Annette Kaiser

Translational control of eIF5A in various diseases

Translational control is a crucial component in the development and progression of different diseases. Translational control may involve selective translation of specific mRNAs, which promote cell proliferation or lead to alterations in translation factor levels and activities. Eukaryotic initiation factor 5A (eIF5A) is the only known protein to contain the unusual amino acid hypusine [Nε- (4-amino-2-hydroxybutyl)-lysine], which is formed from the polyamine spermidine by two catalytic steps. eIF5A is involved in translation, elongation and stimulating peptide bond formation. Hypusination of eIF5A is essential for its activity in promoting cell proliferation. Meanwhile, there is evidence that eIF5A is a key protein in the pathogenicity of different diseases, such as diabetes, several human cancers, malaria and HIV-1 infections. Hitherto, the available data suggest that eIF5A has a role of a cell context-dependent function being more proliferative in the case of several human cancers and being involved under stress conditions in diabetes. Secondly, in HIV-1 infections and in diabetes, eIF5A also has a nuclear function by its sequence-specific binding of mRNAs as an mRNA-shuttle in conjunction with nuclear membrane export proteins. This binding may also influence the half-lives of mRNAs or their sequestration. Based on these data, there is a considerable therapeutic interest in eIF5A as a selective target for drug development through inhibition of hypusination.

In Vitro and In Vivo Antimalarial Activity Assays of Seeds from Balanites aegyptiaca: Compounds of the Extract Show Growth Inhibition and Activity against Plasmodial Aminopeptidase.

Balanites aegyptiaca (Balanitaceae) is a widely grown desert plant with multiuse potential. In the present paper, a crude extract from B. aegyptiaca seeds equivalent to a ratio of 1 : 2000 seeds to the extract was screened for antiplasmodial activity. The determined IC50 value for the chloroquine-susceptible Plasmodium falciparum NF54 strain was 68.26 μg/μL ± 3.5. Analysis of the extract by gas chromatography-mass spectrometry detected 6-phenyl-2(H)-1,2,4-triazin-5-one oxime, an inhibitor of the parasitic M18 Aspartyl Aminopeptidase as one of the compounds which is responsible for the in vitro antiplasmodial activity. The crude plant extract had a K i of 2.35 μg/μL and showed a dose-dependent response. After depletion of the compound, a significantly lower inhibition was determined with a K i of 4.8 μg/μL. Moreover, two phenolic compounds, that is, 2,6-di-tert-butyl-phenol and 2,4-di-tert-butyl-phenol, with determined IC50 values of 50.29 μM ± 3 and 47.82 μM ± 2.5, respectively, were detected. These compounds may contribute to the in vitro antimalarial activity due to their antioxidative properties. In an in vivo experiment, treatment of BALB/c mice with the aqueous Balanite extract did not lead to eradication of the parasites, although a reduced parasitemia at day 12 p.i. was observed.

In vitro and in vivo silencing of plasmodial dhs and eIf-5a genes in a putative, non-canonical RNAi-related pathway

BackgroundDeoxyhypusine synthase (DHS) catalyzes the first step in hypusine biosynthesis of eukaryotic initiation factor 5A (eIF-5A) in Plasmodium falciparum. Target evaluation of parasitic DHS has recently been performed with CNI-1493, a novel selective pro-inflammatory cytokine inhibitor used in clinical phase II for the treatment of Crohns disease. CNI-1493 prevented infected mice from experimental cerebral malaria by decreasing the levels in hypusinated eIF-5A and serum TNF, implicating a link between cytokine signaling and the hypusine pathway.Therefore we addressed the question whether either DHS itself or eIF-5A is required for the outcome of severe malaria. In a first set of experiments we performed an in vitro knockdown of the plasmodial eIF-5A and DHS proteins by RNA interference (RNAi) in 293 T cells. Secondly, transfection of siRNA constructs into murine Plasmodium schizonts was performed which, in turn, were used for infection.Results293 T cells treated with plasmodial DHS- and eIF-5A specific siRNAs or control siRNAs were analyzed by RT-PCR to determine endogenous dhs -and eIF-5A mRNA levels. The expressed DHS-shRNA and EIF-5A-shRNA clearly downregulated the corresponding transcript in these cells. Interestingly, mice infected with transgenic schizonts expressing either the eIF-5A or dhs shRNA showed an elevated parasitemia within the first two days post infection which then decreased intermittently. These results were obtained without drug selection. Blood samples, which were taken from the infected mice at day 5 post infection with either the expressed EIF-5A-shRNA or the DHS-shRNA were analyzed by RT-PCR and Western blot techniques, demonstrating the absence of either the hypusinated form of eIF-5A or DHS.ConclusionsInfection of NMRI mice with schizonts from the lethal P. berghei ANKA wildtype strain transgenic for plasmodial eIF-5A-specific shRNA or DHS-specific shRNA resulted in low parasitemia 2–9 days post infection before animals succumbed to hyperparasitemia similar to infections with the related but non-lethal phenotype P. berghei strain NK65. RT-PCR and Western blot experiments performed with blood from the transfected erythrocytic stages showed that both genes are important for the proliferation of the parasite. Moreover, these experiments clearly demonstrate that the hypusine pathway in Plasmodium is linked to human iNos induction.

Cloning, expression and functional activity of deoxyhypusine synthase from Plasmodium vivax.

BackgroundPlasmodium vivax is the most widespread human malaria parasite. However, genetic information about its pathogenesis is limited at present, due to the lack of a reproducible in vitro cultivation method. Sequencing of the Plasmodium vivax genome suggested the presence of a homolog of deoxyhypusine synthase (DHS) from P. falciparum, the key regulatory enzyme in the first committed step of hypusine biosynthesis. DHS is involved in cell proliferation, and thus a valuable drug target for the human malaria parasite P. falciparum. A comparison of the enzymatic properties of the DHS enzymes between the benign and severe Plasmodium species should contribute to our understanding of the differences in pathogenicity and phylogeny of both malaria parasites.ResultsWe describe the cloning of a 1368 bp putative deoxyhypusine synthase gene (dhs) sequence from genomic DNA of P. vivax PEST strain Salvador I (Accession number AJ549098) after touchdown PCR. The corresponding protein was expressed and functionally characterized as deoxyhypusine synthase by determination of its specific activity and cross-reactivity to human DHS on a Western blot.The putative DHS protein from P. vivax displays a FASTA score of 75 relative to DHS from rodent malaria parasite, P. yoelii, and 74 relative to that from the human parasite, P. falciparum strain 3D7. The ORF encoding 456 amino acids was expressed under control of IPTG-inducible T7 promoter, and expressed as a protein of approximately 50 kDa (theoretically 52.7 kDa) in E. coli BL21 DE3 cells. The N-terminal histidine-tagged protein was purified by Nickel-chelate affinity chromatography under denaturing conditions. DHS with a theoretical pI of 6.0 was present in both eluate fractions. The specific enzymatic activity of DHS was determined as 1268 U/mg protein. The inhibitor, N-guanyl-1, 7-diaminoheptane (GC7), suppressed specific activity by 36-fold. Western blot analysis performed with a polyclonal anti-human DHS antibody revealed cross-reactivity to DHS from P. vivax, despite an amino acid identity of 44% between the proteins.ConclusionWe identify a novel DHS protein in the more benign malaria parasite,P. vivax, on the basis of specific enzymatic activity, cross-reactivity with a polyclonal antibody against human DHS, and amino acid identity with DHS homologs from the rodent malaria parasite, P. yoelii, and human P. falciparum strains.

Piperidones with activity against Plasmodium falciparum

The increasing resistance of the malaria parasites has enforced new strategies of finding new drug targets. We have isolated two genes involved in spermidine metabolism, encoding deoxyhypusine synthase (DHS) and eukaryotic initiation factor 5A (eIF-5A) in the malaria parasites. eIF-5A is activated by the formation of the unusual amino acid hypusine. This process occurs in two steps. DHS transfers an aminobutyl moiety from the triamine spermidine to a specific lysine residue in the eIF-5A precursor protein to form deoxyhypusine. In a second step, deoxyhypusine hydroxylase (DHH), completes hypusine biosynthesis. We used DHH inhibitors, being effective in mammalian cells, to study an antiplasmodicidal effect in Plasmodium falciparum. Experiments with the antifungal drug ciclopiroxolamine, an α-hydroxypyridone, and the plant amino acid l-mimosine, a 4-pyridone, resulted in an antiplasmodial effect in vitro. Using mimosine as a lead structure, alkyl 4-oxo-piperidine 3-carboxylates were found to have the most efficient antiplasmodial effects in vitro and in vivo.

Completing the hypusine pathway in Plasmodium

In searching for new targets for antimalarials we investigated the biosynthesis of hypusine present in eukaryotic initiation factor‐5A (eIF‐5A) in Plasmodium. Here, we describe the cloning and expression of deoxyhypusine hydroxylase (DOHH), which completes the modification of eIF‐5A through hydroxylation of deoxyhypusine. The dohh cDNA sequence revealed an ORF of 1236 bp encoding a protein of 412 amino acids with a calculated molecular mass of 46.45 kDa and an isoelectric point of 4.96. Interestingly, DOHH from Plasmodium has a FASTA SCORE of only 27 compared with its human ortholog and contains several matches similar to E‐Z‐type HEAT‐like repeat proteins (IPR004155 (InterPro), PF03130 (Pfam), SM00567 (SMART) present in the phycocyanin lyase subunits of cyanobacteria. Purified DOHH protein displayed hydroxylase activity in a novel in vitro DOHH assay, but phycocyanin lyase activity was absent. dohh is present as a single‐copy gene and is transcribed in the asexual blood stages of the parasite. A signal peptide at the N‐terminus might direct the protein to a different cellular compartment. During evolution, Plasmodium falciparum acquired an apicoplast that lost its photosynthetic function. It is possible that plasmodial DOHH arose from an E/F‐type phycobilin lyase that gained a new role in hydroxylation.

RNAi in Plasmodium.

RNA interference (RNAi) has quickly proven to be an immensely useful tool for studying gene function and validation of potential drug targets in almost all organisms that possess the required set of proteins of the interference pathway. In protozoan parasites like Plasmodium, Toxoplasma, Entamoeba, Giardia, Trypanosoma, and Leishmania, this set of enzymes is represented divergently. Hitherto, no RNAi-related genes like Dicer and Argonaute have been identified in Plasmodium and Leishmania species, respectively. However, non-canonical RNAi-related pathways might be present in both parasites, as it has been recently demonstrated in Plasmodium. In this review, we discuss existing challenges and future directions for developing RNAi as a tool for studying gene function and as a possible clinical application against Plasmodium.

A Putative Non-Canonical Ras-Like GTPase from P. falciparum: Chemical Properties and Characterization of the Protein

During its development the malaria parasite P. falciparum has to adapt to various different environmental contexts. Key cellular mechanisms involving G-protein coupled signal transduction chains are assumed to act at these interfaces. Heterotrimeric G-proteins are absent in Plasmodium. We here describe the first cloning and expression of a putative, non-canonical Ras-like G protein (acronym PfG) from Plasmodium. PfG reveals an open reading frame of 2736 bp encoding a protein of 912 amino acids with a theoretical pI of 8.68 and a molecular weight of 108.57 kDa. Transcript levels and expression are significantly increased in the erythrocytic phase in particular during schizont and gametocyte formation. Most notably, PfG has GTP binding capacity and GTPase activity due to an EngA2 domain present in small Ras-like GTPases in a variety of Bacillus species and Mycobacteria. By contrast, plasmodial PfG is divergent from any human alpha-subunit. PfG was expressed in E. coli as a histidine-tagged fusion protein and was stable only for 3.5 hours. Purification was only possible under native conditions by Nickel-chelate chromatography and subsequent separation by Blue Native PAGE. Binding of a fluorescent GTP analogue BODIPY® FL guanosine 5’O-(thiotriphosphate) was determined by fluorescence emission. Mastoparan stimulated GTP binding in the presence of Mg2+. GTPase activity was determined colorimetrically. Activity expressed as absolute fluorescence was 50% higher for the human paralogue than the activity of the parasitic enzyme. The PfG protein is expressed in the erythrocytic stages and binds GTP after immunoprecipitation. Immunofluorescence using specific antiserum suggests that PfG localizes to the parasite cytosol. The current data suggest that the putitative, Ras-like G-protein might be involved in a non-canonical signaling pathway in Plasmodium. Research on the function of PfG with respect to pathogenesis and antimalarial chemotherapy is currently under way.

A suggested vital function for eIF‐5A and dhs genes during murine malaria blood‐stage infection

The biological function of the post‐translational modification hypusine in the eukaryotic initiation factor 5A (EIF‐5A) in eukaryotes is still not understood. Hypusine is formed by two sequential enzymatic steps at a specific lysine residue in the precursor protein EIF‐5A. One important biological function of EIF‐5A which was recently identified is the translation of polyproline‐rich mRNA, suggesting its biological relevance in a variety of biological processes. Hypusinated eIF‐5A controls the proliferation of cancer cells and inflammatory processes in malaria. It was shown that pharmacological inhibition of the enzymes involved in this pathway, deoxyhypusine synthase (DHS) and the deoxyhypusine hydroxylase (DOHH), arrested the growth of malaria parasites. Down‐regulation of both the malarial eIF‐5A and dhs genes by in vitro and in vivo silencing led to decreased transcript levels and protein expression and, in turn, to low parasitemia, confirming a critical role of hypusination in eIF‐5A for proliferation in Plasmodium. To further investigate whether eIF‐5A and the activating enzyme DHS are essential for Plasmodium erythrocytic stages, targeted gene disruption was performed in the rodent malaria parasite Plasmodium berghei. Full disruption of both genes was not successful; instead parasites harboring the episome for eIF‐5A and dhs genes were obtained, suggesting that these genes may perform vital functions during the pathogenic blood cell stage. Next, a knock‐in strategy was pursued for both endogenous genes eIF‐5A and dhs from P. berghei. The latter resulted in viable recombinant parasites, strengthening the observation that they might be essential for proliferation during asexual development of the malaria parasite.