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Abstract DHPS deficiency syndrome is an ultra-rare neurodevelopmental disorder (NDD) which results from biallelic mutations in the gene encoding the enzyme deoxyhypusine synthase (DHPS). DHPS is essential to synthesize hypusine, a rare amino acid formed by post-translational modification of a conserved lysine in eukaryotic initiation factor 5 A (eIF5A). DHPS deficiency syndrome causes epilepsy, cognitive and motor impairments, and mild facial dysmorphology. In mice, a brain-specific genetic deletion of Dhps at birth impairs eIF5AHYP-dependent mRNA translation. This alters expression of proteins required for neuronal development and function, and phenotypically models features of human DHPS deficiency. We studied the role of DHPS in early brain development using a zebrafish loss-of-function model generated by knockdown of dhps expression with an antisense morpholino oligomer (MO) targeting the exon 2/intron 2 (E2I2) splice site of the dhps pre-mRNA. dhps knockdown embryos exhibited dose-dependent developmental delay and dysmorphology, including microcephaly, axis truncation, and body curvature. In dhps knockdown larvae, electrophysiological analysis showed increased epileptiform activity, and confocal microscopy analysis revealed reduced arborisation of GABAergic neurons. Our findings confirm that hypusination of eIF5A by DHPS is needed for early brain development, and zebrafish with an antisense knockdown of dhps model features of DHPS deficiency syndrome.

DHPS deficiency syndrome is an ultra-rare neurodevelopmental disorder (NDD) which results from biallelic mutations in the gene encoding the enzyme deoxyhypusine synthase (DHPS). DHPS is essential to synthesize hypusine, a rare amino acid formed by post-translational modification of a conserved lysine in eukaryotic initiation factor 5 A (eIF5A). DHPS deficiency syndrome causes epilepsy, cognitive and motor impairments, and mild facial dysmorphology. In mice, a brain-specific genetic deletion of Dhps at birth impairs eIF5AHYP-dependent mRNA translation. This alters expression of proteins required for neuronal development and function, and phenotypically models features of human DHPS deficiency. We studied the role of DHPS in early brain development using a zebrafish loss-of-function model generated by knockdown of dhps expression with an antisense morpholino oligomer (MO) targeting the exon 2/intron 2 (E2I2) splice site of the dhps pre-mRNA. dhps knockdown embryos exhibited dose-dependent developmental delay and dysmorphology, including microcephaly, axis truncation, and body curvature. In dhps knockdown larvae, electrophysiological analysis showed increased epileptiform activity, and confocal microscopy analysis revealed reduced arborisation of GABAergic neurons. Our findings confirm that hypusination of eIF5A by DHPS is needed for early brain development, and zebrafish with an antisense knockdown of dhps model features of DHPS deficiency syndrome. [ABSTRACT FROM AUTHOR]

Trichomonas vaginalis, the causative agent of trichomoniasis, is a prevalent anaerobic protozoan parasite responsible for the most common nonviral sexually transmitted infection globally. While metronidazole and its derivatives are approved drugs for this infection, rising resistance necessitates the exploration of new antiparasitic therapies. Protein posttranslational modifications (PTMs) play crucial roles in cellular processes, and among them, hypusination, involving eukaryotic translation factor 5A (eIF5A), has profound implications. Despite extensive studies in various organisms, the role of hypusination in T. vaginalis and its potential impact on parasite biology and pathogenicity remain poorly understood. This study aims to unravel the structural basis of the hypusination pathway in T. vaginalis using X‐ray crystallography and cryo‐electron microscopy. The results reveal high structural homology between T. vaginalis and human orthologs, providing insights into the molecular architecture of eIF5A and deoxyhypusine synthase (DHS) and their interaction. Contrary to previous suggestions of bifunctionality, our analyses indicate that the putative hydroxylation site in tvDHS is nonfunctional, and biochemical assays demonstrate exclusive deoxyhypusination capability. These findings challenge the notion of tvDHS functioning as both deoxyhypusine synthase and hydroxylase. The study enhances understanding of the hypusination pathway in T. vaginalis, shedding light on its functional relevance and potential as a drug target, and contributing to the development of novel therapeutic strategies against trichomoniasis. [ABSTRACT FROM AUTHOR]

Rationale: The ubiquitous polyamine spermidine is essential for cell survival and proliferation. One important function of spermidine is to serve as a substrate for hypusination, a posttranslational modification process that occurs exclusively on eukaryotic translation factor 5A (eIF5A) and ensures efficient translation of various gene products. Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive obliteration of the small pulmonary arteries (PAs) caused by excessive proliferation of PA smooth muscle cells (PASMCs) and suppressed apoptosis. Objectives: To characterize the role of hypusine signaling in PAH. Methods: Molecular, genetic, and pharmacological approaches were used both in vitro and in vivo to investigate the role of hypusine signaling in pulmonary vascular remodeling. Measurements and Main Results: Hypusine forming enzymes—deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH)—and hypusinated eukaryotic translation factor 5A are overexpressed in distal PAs and isolated PASMCs from PAH patients and animal models. In vitro, inhibition of DHPS using N1-guanyl-1,7-diaminoheptane or shRNA resulted in a decrease in PAH-PASMC resistance to apoptosis and proliferation. In vivo, inactivation of one allele of Dhps targeted to smooth muscle cells alleviates PAH in mice, and its pharmacological inhibition significantly decreases pulmonary vascular remodeling and improves hemodynamics and cardiac function in two rat models of established PAH. With mass spectrometry, hypusine signaling is shown to promote the expression of a broad array of proteins involved in oxidative phosphorylation, thus supporting the bioenergetic requirements of cell survival and proliferation. Conclusions: These findings support inhibiting hypusine signaling as a potential treatment for PAH. [ABSTRACT FROM AUTHOR]

Pyrrolizidine alkaloids (PAs) are specialized metabolites that are produced by various plant families that act as defense compounds against herbivores. On the other hand, certain lepidopteran insects uptake and utilize these PAs as defense compounds against their predators and as precursors of their sex pheromones. Adult males of Parantica sita, a danaine butterfly, convert PAs into their sex pheromones. In early summer, P. sita swarms over the flowers of Myosotis scorpioides, which belongs to the family Boraginaceae. M. scorpioides produces PAs, but the organs in which PAs are produced and whether P. sita utilizes PAs in M. scorpioides are largely unknown. In the present study, we clarified that M. scorpioides accumulates retronecine-core PAs in N-oxide form in all organs, including flowers. We also identified two M. scorpioides genes encoding homospermidine synthase (HSS), a key enzyme in the PA biosynthetic pathway, and clarified that these genes are expressed in all organs where PAs accumulate. Phylogenetic analysis suggested that these two HSS genes were originated from gene duplication of deoxyhypusine synthase gene like other HSS genes in PA-producing plants. These results suggest that PAs are synthesized and accumulated in the flower of M. scorpioides and provide a possibility for a PA-mediated interaction between P. sita and M. scorpioides. [ABSTRACT FROM AUTHOR]

Salt stress is one of the most harmful stress types affecting our agriculture and food safety. Despite the extensive knowledge of plant responses induced by salt stress, the details of the regulation of metabolite-dependent posttranslational modifications occurring during salt stress have rarely been investigated. Hypusination is polyamine-dependent posttranslational modification of eukaryotic translation factor 5A (eIF5A), which is conserved in all living organisms. Some evidence suggests that this modification plays an important role in salt stress tolerance, but the significance of the enzymes participating in the synthesis of hypusine is unknown. GC7 (N1-guanyl-1,7-diaminoheptane) is a special compound which can inhibit the enzyme activity of deoxyhypusine synthase (DHS). In our study, we applied GC7 in order to examine the applicability and efficiency of this compound and to decipher the modulation of polyamine metabolism during salt stress in plants. Our results support the significance of DHS during salt stress and of GC7 as an efficient tool to study hypusination in plant species.

This study explores a non-kinase effect of extracellular regulated kinases 1/2 (ERK1/2) on the interaction between deoxyhypusine synthase (DHPS) and its substrate, eukaryotic translation initiation factor 5A (eIF5A). We report that Raf/MEK/ERK activation decreases the DHPS-ERK1/2 interaction while increasing DHPS-eIF5A association in cells. We determined the cryoelectron microscopy (cryo-EM) structure of the DHPS-ERK2 complex at 3.5 Å to show that ERK2 hinders substrate entrance to the DHPS active site, subsequently inhibiting deoxyhypusination in vitro. In cells, impairing the ERK2 activation loop, but not the catalytic site, prolongs the DHPS-ERK2 interaction irrespective of Raf/MEK signaling. The ERK2 Ser-Pro-Ser motif, but not the common docking or F-site recognition sites, also regulates this complex. These data suggest that ERK1/2 dynamically regulate the DHPS-eIF5A interaction in response to Raf/MEK activity, regardless of its kinase function. In contrast, ERK1/2 kinase activity is necessary to regulate the expression of DHPS and eIF5A. These findings highlight an ERK1/2-mediated dual kinase-dependent and -independent regulation of deoxyhypusination. [Display omitted] • Identification of a kinase-independent function of ERK1/2 • The structure of ERK2-protein complex addresses kinase-independent ERK2 function • The structure of DHPS complexed with a protein from outside of the hypusination pathway • The mechanism by which the direct DHPS and ERK1/2 interaction regulates hypusination Becker et al. determine the cryo-EM structure of the DHPS-ERK2 complex to show that ERK2 hinders DHPS active site entrance through an atypical interface. Raf/MEK/ERK pathway activation decreases DHPS-ERK1/2 interaction, thereby facilitating increased DHPS-eIF5A association. Kinase function is unnecessary for ERK1/2 to mediate this regulation. [ABSTRACT FROM AUTHOR]

In plants, homospermidine synthase (HSS) is a pathway‐specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA‐producing Convolvulaceae ("morning glories"), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function—the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species. [ABSTRACT FROM AUTHOR]

Abstract In plants, homospermidine synthase (HSS) is a pathway‐specific enzyme initiating the biosynthesis of pyrrolizidine alkaloids (PAs), which function as a chemical defense against herbivores. In PA‐producing Convolvulaceae (“morning glories”), HSS originated from deoxyhypusine synthase at least >50 to 75 million years ago via a gene duplication event and subsequent functional diversification. To study the recruitment of this ancient gene duplicate to PA biosynthesis, the presence of putative hss gene copies in 11 Convolvulaceae species was analyzed. Additionally, various plant parts from seven of these species were screened for the presence of PAs. Although all of these species possess a putative hss copy, PAs could only be detected in roots of Ipomoea neei (Spreng.) O'Donell and Distimake quinquefolius (L.) A.R.Simões & Staples in this study. A precursor of PAs was detected in roots of Ipomoea alba L. Thus, despite sharing high sequence identities, the presence of an hss gene copy does not correlate with PA accumulation in particular species of Convolvulaceae. In vitro activity assays of the encoded enzymes revealed a broad spectrum of enzyme activity, further emphasizing a functional diversity of the hss gene copies. A recently identified HSS specific amino acid motif seems to be important for the loss of the ancestral protein function—the activation of the eukaryotic initiation factor 5A (eIF5A). Thus, the motif might be indicative for a change of function but allows not to predict the new function. This emphasizes the challenges in annotating functions for duplicates, even for duplicates from closely related species.

The translation factor IF5A is highly conserved in Eukarya and Archaea and undergoes a unique post-translational hypusine modification by the deoxyhypusine synthase (DHS) enzyme. DHS transfers the butylamine moiety from spermidine to IF5A using NAD as a cofactor, forming a deoxyhypusine intermediate. IF5A is a key player in protein synthesis, preventing ribosome stalling in proline-rich sequences during translation elongation and facilitating translation elongation and termination. Additionally, human eIF5A participates in various essential cellular processes and contributes to cancer metastasis, with inhibiting hypusination showing anti-proliferative effects. The hypusination pathway of IF5A is therefore an attractive new therapeutic target. We elucidated the 2.0 Å X-ray crystal structure of the archaeal DHS-IF5A complex, revealing hetero-octameric architecture and providing a detailed view of the complex active site including the hypusination loop. This structure, along with biophysical data and molecular dynamics simulations, provides new insights into the catalytic mechanism of the hypusination reaction. [Display omitted] • Structural characterization of the archaeal aIF5A-aDHS complex • The complex presents a 4:4 (aIFA:aDHS) stoichiometry • The archaeal and human aIF5A-aDHS complexes are extremely conserved The research conducted by D'Agostino et al. unveils the high-resolution crystallographic structure of the archaeal protein complex IF5A-DHS revealing octameric architecture. The study offers a comprehensive insight into the enzyme's active site, responsible for initiating the hypusination reaction. [ABSTRACT FROM AUTHOR]

The treatment of a variety of protozoal infections, in particular those causing disabling human diseases, is still hampered by a lack of drugs or increasing resistance to registered drugs. However, in recent years, remarkable progress has been achieved to combat neglected tropical diseases by sequencing the parasites' genomes or the validation of new targets in the parasites by novel genetic manipulation techniques, leading to loss of function. The novel amino acid hypusine is a posttranslational modification (PTM) that occurs in eukaryotic initiation factor 5A (EIF5A) at a specific lysine residue. This modification occurs by two steps catalyzed by deoxyhypusine synthase (dhs) and deoxyhypusine hydroxylase (DOHH) enzymes. dhs from Plasmodium has been validated as a druggable target by small molecules and reverse genetics. Recently, the synthesis of a series of human dhs inhibitors led to 6-bromo-N-(1H-indol-4yl)-1-benzothiophene-2-carboxamide, a potent allosteric inhibitor with an IC50 value of 0.062 µM. We investigated this allosteric dhs inhibitor in Plasmodium. In vitro P. falciparum growth assays showed weak inhibition activity, with IC50 values of 46.1 µM for the Dd2 strain and 51.5 µM for the 3D7 strain, respectively. The antimalarial activity could not be attributed to the targeting of the Pfdhs gene, as shown by chemogenomic profiling with transgenically modified P. falciparum lines. Moreover, in dose-dependent enzymatic assays with purified recombinant P. falciparum dhs protein, only 45% inhibition was observed at an inhibitor dose of 0.4 µM. These data are in agreement with a homology-modeled Pfdhs, suggesting significant structural differences in the allosteric site between the human and parasite enzymes. Virtual screening of the allosteric database identified candidate ligand binding to novel binding pockets identified in P. falciparum dhs, which might foster the development of parasite-specific inhibitors. [ABSTRACT FROM AUTHOR]

Hypusine [Nε-(4-amino-2-hydroxybutyl)lysine] is a derivative of lysine that is formed post-translationally in the eukaryotic initiation factor 5A (eIF5A). Its occurrence at a single site in one cellular protein defines hypusine synthesis as one of the most specific post-translational modifications. Synthesis of hypusine involves two enzymatic steps: first, deoxyhypusine synthase (DHPS) cleaves the 4-aminobutyl moiety of spermidine and transfers it to the ε-amino group of a specific lysine residue of the eIF5A precursor protein to form an intermediate, deoxyhypusine [Nε-(4-aminobutyl)lysine]. This intermediate is subsequently hydroxylated by deoxyhypusine hydroxylase (DOHH) to form hypusine in eIF5A. eIF5A, DHPS, and DOHH are highly conserved in all eukaryotes, and both enzymes exhibit a strict specificity toward eIF5A substrates. eIF5A promotes translation elongation globally by alleviating ribosome stalling and it also facilitates translation termination. Hypusine is required for the activity of eIF5A, mammalian cell proliferation, and animal development. Homozygous knockout of any of the three genes, Eif5a, Dhps, or Dohh, leads to embryonic lethality in mice. eIF5A has been implicated in various human pathological conditions. A recent genetic study reveals that heterozygous germline EIF5A variants cause Faundes–Banka syndrome, a craniofacial–neurodevelopmental malformations in humans. Biallelic variants of DHPS were identified as the genetic basis underlying a rare inherited neurodevelopmental disorder. Furthermore, biallelic DOHH variants also appear to be associated with neurodevelopmental disorder. The clinical phenotypes of these patients include intellectual disability, developmental delay, seizures, microcephaly, growth impairment, and/or facial dysmorphisms. Taken together, these findings underscore the importance of eIF5A and the hypusine modification pathway in neurodevelopment in humans. [ABSTRACT FROM AUTHOR]

Summary: Mitochondrial function declines during brain aging and is suspected to play a key role in age-induced cognitive decline and neurodegeneration. Supplementing levels of spermidine, a body-endogenous metabolite, has been shown to promote mitochondrial respiration and delay aspects of brain aging. Spermidine serves as the amino-butyl group donor for the synthesis of hypusine (Nε-[4-amino-2-hydroxybutyl]-lysine) at a specific lysine residue of the eukaryotic translation initiation factor 5A (eIF5A). Here, we show that in the Drosophila brain, hypusinated eIF5A levels decline with age but can be boosted by dietary spermidine. Several genetic regimes of attenuating eIF5A hypusination all similarly affect brain mitochondrial respiration resembling age-typical mitochondrial decay and also provoke a premature aging of locomotion and memory formation in adult Drosophilae. eIF5A hypusination, conserved through all eukaryotes as an obviously critical effector of spermidine, might thus be an important diagnostic and therapeutic avenue in aspects of brain aging provoked by mitochondrial decline.

The treatment of a variety of protozoal infections, in particular those causing disabling human diseases, is still hampered by a lack of drugs or increasing resistance to registered drugs. However, in recent years, remarkable progress has been achieved to combat neglected tropical diseases by sequencing the parasites’ genomes or the validation of new targets in the parasites by novel genetic manipulation techniques, leading to loss of function. The novel amino acid hypusine is a posttranslational modification (PTM) that occurs in eukaryotic initiation factor 5A (EIF5A) at a specific lysine residue. This modification occurs by two steps catalyzed by deoxyhypusine synthase (dhs) and deoxyhypusine hydroxylase (DOHH) enzymes. dhs from Plasmodium has been validated as a druggable target by small molecules and reverse genetics. Recently, the synthesis of a series of human dhs inhibitors led to 6-bromo-N-(1H-indol-4yl)-1-benzothiophene-2-carboxamide, a potent allosteric inhibitor with an IC50 value of 0.062 µM. We investigated this allosteric dhs inhibitor in Plasmodium. In vitro P. falciparum growth assays showed weak inhibition activity, with IC50 values of 46.1 µM for the Dd2 strain and 51.5 µM for the 3D7 strain, respectively. The antimalarial activity could not be attributed to the targeting of the Pfdhs gene, as shown by chemogenomic profiling with transgenically modified P. falciparum lines. Moreover, in dose-dependent enzymatic assays with purified recombinant P. falciparum dhs protein, only 45% inhibition was observed at an inhibitor dose of 0.4 µM. These data are in agreement with a homology-modeled Pfdhs, suggesting significant structural differences in the allosteric site between the human and parasite enzymes. Virtual screening of the allosteric database identified candidate ligand binding to novel binding pockets identified in P. falciparum dhs, which might foster the development of parasite-specific inhibitors.

Deoxyhypusine synthase transfers an aminobutyl moiety from spermidine to the eukaryotic translation initiation factor 5A (eIF5A) in the first step of eIF5A activation. This exclusive post‐translational modification is conserved in all eukaryotes. Activated eIF5A has been shown to be essential for cell proliferation and viability. Recent reports have linked the activation of eIF5A to several human diseases. Deoxyhypusine synthase, which is encoded by a single gene copy in most eukaryotes, was duplicated in several plant lineages during evolution, the copies being repeatedly recruited to pyrrolizidine alkaloid biosynthesis. However, the function of many of these duplicates is unknown. Notably, deoxyhypusine synthase is highly promiscuous and can catalyze various reactions, often of unknown biological relevance. To facilitate in‐depth biochemical studies of this enzyme, we report here the development of a simple and robust in vitro enzyme assay. It involves precolumn derivatization of the polyamines taking part in the reaction and avoids the need for the previously used radioactively labeled tracers. The derivatized polyamines are quantified after high‐performance liquid chromatography coupled to diode array and fluorescence detectors. By performing kinetic analyses of deoxyhypusine synthase and its paralog from the pyrrolizidine alkaloid‐producing plant Senecio vernalis, we demonstrate that the assay unequivocally differentiates the paralogous enzymes. Furthermore, it detects and quantifies, in a single assay, the side reactions that occur in parallel to the main reaction. The presented assay thus provides a detailed biochemical characterization of deoxyhypusine synthase and its paralogs. [ABSTRACT FROM AUTHOR]

Abstract Background Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites that function as feeding deterrents in a range of different plant species. In perennial ryegrass (Lolium perenne L.) the only PAs that have been identified are the thesinine-rhamnoside group, which displays significant genetic variation. Homospermidine synthase (HSS) has evolved from deoxyhypusine synthase (DHS) and catalyses the first step in the PA pathway, making it a key candidate for the investigation of genes influencing observed PA trait variation. Results During PCR amplification and sequence analysis of DHS we identified two putative HSS genes in perennial ryegrass. One of the genes (LpHSS1) was absent in some perennial ryegrass plants. Thesinine-rhamnoside levels were measured using liquid chromatography coupled with mass spectrometry in a diverse association mapping population, consisting of 693 plants free of fungal endophytic symbionts. Association tests that accounted for population structure identified a significant association of absence of the LpHSS1 gene with lower levels of thesinine-rhamnoside PAs. HSS-like gene sequences were identified for other grass species of the Poaceae, including tall fescue, wheat, maize and sorghum. Conclusion HSS is situated at the crucial first step in the PA pathway making it an important candidate gene for investigation of involvement in PA phenotypic variation. In this study, PA level in perennial ryegrass was strongly associated with the presence or absence of the LpHSS1 gene. A genetic marker, developed for the presence/absence of LpHSS1, may be used for marker-assisted breeding to either lower or increase PAs in breeding populations of perennial or Italian ryegrass to investigate a potential role in the deterrence of herbivore pests. The presence of HSS-like genes in several other Poaceae species suggests that PA biosynthesis may occur in plant family members beyond perennial ryegrass and tall fescue and identifies a potential route for manipulating PA levels.

Background Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs. Methods Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling. Results Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated PfDHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) expression. PfDHS-attenuated parasites showed increased sensitivity to N1-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes. Discussion Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds.

Deoxyhypusine synthase (DHS) is encoded by a nuclear gene and is the key enzyme involved in the post-translational activation of the eukaryotic translation initiation factor eIF5A. DHS plays important roles in plant growth and development. To gain a better understanding of DHS, the petunia (Petunia hybrida) PhDHS gene was isolated, and the role of PhDHS in plant growth was analyzed. PhDHS protein was localized to the nucleus and cytoplasm. Virus-mediated PhDHS silencing caused a sectored chlorotic leaf phenotype. Chlorophyll levels and photosystem II activity were reduced, and chloroplast development was abnormal in PhDHS-silenced leaves. In addition, PhDHS silencing resulted in extended leaf longevity and thick leaves. A proteome assay revealed that 308 proteins are upregulated and 266 proteins are downregulated in PhDHS-silenced plants compared with control, among the latter, 21 proteins of photosystem I and photosystem II and 12 thylakoid (thylakoid lumen and thylakoid membrane) proteins. In addition, the mRNA level of PheIF5A-1 significantly decreased in PhDHS-silenced plants, while that of another three PheIF5As were not significantly affected in PhDHS-silenced plants. Thus, silencing of PhDHS affects photosynthesis presumably as an indirect effect due to reduced expression of PheIF5A-1 in petunia.Significance:PhDHS-silenced plants develop yellow leaves and exhibit a reduced level of photosynthetic pigment in mesophyll cells. In addition, arrested development of chloroplasts is observed in the yellow leaves.

Hypusination is a unique posttranslational modification of eIF5A, a eukaryotic translation factor. Hypusine is a rare amino acid synthesized in this process and is mediated by two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Despite the essential participation of this conserved eIF5A protein in plant development and stress responses, our knowledge of its proper function is limited. In this review, we demonstrate the main findings regarding how eIF5A and hypusination could contribute to plant-specific responses in growth and stress-related processes. Our aim is to briefly discuss the plant-specific details of hypusination and decipher those signal pathways which can be effectively modified by this process. The diverse functions of eIF5A isoforms are also discussed in this review.

Background: Hypusination is an essential post-translational modification in eukaryotes. The two enzymes required for this modification, namely deoxyhypusine synthase (DHS) and deoxyhypusine hydrolase are also conserved. Plasmodium falciparum human malaria parasites possess genes for both hypusination enzymes, which are hypothesized to be targets of antimalarial drugs. Methods: Transgenic P. falciparum parasites with modification of the PF3D7_1412600 gene encoding PfDHS enzyme were created by insertion of the glmS riboswitch or the M9 inactive variant. The PfDHS protein was studied in transgenic parasites by confocal microscopy and Western immunoblotting. The biochemical function of PfDHS enzyme in parasites was assessed by hypusination and nascent protein synthesis assays. Gene essentiality was assessed by competitive growth assays and chemogenomic profiling. Results: Clonal transgenic parasites with integration of glmS riboswitch downstream of the PfDHS gene were established. PfDHS protein was present in the cytoplasm of transgenic parasites in asexual stages. The PfDHS protein could be attenuated fivefold in transgenic parasites with an active riboswitch, whereas PfDHS protein expression was unaffected in control transgenic parasites with insertion of the riboswitch-inactive sequence. Attenuation of PfDHS expression for 72 h led to a significant reduction of hypusinated protein; however, global protein synthesis was unaffected. Parasites with attenuated Pf DHS expression showed a significant growth defect, although their decline was not as rapid as parasites with attenuated dihydrofolate reductase-thymidylate synthase (Pf DHFR-TS) expression. Pf DHS-attenuated parasites showed increased sensitivity to N¹-guanyl-1,7-diaminoheptane, a structural analog of spermidine, and a known inhibitor of DHS enzymes. Discussion: Loss of PfDHS function leads to reduced hypusination, which may be important for synthesis of some essential proteins. The growth defect in parasites with attenuated Pf DHS expression suggests that this gene is essential. However, the slower decline of PfDHS mutants compared with PfDHFR-TS mutants in competitive growth assays suggests that PfDHS is less vulnerable as an antimalarial target. Nevertheless, the data validate PfDHS as an antimalarial target which can be inhibited by spermidine-like compounds. [ABSTRACT FROM AUTHOR]

Deoxyhypusine synthase (DHS) is encoded by a nuclear gene and is the key enzyme involved in the post-translational activation of the eukaryotic translation initiation factor eIF5A. DHS plays important roles in plant growth and development. To gain a better understanding of DHS, the petunia (Petunia hybrida) PhDHS gene was isolated, and the role of PhDHS in plant growth was analyzed. PhDHS protein was localized to the nucleus and cytoplasm. Virus-mediated PhDHS silencing caused a sectored chlorotic leaf phenotype. Chlorophyll levels and photosystem II activity were reduced, and chloroplast development was abnormal in PhDHS -silenced leaves. In addition, PhDHS silencing resulted in extended leaf longevity and thick leaves. A proteome assay revealed that 308 proteins are upregulated and 266 proteins are downregulated in PhDHS -silenced plants compared with control, among the latter, 21 proteins of photosystem I and photosystem II and 12 thylakoid (thylakoid lumen and thylakoid membrane) proteins. In addition, the mRNA level of PheIF5A-1 significantly decreased in PhDHS -silenced plants, while that of another three PheIF5As were not significantly affected in PhDHS -silenced plants. Thus, silencing of PhDHS affects photosynthesis presumably as an indirect effect due to reduced expression of PheIF5A-1 in petunia. Significance: PhDHS -silenced plants develop yellow leaves and exhibit a reduced level of photosynthetic pigment in mesophyll cells. In addition, arrested development of chloroplasts is observed in the yellow leaves. [ABSTRACT FROM AUTHOR]

Hypusine is formed post-translationally from lysine and is found in a single cellular protein, eukaryotic translation initiation factor-5A (eIF5A), and its homolog eIF5A2. Biosynthesis of hypusine is a two-step reaction involving the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). eIF5A is highly conserved throughout eukaryotic evolution and plays a role in mRNA translation, cellular proliferation, cellular differentiation, and inflammation. DHPS is also highly conserved and is essential for life, as Dhps -null mice are embryonic lethal. Using exome sequencing, we identified rare biallelic, recurrent, predicted likely pathogenic variants in DHPS segregating with disease in five affected individuals from four unrelated families. These individuals have similar neurodevelopmental features that include global developmental delay and seizures. Two of four affected females have short stature. All five affected individuals share a recurrent missense variant (c.518A>G [p.Asn173Ser]) in trans with a likely gene disrupting variant (c.1014+1G>A, c.912_917delTTACAT [p.Tyr305_Ile306del], or c.1A>G [p.Met1?]). cDNA studies demonstrated that the c.1014+1G>A variant causes aberrant splicing. Recombinant DHPS enzyme harboring either the p.Asn173Ser or p.Tyr305_Ile306del variant showed reduced (20%) or absent in vitro activity, respectively. We co-transfected constructs overexpressing HA-tagged DHPS (wild-type or mutant) and GFP-tagged eIF5A into HEK293T cells to determine the effect of these variants on hypusine biosynthesis and observed that the p.Tyr305_Ile306del and p.Asn173Ser variants resulted in reduced hypusination of eIF5A compared to wild-type DHPS enzyme. Our data suggest that rare biallelic variants in DHPS result in reduced enzyme activity that limits the hypusination of eIF5A and are associated with a neurodevelopmental disorder. [ABSTRACT FROM AUTHOR]

The discovery of new therapeutic strategies for diseases is essential for drug research. Deoxyhypusine synthase (DHPS) is a critical enzyme that modifies the conversion of the eukaryotic translation initiation factor 5A (eIF5A) precursor into physiologically active eIF5A (eIF5A-Hyp). Recent studies have revealed that the hypusine modifying of DHPS on eIF5A has an essential regulatory role in human diseases. The hypusination-induced DHPS/eIF5A pathway has been shown to play an essential role in various cancers, and it could regulate immune-related diseases, glucose metabolism-related diseases, neurological-related diseases, and aging. In addition, DHPS has a more defined substrate and a well-defined structure within the active pocket than eIF5A. More and more researchers are focusing on the prospect of advanced development of DHPS inhibitors. This review summarizes the regulatory mechanisms of the hypusination-induced DHPS/eIF5A pathway in a variety of diseases in addition to the inhibitors related to this pathway; it highlights and analyzes the structural features and mechanisms of action of DHPS inhibitors and expands the prospects of future drug development using DHPS as an anticancer target. [Display omitted] • DHPS/eIF5A could be a new strategy for cancer therapy. • DHPS/eIF5A is involved in the regulatory process of diverse diseases. • DHPS allosteric inhibitors have superiority over classical inhibitors. [ABSTRACT FROM AUTHOR]

Deoxyhypusine synthase (DHS) is a transferase enabling the formation of deoxyhypusine, which is the first, rate-limiting step of a unique post-translational modification: hypusination. DHS catalyses the transfer of a 4-aminobutyl moiety of polyamine spermidine to a specific lysine of eukaryotic translation factor 5A (eIF5A) precursor in a nicotinamide adenine dinucleotide (NAD)-dependent manner. This modification occurs exclusively on one protein, eIF5A, and it is essential for cell proliferation. Malfunctions of the hypusination pathway, including those caused by mutations within the DHS encoding gene, are associated with conditions such as cancer or neurodegeneration. Here, we present a series of high-resolution crystal structures of human DHS. Structures were determined as the apoprotein, as well as ligand-bound states at high-resolutions ranging from 1.41 to 1.69 Å. By solving DHS in complex with its natural substrate spermidine (SPD), we identified the mode of substrate recognition. We also observed that other polyamines, namely spermine (SPM) and putrescine, bind DHS in a similar manner as SPD. Moreover, we performed activity assays showing that SPM could to some extent serve as an alternative DHS substrate. In contrast to previous studies, we demonstrate that no conformational changes occur in the DHS structure upon spermidine-binding. By combining mutagenesis and a light-scattering approach, we show that a conserved “ball-and-chain” motif is indispensable to assembling a functional DHS tetramer. Our study substantially advances our knowledge of the substrate recognition mechanism by DHS and may aid the design of pharmacological compounds for potential applications in cancer therapy.

In an extreme thermophile, Thermus thermophilus, sym-homospermidine is synthesized by the actions of two enzymes. The first enzyme coded by dhs gene (annotated to be deoxyhypusine synthase gene) catalyzes synthesis of an intermediate, supposed to be 1,9-bis(guanidino)-5-aza-nonane (=N 1 , N 11 -bis(amidino)-sym-homospermidine), from two molecules of agmatine in the presence of NAD. The second enzyme (aminopropylagmatinase) coded by speB gene catalyzes hydrolysis of the intermediate compound to sym-homospermidine releasing two molecules of urea.

Summary: Polyamines are primordial, small organic polycations present in almost all cells, but their roles in bacteria are poorly understood. sym‐Homospermidine is the dominant polyamine in the filamentous, N2‐fixing, heterocyst‐forming cyanobacterium Anabaena sp. PCC 7120. Synthesis of homospermidine was dependent on speA (encoding arginine decarboxylase), speB (agmatinase) and speY (deoxyhypusine synthase homologue), which in bacteria is an unprecedented pathway. Inactivation of any of these genes impaired diazotrophic growth. Heterocyst differentiation in the speA mutant was blocked at an early step, after induction of the regulatory gene hetR but before production of heterocyst‐specific glycolipids (HGL). In contrast, the speY mutant produced HGL and showed slow diazotrophic growth. Analysis of fusions to green fluorescent protein revealed that SpeA (like SpeB previously described) accumulates at higher levels in vegetative cells than in heterocysts, and that SpeY accumulates in vegetative cells but also at significant levels in heterocysts. The homospermidine biosynthetic pathway is therefore active primarily in vegetative cells but the last step can be completed in heterocysts. Our findings indicate an important role for polyamines in the diazotrophic biology of Anabaena. Furthermore, inactivation of a gene cluster (potADB) encoding a polyamine ABC transporter disrupted diazotrophic growth, corroborating the importance of polyamine homeostasis in Anabaena. Different pathways for polyamine biosynthesis have evolved in different organisms. The heterocyst‐forming cyanobacterium Anabaena synthesizes the polyamine sym‐homospermidine through a pathway that involves arginine decarboxylase (SpeA), agmatinase (SpeB) and a deoxyhypusine synthase‐like enzyme (SpeY). Production of a polyamine (mainly homospermidine) is needed for heterocyst differentiation and expression of nitrogenase activity in Anabaena. As shown with GFP fusions, the pathway is in part differentially expressed between vegetative cells and heterocysts (indicated by arrows in the micrograph). [ABSTRACT FROM AUTHOR]

Eukaryotic eIF5A and its bacterial orthologue EF-P are translation elongation factors whose task is to rescue ribosomes from stalling during the synthesis of proteins bearing particular sequences such as polyproline stretches. Both proteins are characterized by unique post-translational modifications, hypusination and lysinylation, respectively, which are essential for their function. An orthologue is present in all Archaea but its function is poorly understood. Here, we show that aIF5A of the crenarchaeum Sulfolobus solfataricus is hypusinated and forms a stable complex with deoxyhypusine synthase, the first enzyme of the hypusination pathway. The recombinant enzyme is able to modify its substrate in vitro resulting in deoxyhypusinated aIF5A. Moreover, with the aim to identify the enzyme involved in the second modification step, i.e. hypusination, a set of proteins interacting with aIF5A was identified. [ABSTRACT FROM AUTHOR]

Translation factor eIF5A participates in protein synthesis at the stage of polypeptide chain elongation. Two eIF5A isoforms are known that are encoded by related genes whose expression varies significantly in different tissues. The eIF5A1 isoform is a constitutively and ubiquitously expressed gene, while the eIF5A2 isoform is expressed in few normal tissues and is an oncogene by a number of parameters. Unique feature of eIF5A isoforms is that they are the only two proteins that contain amino acid hypusine. Modification with hypusine is critical requirement for eIF5A activity. Another distinctive feature of eIF5A is its involvement in the translation of only a subset of the total population of cell mRNAs. The genes for which mRNAs translation requires eIF5A are the members of certain functional groups and are involved in cell proliferation, apoptosis, inflammatory processes, and regulation of transcription and RNA metabolism. The involvement of eIF5A is necessary for the translation of proteins containing oligoproline fragments and some other structures. Modification of eIF5A by hypusine is implemented by two highly specialized enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), which are not involved in other biochemical reactions. Intracellular activity of these enzymes is closely associated with systems of protein acetylation, polyamine metabolism and other biochemical processes. Inhibition of DHS and DOHH activity provides the possibility of pharmacological control of eIF5A activity and expression of eIF5A-dependent genes. [ABSTRACT FROM AUTHOR]

B3LYP and M06–2X optimization and MP2 single point calculations are reported for the 4 H 5 and 5 H 4 conformations of 3,4,6-tri- O -acetyl-D-allal, 3,4,6-tri- O -acetyl-D-galactal, 3,4,6-tri- O -acetyl-D-glucal, and 3,4,6-tri- O -acetyl-D-gulal. Significant discrepancies in predictions of relative energies and conformers' population for B3LYP and M06–2X optimized geometries are observed. Generally, B3LYP overestimates the conformers' energies with respect to MP2, whereas M06–2X slightly underestimates the conformers' energies. B3LYP failed to estimate the 4 H 5 ⇄ 5 H 4 conformational equilibrium for 3,4,6-tri- O -acetyl-D-galactal and 3,4,6-tri- O -acetyl-D-glucal. The M06–2X functional showed good agreement with experimental results for all glycals studied. The 4 H 5 ⇄ 5 H 4 conformational equilibrium for 3,4,6-tri- O -acetyl-D-allal and 3,4,6-tri- O -acetyl-D-gulal is governed by the vinylogous anomeric effect (VAE), whereas competition between the VAE and quasi 1,3-diaxial interactions influence this equilibrium for 3,4,6-tri- O -acetyl-D-galactal and 3,4,6-tri- O -acetyl-D-glucal. The orientation of the 4-OAc group influences the strength of the quasi 1,3-diaxial interactions between the 3-OAc and 5-CH 2 OAc groups. AIM analysis shows weak bonding interaction between the 3-OAc and 5-CH 2 OAc groups. [ABSTRACT FROM AUTHOR]

A new 3D metal–organic framework {[Cd3(BDC)3(OPP)(DMF)2]·2DMA}n (TMU-33) has been synthesized using a novel V-shaped imine-functionalized ligand, N,N′-(oxybis(4,1-phenylene))bis(1-(pyridin-4-yl)methanimine) (OPP), terephthalic acid (H2BDC), and Cd(NO3)2·4H2O and has been fully characterized. The function of the imine moiety of the OPP ligand as a Brønsted base and open Cd site as a Lewis acid in this framework was investigated to determine the role of the cooperative catalyst in the synthesis of Hantzsch 1,4-dihydropyridines simultaneously and separately at ambient temperature. In addition, nanorods and nanoplates of TMU-33 were synthesized via a sonochemical reaction by the coordination modulation method, and the effect of the nanostructure on the relative accessibility of the catalytic site was studied. Moreover, the catalytic performance of TMU-33 with different morphologies was evaluated in this reaction. Efficient catalytic activity was observed for the nanoscale catalysts at ambient temperature, and the acidic sites in the cooperative catalyst were found to be more effective than the existing basic sites in the structure. TMU-33 can be reused by washing with ethanol at least four times with no significant degradation of the catalytic activity (less than 7%). [ABSTRACT FROM AUTHOR]

Deoxyhypusine synthase (DHS) catalyzes the post-translational modification of eukaryotic translation factor 5A (eIF5A) by the polyamine, spermidine, that converts one specific lysine residue to deoxyhypusine [N-4-aminobutyl(lysine)], which is subsequently hydroxylated to hypusine [N-4-amino-2-hydroxybutyl(lysine)]. Hypusine synthesis represents the most critical function of polyamine. As eIF5A has been implicated in various human diseases, identification of specific inhibitors of hypusine modification is of vital importance. DHS catalyzes a complex reaction that occurs in two stages, first, the NAD-dependent cleavage of spermidine to form an enzyme-butylimine intermediate and enzyme-bound NADH, and second, the transfer of the butylimine moiety from the enzyme intermediate to the eIF5A precursor and subsequent reduction of the eIF5A-butylimine intermediate by enzyme-bound NADH to form deoxyhypusine [N-4-aminobutyl(lysine)]. Our data demonstrate that there is a measurable release of enzyme-bound NADH in the absence of eIF5A precursor and that the DHS activity can be determined by coupling the first phase reaction with the NADH-Glo assay in which the generation of luminescence is dependent on NADH derived from the DHS partial reaction. The conventional DHS assay that measures the incorporation of radioactivity from [1,8-H]spermidine into the eIF5A precursor in the complete reaction cannot be readily adapted for high throughput screening (HTS). In contrast, the non-radioactive DHS/NADH-Glo coupled assay is highly specific, sensitive and reproducible and could be configured for HTS of small molecule libraries for the identification of new inhibitors of DHS. Furthermore, the coupled assay provides new insights into the dynamics of the DHS reaction especially regarding the fate of NADH. [ABSTRACT FROM AUTHOR]

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignancy due to its broad resistance to chemotherapy. Gemcitabine is used as a standard chemotherapeutic drug for PDAC treatment, either alone or in combination with other chemotherapeutics. However, in patients with advanced disease, survival is rarely improved. This study aimed to investigate the therapeutic efficacy of N1-guanyl-1, 7-diaminoheptane (GC7) combined with gemcitabine in PDAC therapy. We measured eukaryotic translation initiation factor 5A2 (eIF5A2) expression and gemcitabine sensitivity in different PDAC cell lines (Panc-1, BxPC-3, and T3-M4). The synergistic cytotoxic effects of gemcitabine combined with GC7 were measured using Cell Counting Kit-8 assays. Western blots were performed to measure eIF5A2 and multi-drug resistance 1 (MDR1) protein expression in PDAC cells. The present findings demonstrated that combined treatment with GC7 and gemcitabine significantly inhibited PDAC cell line viability (P<0.05). EdU incorporation assays also indicated that GC7 co-treatment remarkably enhanced gemcitabine sensitivity in PDAC cells. Furthermore, downregulation of eIF5A2 diminished the regulatory role of GC7 in gemcitabine cytotoxicity. Western blotting data indicated that GC7 downregulated the expression of MDR1 while gemcitabine induced MDR1 upregulation. These findings showed that GC7 combination therapy may enhance the therapeutic efficacy of gemcitabine in PDAC by downregulating MDR1 expression. [ABSTRACT FROM AUTHOR]

Replication of the human immunodeficiency virus type 1 (HIV-1) is dependent on eIF5A hypusination. Hypusine is formed post-translationally on the eIF5A precursor by two consecutive enzymatic steps; a reversible reaction involving the enzyme deoxyhypusine synthase (DHS) and an irreversible step involving the enzyme deoxyhypusine hydroxylase (DOHH). In this study we explored the effect of inhibiting DOHH activity and therefore eIF5A hypusination, on HIV-1 gene expression. Results show that the expression of proteins from an HIV-1 molecular clone is reduced when DOHH activity is inhibited by Deferiprone (DFP) or Ciclopirox (CPX). Next we evaluated the requirement of DOHH activity for internal ribosome entry site (IRES)-mediated translation initiation driven by the 5'untranslated region (5′UTR) of the full length HIV-1 mRNA. Results show that HIV-1 IRES activity relies on DOHH protein concentration and enzymatic activity. Similar results were obtained for IRES-dependent translation initiation mediated by 5′UTR of the human T-cell lymphotropic virus type 1 (HTLV-1) and the mouse mammary tumor virus (MMTV) mRNAs. Interestingly, activity of the poliovirus IRES, was less sensitive to the targeting of DOHH suggesting that not all viral IRESs are equally dependent on the cellular concentration or the activity of DOHH. In summary we present evidence indicating that the cellular concentration of DOHH and its enzymatic activity play a role in HIV-1, HTLV-1 and MMTV IRES-mediated translation initiation. [ABSTRACT FROM AUTHOR]

The unique amino acid hypusine is present in only two proteins in eukaryotic cells, eukaryotic translation initiation factor 5A-1 (eIF5A1), and eIF5A2, where it is covalently linked to the lysine-50 residue of these proteins via a post-translational modification coined hypusination. This unique modification is directed by two highly conserved and essential enzymes, deoxyhypusine synthase (DHPS), and deoxyhypusine hydroxylase (DOHH), which selectively use the polyamine spermidine as a substrate to generate hypusinated eIF5A. Notably, elevated levels of polyamines are a hallmark of most tumor types, and increased levels of polyamines can also be detected in the urine and blood of cancer patients. Further, in-clinic agents that block the function of key biosynthetic enzymes in the polyamine pathway markedly impair tumor progression and maintenance of the malignant state. Thus, the polyamine pathway is attractive as a prognostic, prevention and therapeutic target. As we review, recent advances in our understanding of the specific functions of hypusinated eIF5A and its role in tumorigenesis suggest that the polyamine-hypusine circuit is a high priority target for cancer therapeutics. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Earlier unknown hydroxylamine-containing analogues of 1-guanidino-7-aminohepane (GC7) containing the free aminooxy group (1-aminooxy-6-guanidinohexane), bis-guanyl derivatives of 1-aminooxy-6-aminohexane, and 1-aminooxy-5-aminopentane, and mono-guanidinooxy analogue (1-guanidinooxy-6-aminohexane) were synthesized in high overall yields from commercially available 5-(Boc-amino)1-pentanol and 6-(Boc-amino)-1-hexanol. It was demonstrated that 1,3-di-Boc-2-(trifluoromethylsulfonyl)guanidine is able to specifically amidinate the amino group in the presence of the free aminooxy group. The application of newly synthesized GC7 analogues for the investigation of the peculiarities of their interaction with the active site of deoxyhypusine synthase was discussed. [ABSTRACT FROM AUTHOR]

The Trichomonas vaginalis genome analysis suggested the presence of a putative deoxyhypusine synthase (TvDHS) that catalyzes the posttranslational modification of eIF-5A. Herein, we expressed and purified the recombinant TvDHS (rTvDHS) protein (43 kDa) and the recombinant TveIF-5A (rTveIF-5A) precursor protein (46 kDa). A 41 kDa band of the native TvDHS was recognized by western blot analysis in T. vaginalis total protein extract by a mouse polyclonal anti-rTvDHS antibody. The enzymatic activity of rTvDHS was determined by in vitro rTveIF-5A precursor modification. The modification reaction was performed by using ( 3 H)-spermidine, and the biochemical analysis showed that rTvDHS exhibited Km value of 0.6 μM. The rTvDHS activity was inhibited by the spermidine analog, N ″-guanyl-1,7-diamino-heptane (GC7). Native gel electrophoresis analysis showed two bands corresponding to an rTvDHS-rTveIF-5A complex and an intermediate form of rTveIF-5A. The two forms were subsequently separated by ion exchange chromatography to identify the hypusine residue by MS/MS analysis. Moreover, mutations in TvDHS showed that the putative HE motif present in this enzyme is involved in the hydroxylation of TveIF-5A. We observed that only hypusine-containing TveIF-5A was bound to an RNA hairpin ERE structure from the cox-2 gene, which contains the AAAUGUCACAC consensus sequence. Interestingly, 2DE-WB assays, using parasites that were grown in DAB-culture conditions and transferred to exogenous putrescine, showed the new isoform of TveIF-5A. In summary, our results indicate that T. vaginalis contains an active TvDHS capable of modifying the precursor TveIF-5A protein, which subsequently exhibits RNA binding activity. [ABSTRACT FROM AUTHOR]