zed in L. donovani (LinJ.26.1920) [38]. Pfam domain assignments ofOHH show two HEAT repeat domains (http://pfam.sanger.ac.uk)

Table 1). The DOHH inhibitor, ciclopirox, inhibited cell prolifer-tion of AG83 promastigotes as well as intracellular amastigotes,ndicating that DOHH inhibition might lead to decreased levels ofctive eIF5A in the cell, thus compromising growth of parasites.

An ODC null mutant of L. donovani lost the ability to sustainnfection. Parasitemia load in murine macrophages was reduced by0% compared to wild-type cells [39]. This reduced infectivity wasestored either by episomal complementation of the chromosomalesion in ODC [39] or by external administration of putrescine [40].imilarly, null mutants of spermidine synthase made the parasiteuxotrophic for polyamines. There was a substantial reduction inarasite burden in mice compared to the wild type, i.e. a reductiony 3 orders of magnitude in liver and 2 orders of magnitude in thepleen [41]. These experiments show that a reduction in polyamineiosynthetic capacity of the parasite results in decreased ability ofhe parasite to infect the mammalian host. The reduced virulencef the parasite can be correlated to reduced levels of putrescine andpermidine, ultimately affecting the levels of hypusinated eIF5A.

At present, little is known about eIF5A hypusination in try-anosomes. Candidate DHS genes have been annotated in theenomes of Trypanosoma brucei (Tb927.1.870) and Trypanosomaruzi (Tc00.1047053511421.60) (http://tritrypdb.org/tritrypdb)42]. In addition, two putative DHS-like sequences (Tb927.1.870nd Tb927.10.2750) have been identified in the genome of T.rucei 927 strain. Multiple amino acid sequence alignments of theutative protein sequences reveal that they share 38% and 37%equence identity to human DHS. At present we are characterizinghe proteins in trypanosomes.

In contrast, the entire hypusine synthetic pathwayas been characterized in Plasmodium. eIF5A (Q8MX76)http://www.uniprot.org/) was cloned by screening a cDNAibrary of P. falciparum Dd2 strain. The cDNA open reading frameORF) of 486 bp encodes a predicted protein of 161 amino acids43]. Unlike other eukaryotes, Plasmodium has only one geneor eIF5A, which is expressed at all erythrocytic phases of thearasite [43]. Quantitative time course analysis of relative proteinbundance for schizont-stage parasites (34–46 h) after invasionxhibited a 15-fold increase [44]. Two isoforms of eIF5A were iden-ified, showing a modest decrease in expression during schizontevelopment and correlating well with the mRNA levels with aelay. In addition, DHS (Q0KHM1) (http://www.uniprot.org/) from. vivax was cloned and functionally characterized. Amino acidequence alignment of Plasmodium DHS protein with homolo-ous sequence from human DHS showed 44% identity [45]. Thective site and the residue responsible for binding spermidinend mono guanyl diamine GC7 are highly conserved betweenuman and all Plasmodium DHS sequences. The DOHH (C9QNK6)http://www.uniprot.org/) cDNA sequence revealed an ORF of236 bp encoding a protein of 412 amino acids. DOHH proteinsrom Plasmodium and humans share only 27% sequence identity46].

Polyamines, the precursors of hypusine, are also importantor growth of malarial parasites. There are two key enzymesn the polyamine pathway, ODC, which forms putrescine, and-adenosylmethionine (SAM) decarboxylase, which decarboxy-

ates SAM to form the intermediate for subsequent productionf spermidine. This spermidine acts as the source of the 4-minobutyl moiety for hypusine formation. Parasites treated with-difluoromethylornithine had their development completely

al Parasitology 187 (2013) 21– 31 25

blocked, i.e. they were unable to transform from trophozoiteto schizont stage [47]. P. falciparum treated with the SAMdecarboxylase inhibitor, SAM486A, had reduced hypusine for-mation due to suppression of spermidine levels. The drug alsoprotected infected mice from cerebral malaria and prolonged thesurvival time in comparison to untreated mice. The parasitemiaload was also reduced to 10% after 8–10 days of treatment withthe inhibitor [48]. These findings indicate that reduced hypusina-tion of eIF5A affects the ability of the parasite to cause the disease.Similar results were obtained when chloroquine-susceptible strainNF54 and chloroquine-resistant R strain of P. falciparum weretreated with 1,7-diaminoheptane, a competitive inhibitor of DHS.The inhibitory effect of the compound was most prominent after48 h of treatment, which coincides with the observation that theactivity of DHS is maximal in rapidly proliferating cells due to onsetof nucleic acid synthesis, indicating that the effect of the inhibitorwas due to the inhibition of DHS activity [49].

Further characterization of the hypusine biosynthetic pathwayin kinetoplastids and Plasmodium would help in evaluating the roleof this pathway in virulence and pathogenesis.

3. Diphthamide modification of eEF2

Diphthamide (2-[3-carboxyamido-3-(trimethylammonio)-propyl] histidine) is a unique amino acid formed byposttranslational modification of a strictly conserved histi-dine residue on eEF2 or its archaeal ortholog, aEF2. Diphtheriatoxin from Corneybacterium diphtheriae and Pseudomonas exotoxinA target eEF2 by transfer of ADP-ribose from NAD+ to the diph-thamide residue of eEF2 [50]. This modification is well conserved inarchaea and eukaryotes, but absent from the eubacterial ortholog,EF-G, suggesting that eubacterial toxins have evolved a mecha-nism of specifically targeting diphthamide without inactivatingendogenous EF-G.

Biosynthesis of diphthamide involves a complex pathway,which includes six different enzymes acting in three steps (Fig. 3a).The first step involves the transfer of a 3-amino-3-carboxypropylgroup from SAM to the C-2 of the imidazole of a specific histi-dine residue (His699 in S. cerevisiae). This step is catalyzed bythe iron-sulfur cluster enzyme Dph2 in archaea while yeast andother eukaryotes require four enzymes (Dph1-4). The second step,involving trimethylation of the amino group resulting in a diph-thine intermediate, is catalyzed by diphthine synthase (Dph5)using SAM as methyl group donor. The last step involves the ATP-dependent amidation of the carboxylate group resulting in theformation of diphthamide and is catalyzed by diphthine-ammonialigase [51]. Out of the six enzymes involved, five have been identi-fied by screening of recessive yeast mutants and CHO cells whichwere resistant to DT [52]. These mutants are sorted into five com-plementation groups (Dph1, Dph2, Dph3, Dph4 and Dph5) in yeast[51] and three groups (CG-1, CG-2 and CG-3) in CHO cells. The lastenzyme involved in amidation was only recently identified in yeast(YBR246W or Dph7) by showing that a mutant of this ORF accu-mulated diphthine, suggesting that the product of this ORF maybe involved in the last step of diphthamide synthesis [52]. How-ever, this enzyme is not conserved in archaea and it contains WDdomains. Using integrated data-mining, de Crécy-Lagard et al. [53]have demonstrated that the DUF71/COG2102 family contains atleast two subfamilies, one of which was predicted to be the missingdiphthine-ammonia ligase (Dph6), the last enzyme in the diph-thamide biosynthesis pathway. Dph6 orthologs are present in all

archaea and eukaryotes.

eEF2 belongs to the family of GTP-binding proteins. It catalysesthe coordinated movement of the ribosome, mRNA and bound tRNAduring translation, thus maintaining the reading frame. Structure

26 N. Mittal et al. / Molecular & Biochemical Parasitology 187 (2013) 21– 31

Fig. 3. Post-translational modification of eEF2 showing diphthamide formation (a) proposed pathway of diphthamide biosynthesis showing all the three steps. Dph7 in yeastr ified hg n in grt .)

oimfiIynidicitttmflsi[

efers to YBR246W. (b) Structure of yeast EF2 (PDB: 1N0U). Post-translationally modreen sticks. The four domains GTP EFTU, GTP EFTU D2, EFG IV and EFG C are showo color in this figure legend, the reader is referred to the web version of this article

f yeast eEF2 (PDB:1NOU) with the conserved histidine involvedn diphthamide biosynthesis (His699) is shown in Fig. 3b. The

olecule has five structural domains with the diphthamide modi-cation being present at a histidine residue at the tip of domain

V [54]. Three dimensional cryo electron microscopy studies ofeast eEF2 revealed that the tip of domain IV lies close to the cog-ate tRNA. Domain IV reaches deeper into the ribosome, where it

nteracts with both the 40S and 60S subunit in the region of theecoding center [55]. Within the decoding center there are two

mportant adenine residues, A1492 and A1493, which are highlyonserved in helix 44 of both 16S and 18S rRNA. These two adeninesnteract with the first two base pairs of the codon anticodon helix,hereby allowing the ribosome to closely monitor the match andhe mismatch between the anticodon of the incoming tRNA andhe codon of the mRNA [56]. This interaction involves a confor-

ational cycle of stacked adenines in the absence of tRNA and

ipped bases in the presence of tRNA. Diphthamide is thought totabilize these adenines in their stacked position, thereby stabiliz-ng codon anticodon pairing, hence maintaining the reading frame57] and preventing -1 frame shifting during translation [58]. As a

istidine His699 residue is shown in white color. GTP binding residues are shown aseen, red, violet and yellow colors, respectively. (For interpretation of the references

consequence diphthamide modification promotes the progressionof the translation process.

3.1. Functional significance of eEF2 and diphthamidemodification

The exact role of diphthamide in cellular physiology is still anenigma, but the conservation of all six enzymes, Dph1-Dph6, inall eukaryotes suggests an important function for diphthamide.Yeast mutants of Dph1, Dph2, Dph4 and Dph5 show only subtlechanges in phenotypes when compared to wild type cells. OnlyDph3 mutant of yeast showed varied growth defects indicatingthat Dph3 might also have other roles in cellular physiology [59].In mammals, Dph1 was identified as OVCA1 [59,60], which is acandidate tumor suppressor gene whose deletion leads to ovarianmalignancies. In addition, it is required for embryonic and postna-

tal viability [61]. It shares ∼53% amino acid sequence identity withyeast Dph1. Mouse mutants of OVCA1 have a high risk of develop-ing tumors [61], suggesting a role for dph1 in tumorigenesis. Resultsfrom studies using Dph3 knock-out mice revealed that Dph3 may

N. Mittal et al. / Molecular & Biochemical Parasitology 187 (2013) 21– 31 27

Table 2Candidate proteins involved in diphthamide biosynthesis in L. infantum, T. brucei gambiense and P. falciparum 3D7.

btIdc

3

trhnpHspftcbDDasrtatH

e involved in development. Dph3 heterozygous mice were pheno-ypically normal but the null mutant showed embryonic lethality.n addition, Dph3 null mutants showed abnormalities in placentaevelopment, wherein there is a lack of fusion of allantois with thehorion [62].

.2. Diphthamide modification of eEF2 in protozoan parasites

eEF2 has been reported as a T-helper cell Th1-stimulatory pro-ein of Leishmania donovani, generating strong IFN-� and IL-12esponses in cured Leishmania-infected patients and protectingamsters against Leishmania challenge [63]. At present, there areo reports on diphthamide modification of eEF2 in protozoanarasites. The histidine residue of eEF2 (His715 in humans andis699 in yeast) carrying the diphthamide modification is con-

erved in eEF2 of kinetoplastids and Plasmodium, indicating thatrotozoan parasites may also make this modification. Our bioin-ormatic survey using Hidden Markov Models [64] (HMMs) againsthe L. infantum genome database shows that the genome containsandidate ORFs for all six enzymes involved in the diphthamideiosynthetic pathway, Dph1 (LinJ.29.1900), Dph2 (LinJ.33.2540),ph3 (LinJ.26.2660), Dph4 (LinJ.18.1470), Dph5 (LinJ.31.1640) andph6 (LinJ.25.0300) (Table 2). While LinJ.29.1900, LinJ.31.1640nd LinJ.18.1470 have already been annotated as diphthamideynthetase, diphthine synthase and chaperone DnaJ-like protein,espectively, in the Tritryp database, the hypothetical pro-

eins LinJ.33.2540, LinJ.26.2660 and LinJ.25.0300 have now beennnotated based on our bioinformatics analysis. While several pro-eins encoding DnaJ domains are present in L. infantum, PFAMMM-based domain assignments revealed the presence of a DnaJ

domain and a Zf-CSL zinc finger domain in LinJ.18.1470, suggestingthat it may represent Dph4 (Table 2). Furthermore, the last enzymein the diphthamide biosynthetic pathway in yeast, encoded byYBR246W (Dph7), contains WD repeats. However, a recent reportsuggests the presence of Dph6 which contains an ATP bindingdomain which is often accompanied by an Endoribonuclease L-PSPdomain at the C-terminus. Unlike yeasts, kinetoplastids (Leishmaniaand Trypanosoma) encode Dph6 (LinJ.25.0300 and Tbg972.11.240,respectively) containing an ATP binding domain. Similar can-didate genes are also present in the genome of P. falciparum(Table 2).

Multiple amino acid sequence alignments of Dph1-6 from L.infantum with T. brucei, T. cruzi, human and S. cerevisiae usingCLUSTALW [80] are shown as Suppl. Figs. 1–6. Multiple sequencealignment of Dph3 shows the four strictly conserved cysteines aswell as conservation of the “CSL” motif, confirming it being a CSLtype Zinc finger. Furthermore, all Dph4 homologs have a CSL-typeZinc finger domain tethered C-terminally to a DnaJ domain, whichcontains the conserved tripeptide “HPD” motif found in all func-tional DnaJ proteins. The four cysteines essential for zinc bindingand the “CSL” motif are conserved in the Zinc finger domain. Mul-tiple sequence alignment of Dph6 shows the conservation of the“SGGKDS” motif found in all PP-loop ATPases essential for ATPbinding. While the human homolog contains only the ATP bindingdomain, Leishmania and Trypanosoma Dph6 have an unassigned C-terminal domain. In contrast, the yeast homolog has an N-terminal

ATP binding domain and a C-terminal ribonuclease L-PSP (endori-bonuclease L-PSP) domain. In silico identification of the enzymesinvolved in the diphthamide modification of eEF2 may open up anentirely new area of research in kinetoplastids and apicomplexans.

28 N. Mittal et al. / Molecular & Biochemical Parasitology 187 (2013) 21– 31

Fig. 4. Post-translational modification of eEF1A showing EPG formation (a) theoretical structural model of human EF1A (PDBe:1SYW). The three domains GTP EFTU,GTP EFTU D2 and GTP EFTU D3 are shown in pink, orange and violet colors, respectively. Modified Glu residues (301 and 374) are shown in white color. GTP bindingr achmd

4

ob

esidues are shown as green ball and stick models. (b) Proposed pathway of EPG atteacylation to EPG.

. EPG modification of eEF1A

eEF1A is an essential protein involved in the elongation stepf peptide synthesis in all eukaryotes. It mediates GTP-dependentinding of aminoacylated tRNA to the acceptor site of a ribosome.

ent involving modification of eEF1A by phosphatidylethanolamine and subsequent

Following GTP hydrolysis, eEF1A dissociates from the ribosome

and is reactivated by nucleotide exchange factor eEF1B [65]. Theactivity of eEF1A is modulated by protein modifications, includ-ing phosphorylation [7], lysine methylation [8] and C-terminalmethyl esterification [66]. In addition, numerous reports indicate

hemic

toa

Iwgi[rislcpscfa

wemwbonioefaqs

4

imoilebli

4

dIbstttpsofeitd

N. Mittal et al. / Molecular & Bioc

hat eEF1A may fulfill various other functions in the cell, such asrganization of the cytoskeleton, regulation of protein degradation,nd viral propagation [67].

EPG is a unique protein modification attached to eEF1A.t was discovered when mammalian cells were labeled

ith [3H]ethanolamine, a precursor often used to identifylycosylphosphatidylinositol-anchored proteins. Subsequentsolation and chemical and mass spectrometric analyses of a3H]ethanolamine-labeled 49 kDa protein revealed that theadioactivity was not associated with a glycosylphosphatidylinos-tol structure but with a previously unknown EPG moiety [68]. Theites of EPG attachment were identified as two glutamate residuesocated in domains II and III of eEF1A from murine and humanells [68,69] (Fig. 4a). Subsequent studies in plants confirmed theresence of EPG in eEF1A and demonstrated that the modificationites were conserved between mammals and plants [70]. Inontrast, no evidence for EPG modification was found in eEF1Arom S. cerevisiae [8]. In addition, the EPG modification is absent inrchaeal or bacterial eEF1A orthologs (reviewed in [3]).

The pathways for EPG synthesis and attachment to eEF1A, asell as the enzymes involved in these events, have remained

lusive. An early study using a cell-free cytosolic extract from aurine lymphocyte cell line showed that free [3H] ethanolamineas incorporated into eEF1A, suggesting that EPG may be assem-

led stepwise [71]. However, since the modification lacked thether two components of the EPG moiety, glycerol and phosphate,o reaction sequence was proposed [71]. More recently, studies

n T. brucei procyclic forms demonstrated that the cellular poolf phosphatidylethanolamine, a major phospholipid class in mostukaryotic cells, represents the donor of the ethanolamine residueor eEF1A [72]. Based on this finding, a reaction sequence involvingttachment of phosphatidylethanolamine to eEF1A with subse-uent cleavage of the two glycerol-bound acyl chains has beenuggested (Fig. 4b).

.1. Functional significance of eEF1A and EPG modification

Besides its canonical role during protein synthesis, eEF1A is alsonvolved in many other cellular processes, such as binding to and

ediating interactions of cytoskeletal proteins [73], nuclear exportf proteins [74] and mitochondrial tRNA import [75]. Because ofts high amino acid sequence conservation among eukaryotes, it isikely that non-canonical roles of eEF1A are also conserved in otherukaryotes. Remarkably, although attachment of EPG to eEF1A haseen described for the first time more than 20 years ago, the bio-

ogical function of this unique modification has not been addressedn mammalian cells and plants.

.2. EPG modification of eEF1A in protozoan parasites

So far, EPG attachment to eEF1A in parasites has only beenemonstrated in T. brucei procyclic and bloodstream forms [72].

nterestingly, and in contrast to mammalian and plant eEF1A, T.rucei eEF1A was found to contain a single EPG attached to a con-erved glutamate residue, Glu362, in domain III [72]. More recently,rypanosomes have been used as model eukaryote to investigatehe role of EPG in eEF1A function. Amino acid point mutations ofhe EPG attachment site, Glu362, of T. brucei eEF1A, were shown torevent EPG linkage, even when Glu was replaced by Asp, demon-trating that EPG attachment is strictly dependent on the presencef Glu [76]. In addition, conditional expression in T. brucei procyclicorms of an EPG-deficient form of eEF1A, after down-regulation of

ndogenous eEF1A, demonstrated that attachment of EPG to eEF1As not essential for growth of parasites in culture [77]. However,he fact that the EPG glutamate modification has been preserveduring eukaryotic evolution, occurring in mammals, plants and

[

[

al Parasitology 187 (2013) 21– 31 29

protozoa, suggests that it may provide a clear advantage, or rep-resent an essential function, for the organisms under certainconditions. Interestingly, in a recent report, attachment of EPG wasshown to be evolutionary conserved, i.e. human eEF1A expressedin T. brucei procyclic forms was modified with EPG [78].

5. Concluding remarks and future prospects

We have discussed the unique modifications of eIF5A, eEF2and eEF1A and their roles in viability and pathogenesis of proto-zoan parasites. All three proteins are important factors requiredfor the basic cell survival process of translation. Hence, they arewell conserved among eukaryotes, including parasitic protozoa.The hypusine modification of eIF5A and its synthesis have beenwell characterized in kinetoplastids and Plasmodium. Various knockout studies have shown that the protein as well as its hypusinemodification are essential for parasite survival. The physiologicalfunctions of the other two modifications, diphthamide on eEF2 andEPG glutamate on eEF1A, have not been characterized in kineto-plastids and apicomplexans. Further studies on the roles of theseunique modifications in physiology as well as pathogenesis of pro-tozoan parasites will provide new insights in parasitic biology,which in turn may facilitate identification of new drug targets anddevelopment of novel therapeutic interventions.

Acknowledgements

The work on hypusine and diphthamide is supported by a grantfrom the Council of Scientific and Industrial Research, India, to R.M.Work in P.B.’s laboratory on EPG modification of eEF1A is supportedby grant 31003A-130815 from the Swiss National Science Foun-dation. P.B. wishes to thank R. Tedder for stimulation. N.M. is arecipient of funding from the Council of Scientific and IndustrialResearch. V.S.G. is a Kothari post-doctoral fellow supported by theUniversity Grants Commission, India.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.molbiopara.2012.11.001.

References

[1] Seet BT, Dikic I, Zhou MM, Pawson T. Reading protein modifications with inter-action domains. Nature Reviews Molecular Cell Biology 2006;7:473–83.

[2] Ahrne E, Muller M, Lisacek F. Unrestricted identification of modified proteinsusing MS/MS. Proteomics 2010;10:671–86.

[3] Greganova E, Altmann M, Bütikofer P. Unique modifications of translation elon-gation factors. The FEBS Journal 2011;278:2613–24.

[4] Pestova TV, Kolupaeva VG, Lomakin IB, Pilipenko EV, Shatsky IN, Agol VI, et al.Molecular mechanisms of translation initiation in eukaryotes. Proceedings ofthe National Academy of Sciences 2001;98:7029–36.

[5] Proud CG. Peptide-chain elongation in eukaryotes. Molecular Biology Reports1994;19:161–70.

[6] Pape T, Wintermeyer W, Rodnina MV. Complete kinetic mechanism of elonga-tion factor Tu-dependent binding of aminoacyl-tRNA to the A site of the E. coliribosome. The EMBO Journal 1998;17:7490–7.

[7] Traugh JA. Insulin, phorbol ester and serum regulate the elongationphase of protein synthesis. Progress in Molecular and Subcellular Biology2001;26:33–48.

[8] Cavallius J, Zoll W, Chakraburtty K, Merrick WC. Characterization of yeast EF-1 alpha: non-conservation of post-translational modifications. Biochimica etBiophysica Acta 1993;1163:75–80.

[9] Ortiz PA, Ulloque R, Kihara GK, Zheng H, Kinzy TG. Translation elongation fac-tor 2 anticodon mimicry domain mutants affect fidelity and diphtheria toxin

resistance. The Journal of Biological Chemistry 2006;281:32639–48.

10] Ryazanov AG. Ca2+/calmodulin-dependent phosphorylation of elongation fac-tor 2. FEBS Letters 1987;214:331–4.

11] Lee SB, Park JH, Folk JE, Deck JA, Pegg AE, Sokabe M, et al. Inactivation ofeukaryotic initiation factor 5A (eIF5A) by specific acetylation of its hypusine

3 hemic

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

0 N. Mittal et al. / Molecular & Bioc

residue by spermidine/spermine acetyltransferase 1 (SSAT1). Biochemical Jour-nal 2011;433:205–13.

12] Jortzik E, Wang L, Becker K. Thiol-based posttranslational modifications in para-sites. Antioxidants & Redox Signaling 2012;17:657–73.

13] Wolff EC, Kang KR, Kim YS, Park MH. Posttranslational synthesis of hypusine:evolutionary progression and specificity of the hypusine modification. AminoAcids 2007;33:341–50.

14] Shiba T, Mizote H, Kaneko T, Nakajima T, Kakimoto Y. Hypusine a new aminoacid occurring in bovine brain. Isolation and structural determination. Biochim-ica et Biophysica Acta 1971;244:523–31.

15] Park MH, Cooper HL, Folk JE. Identification of hypusine, an unusual amino acid,in a protein from human lymphocytes and of spermidine as its biosyntheticprecursor. Proceedings of the National Academy of Sciences 1981;78:2869–73.

16] Cooper HL, Park MH, Folk JE, Safer B, Braverman R. Identification of thehypusine-containing protein hy+ as translation initiation factor eIF-4D.Proceedings of the National Academy of Sciences 1983;80:1854–7.

17] Tong Y, Park I, Hong BS, Nedyalkova L, Tempel W, Park HW. Crystal structure ofhuman eIF5A1: insight into functional similarity of human eIF5A1 and eIF5A2.Proteins 2009;75:1040–5.

18] Schnier J, Schwelberger HG, Smit-McBride Z, Kang HA, Hershey JW. Transla-tion initiation factor 5A and its hypusine modification are essential for cellviability in the yeast Saccharomyces cerevisiae. Molecular and Cellular Biology1991;11:3105–14.

19] Zanelli CF, Valentini SR. Is there a role for eIF5A in translation. Amino Acids2007;33:351–8.

20] Park MH, Nishimura K, Zanelli CF, Valentini SR. Functional significance of eIF5Aand its hypusine modification in eukaryotes. Amino Acids 2010;38:491–500.

21] Benne R, Hershey JW. The mechanism of action of protein synthesis initi-ation factors from rabbit reticulocytes. The Journal of Biological Chemistry1978;253:3078–87.

22] Kemper WM, Berry KW, Merrick WC. Purification and properties of rabbit retic-ulocyte protein synthesis initiation factors M2Balpha and M2Bbeta. The Journalof Biological Chemistry 1976;251:5551–7.

23] Zanelli CF, Maragno AL, Gregio AP, Komili S, Pandolfi JR, Mestriner CA,et al. eIF5A binds to translational machinery components and affects trans-lation in yeast. Biochemical and Biophysical Research Communications2006;348:1358–66.

24] Saini P, Eyler DE, Green R, Dever TE. Hypusine-containing protein eIF5A pro-motes translation elongation. Nature 2009;459:118–21.

25] Gregio AP, Cano VP, Avaca JS, Valentini SR, Zanelli CF. eIF5A has a function in theelongation step of translation in yeast. Biochemical and Biophysical ResearchCommunications 2009;380:785–90.

26] Jenkins ZA, Haag PG, Johansson HE. Human eIF5A2 on chromosome 3q25-q27is a phylogenetically conserved vertebrate variant of eukaryotic translationinitiation factor 5A with tissue-specific expression. Genomics 2001;71:101–9.

27] Mehta KD, Leung D, Lefebvre L, Smith M. The ANB1 locus of Saccharomycescerevisiae encodes the protein synthesis initiation factor eIF-4D. The Journal ofBiological Chemistry 1990;265:8802–7.

28] Lee SB, Park JH, Kaevel J, Sramkova M, Weigert R, Park MH. The effect of hypu-sine modification on the intracellular localization of eIF5A. Biochemical andBiophysical Research Communications 2009;383:497–502.

29] Guan XY, Fung JM, Ma NF, Lau SH, Tai LS, Xie D, et al. Oncogenic role of eIF-5A2in the development of ovarian cancer. Cancer Research 2004;64:4197–200.

30] Parreiras-e-Silva LT, Luchessi AD, Reis RI, Oliver C, Jamur MC, Ramos RG, et al.Evidences of a role for eukaryotic translation initiation factor 5A (eIF5A) inmouse embryogenesis and cell differentiation. Journal of Cellular Physiology2010;225:500–5.

31] Park MH, Wolff EC, Lee YB, Folk JE. Antiproliferative effects of inhibitorsof deoxyhypusine synthase. Inhibition of growth of Chinese hamster ovarycells by guanyl diamines. The Journal of Biological Chemistry 1994;269:27827–32.

32] Taylor CA, Sun Z, Cliche DO, Ming H, Eshaque B, Jin S, et al. Eukaryotic transla-tion initiation factor 5A induces apoptosis in colon cancer cells and associateswith the nucleus in response to tumour necrosis factor alpha signalling. Exper-imental Cell Research 2007;313:437–49.

33] Sun Z, Cheng Z, Taylor CA, McConkey BJ, Thompson JE. Apoptosis induction byeIF5A1 involves activation of the intrinsic mitochondrial pathway. Journal ofCellular Physiology 2010;223:798–809.

34] Kaiser A, Ulmer D, Goebel T, Holzgrabe U, Saeftel M, Hoerauf A. Inhibition ofhypusine biosynthesis in plasmodium: a possible, new strategy in preventionand therapy of malaria. Mini-Reviews in Medicinal Chemistry 2006;6:1231–41.

35] Pegg AE. Polyamine metabolism and its importance in neoplastic growth anda target for chemotherapy. Cancer Research 1988;48:759–74.

36] Gerner EW, Mamont PS, Bernhardt A, Siat M. Post-translational modification ofthe protein-synthesis initiation factor eIF-4D by spermidine in rat hepatomacells. Biochemical Journal 1986;239:379–86.

37] Chawla B, Jhingran A, Singh S, Tyagi N, Park MH, Srinivasan N, et al. Identifi-cation and characterization of a novel deoxyhypusine synthase in Leishmaniadonovani. The Journal of Biological Chemistry 2010;285:453–63.

38] Chawla B, Kumar RR, Tyagi N, Subramanian G, Srinivasan N, Park MH, et al.A unique modification of the eukaryotic initiation factor 5A shows the pres-

ence of the complete hypusine pathway in Leishmania donovani. PLoS ONE2012;7:e33138.

39] Boitz JM, Yates PA, Kline C, Gaur U, Wilson ME, Ullman B, et al. Leishmaniadonovani ornithine decarboxylase is indispensable for parasite survival in themammalian host. Infection and Immunity 2009;77:756–63.

[

[

al Parasitology 187 (2013) 21– 31

40] Olenyik T, Gilroy C, Ullman B. Oral putrescine restores virulence of ornithinedecarboxylase-deficient Leishmania donovani in mice. Molecular and Biochem-ical Parasitology 2011;176:109–11.

41] Gilroy C, Olenyik T, Roberts SC, Ullman B. Spermidine synthase is required forvirulence of Leishmania donovani. Infection and Immunity 2011;79:2764–9.

42] El-Sayed NM, Hegde P, Quackenbush J, Melville SE, Donelson JE. The Africantrypanosome genome. International Journal for Parasitology 2000;30:329–45.

43] Molitor IM, Knobel S, Dang C, Spielmann T, Allera A, Konig GM. Translation ini-tiation factor eIF-5A from Plasmodium falciparum. Molecular and BiochemicalParasitology 2004;137:65–74.

44] Foth BJ, Zhang N, Mok S, Preiser PR, Bozdech Z. Quantitative proteinexpression profiling reveals extensive post-transcriptional regulation andpost-translational modifications in schizont-stage malaria parasites. GenomeBiology 2008;9:R177.

45] Njuguna JT, Nassar M, Hoerauf A, Kaiser AE. Cloning expression and functionalactivity of deoxyhypusine synthase from Plasmodium vivax. BMC Microbiology2006;6:91.

46] Frommholz D, Kusch P, Blavid R, Scheer H, Tu JM, Marcus K, et al. Com-pleting the hypusine pathway in Plasmodium. The FEBS Journal 2009;276:5881–91.

47] Assaraf YG, Golenser J, Spira DT, Bachrach U. Polyamine levels and the activityof their biosynthetic enzymes in human erythrocytes infected with the malarialparasite, Plasmodium falciparum. Biochemical Journal 1984;222:815–9.

48] Blavid R, Kusch P, Hauber J, Eschweiler U, Sarite SR, Specht S, et al. Down-regulation of hypusine biosynthesis in Plasmodium by inhibition of S-adenosyl-methionine-decarboxylase. Amino Acids 2010;38:461–9.

49] Kaiser A, Gottwald A, Wiersch C, Lindenthal B, Maier W, Seitz HM. Effect ofdrugs inhibiting spermidine biosynthesis and metabolism on the in vitro devel-opment of Plasmodium falciparum. Parasitology Research 2001;87:963–72.

50] Liu S, Milne GT, Kuremsky JG, Fink GR, Leppla SH. Identification of theproteins required for biosynthesis of diphthamide, the target of bacterial ADP-ribosylating toxins on translation elongation factor 2. Molecular and CellularBiology 2004;24:9487–97.

51] Chen JY, Bodley JW. Biosynthesis of diphthamide in Saccharomycescerevisiae. Partial purification and characterization of a specific S-adenosylmethionine:elongation factor 2 methyltransferase. The Journalof Biological Chemistry 1988;263:11692–6.

52] Chen JY, Bodley JW, Livingston DM. Diphtheria toxin-resistant mutants of Sac-charomyces cerevisiae. Molecular and Cellular Biology 1985;5:3357–60.

53] de Crecy-Lagard V, Forouhar F, Brochier-Armanet C, Tong L, Hunt JF. Com-parative genomic analysis of the DUF71/COG2102 family predicts roles indiphthamide biosynthesis and B12 salvage. Biology Direct 2012;7:32.

54] Jorgensen R, Merrill AR, Andersen GR. The life and death of translation elonga-tion factor 2. Biochemical Society Transactions 2006;34:1–6.

55] Gomez-Lorenzo MG, Spahn CM, Agrawal RK, Grassucci RA, Penczek P,Chakraburtty K, et al. Three-dimensional cryo-electron microscopy localiza-tion of EF2 in the Saccharomyces cerevisiae 80S ribosome at 17.5 A resolution.The EMBO Journal 2000;19:2710–8.

56] Ogle JM, Brodersen DE, Clemons Jr WM, Tarry MJ, Carter AP, RamakrishnanV. Recognition of cognate transfer RNA by the 30S ribosomal subunit. Science2001;292:897–902.

57] Jorgensen R, Merrill AR, Yates SP, Marquez VE, Schwan AL, Boesen T, et al.Exotoxin A-eEF2 complex structure indicates ADP ribosylation by ribosomemimicry. Nature 2005;436:979–84.

58] Liu S, Bachran C, Gupta P, Miller-Randolph S, Wang H, Crown D, et al. Diph-thamide modification on eukaryotic elongation factor 2 is needed to assurefidelity of mRNA translation and mouse development. Proceedings of theNational Academy of Sciences 2012;109:13817–22.

59] Phillips NJ, Ziegler MR, Radford DM, Fair KL, Steinbrueck T, Xynos FP, et al. Allelicdeletion on chromosome 17p13.3 in early ovarian cancer. Cancer Research1996;56:606–11.

60] Phillips NJ, Zeigler MR, Deaven LL. A cDNA from the ovarian cancer criticalregion of deletion on chromosome 17p13.3. Cancer Letters 1996;102:85–90.

61] Chen CM, Behringer RR. Ovca1 regulates cell proliferation, embryonic develop-ment, and tumorigenesis. Genes and Development 2004;18:320–32.

62] Liu S, Wiggins JF, Sreenath T, Kulkarni AB, Ward JM, Leppla SH. Dph3 a small pro-tein required for diphthamide biosynthesis, is essential in mouse development.Molecular and Cellular Biology 2006;26:3835–41.

63] Kushawaha PK, Gupta R, Sundar S, Sahasrabuddhe AA, Dube A. Elonga-tion factor-2, a Th1 stimulatory protein of Leishmania donovani, generatesstrong IFN-gamma and IL-12 response in cured Leishmania-infectedpatients/hamsters and protects hamsters against Leishmania challenge. TheJournal of Immunology 2011;187:6417–27.

64] Hughey R, Krogh A. Hidden Markov models for sequence analysis: extensionand analysis of the basic method. Computer Applications in the Biosciences1996;12:95–107.

65] Andersen GR, Nissen P, Nyborg J. Elongation factors in protein biosynthesis.Trends in Biochemical Sciences 2003;28:434–41.

66] Zobel-Thropp P, Yang MC, Machado L, Clarke S. A novel post-translational mod-ification of yeast elongation factor 1A. Methylesterification at the C terminus.The Journal of Biological Chemistry 2000;275:37150–8.

67] Mateyak MK, Kinzy TG. eEF1A: thinking outside the ribosome. The Journal ofBiological Chemistry 2010;285:21209–13.

68] Whiteheart SW, Shenbagamurthi P, Chen L, Cotter RJ, Hart GW. Murineelongation factor 1 alpha (EF-1 alpha) is posttranslationally modified bynovel amide-linked ethanolamine-phosphoglycerol moieties. Addition of

hemic

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[

[

[

[

[

[

[

[

[

[

N. Mittal et al. / Molecular & Bioc

ethanolamine-phosphoglycerol to specific glutamic acid residues on EF-1alpha. The Journal of Biological Chemistry 1989;264:14334–41.

69] Rosenberry TL, Krall JA, Dever TE, Haas R, Louvard D, Merrick WC. Biosyntheticincorporation of [3H]ethanolamine into protein synthesis elongation factor 1alpha reveals a new post-translational protein modification. The Journal ofBiological Chemistry 1989;264:7096–9.

70] Ransom WD, Lao PC, Gage DA, Boss WF. Phosphoglycerylethanolamine post-translational modification of plant eukaryotic elongation factor 1alpha. PlantPhysiology 1998;117:949–60.

71] Whiteheart SW, Hart GW. Incorporation of [3H]ethanolamine into a singlecytosolic protein in a cell free system: ethanolaminylation of EF-1 alpha invitro. Archives of Biochemistry and Biophysics 1994;309:387–91.

72] Signorell A, Jelk J, Rauch M, Bütikofer P. Phosphatidylethanolamine is theprecursor of the ethanolamine phosphoglycerol moiety bound to eukaryoticelongation factor 1A. The Journal of Biological Chemistry 2008;283:20320–9.

73] Gross SR, Kinzy TG. Improper organization of the actin cytoskeleton affects pro-tein synthesis at initiation. Molecular and Cellular Biology 2007;27:1974–89.

74] Khacho M, Mekhail K, Pilon-Larose K, Pause A, Cote J, Lee S. eEF1A is a novelcomponent of the mammalian nuclear protein export machinery. MolecularBiology of the Cell 2008;19:5296–308.

[

al Parasitology 187 (2013) 21– 31 31

75] Bouzaidi-Tiali N, Aeby E, Charriere F, Pusnik M, Schneider A. Elongation factor1a mediates the specificity of mitochondrial tRNA import in T. brucei. The EMBOJournal 2007;26:4302–12.

76] Greganova E, Heller M, Bütikofer P. A structural domain mediates attachmentof ethanolamine phosphoglycerol to eukaryotic elongation factor 1A in Try-panosoma brucei. PLoS ONE 2010;5:e9486.

77] Greganova E, Bütikofer P. Ethanolamine phosphoglycerol attachment to eEF1Ais not essential for normal growth of Trypanosoma brucei. Surface ScienceReports 2012;2:254.

78] Eltschinger S, Greganova E, Heller M, Bütikofer P, Altmann M. Eukaryotic trans-lation elongation factor 1A (eEF1A) domain I from S. cerevisiae is requiredbut not sufficient for inter-species complementation. PLoS ONE 2012;7:e42338.

79] Nishimura K, Lee SB, Park JH, Park MH. Essential role of eIF5A-1 anddeoxyhypusine synthase in mouse embryonic development. Amino Acids

2012;42:703–10.

80] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitiv-ity of progressive multiple sequence alignment through sequence weighting,position-specific gap penalties and weight matrix choice. Nucleic AcidsResearch 1994;22:4673–80.