Hypusine Modification Research Articles

Crystal structure of archaeal IF5A-DHS complex reveals insights into the hypusination mechanism

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 2.0Å 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.

The Deficiency of Hypusinated eIF5A Decreases the Putrescine/Spermidine Ratio and Inhibits +1 Programmed Ribosomal Frameshifting during the Translation of Ty1 Retrotransposon in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.

Hypusinated eIF5A Promotes Ribosomal Frameshifting during Decoding of ODC Antizyme mRNA in Saccharomyces cerevisiae

Polyamines are essential biogenic poly-cations with important roles in many cellular processes and diseases such as cancer. A rate-limiting step early in the biosynthesis of polyamines is the conversion of ornithine to putrescine by the homodimeric enzyme ornithine decarboxylase (ODC). In a conserved mechanism of posttranslational regulation, ODC antizyme (OAZ) binds to ODC monomers promoting their ubiquitin-independent degradation by the proteasome. Decoding of OAZ mRNA is unusual in that it involves polyamine-regulated bypassing of an internal translation termination (STOP) codon by a ribosomal frameshift (RFS) event. Using Saccharomyces cerevisiae, we earlier showed that high polyamine concentrations lead to increased efficiency of OAZ1 mRNA translation by binding to nascent Oaz1 polypeptide. The binding of polyamines prevents stalling of the ribosomes on OAZ1 mRNA caused by nascent Oaz1 polypeptide thereby promoting synthesis of full-length Oaz1. Polyamine depletion, however, also inhibits RFS during the decoding of constructs bearing the OAZ1 shift site lacking sequences encoding the Oaz1 parts implicated in polyamine binding. Polyamine depletion is known to impair hypusine modification of translation factor eIF5A. Using a novel set of conditional mutants impaired in the function of eIF5A/Hyp2 or its hypusination, we show here that hypusinated eIF5A is required for efficient translation across the OAZ1 RFS site. These findings identify eIF5A as a part of Oaz1 regulation, and thereby of polyamine synthesis. Additional experiments with DFMO, however, show that depletion of polyamines inhibits translation across the OAZ1 RFS site not only by reducing Hyp2 hypusination, but in addition, and even earlier, by affecting RFS more directly.

Global analyses of mRNA expression in human sensory neurons reveal eIF5A as a conserved target for inflammatory pain.

Nociceptors are a type of sensory neuron that are integral to most forms of pain. Targeted disruption of nociceptor sensitization affords unique opportunities to prevent pain. An emerging model for nociceptors are sensory neurons derived from human stem cells. Here, we subjected five groups to high-throughput sequencing: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. Co-culture of nociceptors and astrocytes promotes expression of transcripts enriched in DRG tissues. Comparisons of the hiPSC models to tissue samples reveal that many key transcripts linked to pain are present. Markers indicative of a range of neuronal subtypes present in the DRG were detected in mature hiPSCs. Intriguingly, translation factors were maintained at consistently high expression levels across species and culture systems. As a proof of concept for the utility of this resource, we validated expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissues and hiPSC samples. eIF5A is subject to a unique posttranslational hypusine modification required for its activity. Inhibition of hypusine biosynthesis prevented hyperalgesic priming by inflammatory mediators in vivo and diminished hiPSC activity in vitro. Collectively, our results illuminate the transcriptomes of hiPSC sensory neuron models. We provide a demonstration for this resource through our investigation of eIF5A. Our findings reveal hypusine as a potential target for inflammation associated pain in males.

259-LB: Inhibition of the Polyamine-Hypusine Circuit Delimits Islet ß-Cell Stress in the Context of T1D via Suppression of IRE-1a Signaling

Recent data suggest that stress-induced molecular pathways in the β cell may trigger the display of neoantigens that initiate autoimmunity in type 1 diabetes (T1D) . Polyamines (putrescine, spermidine, and spermine) are biogenic amines that participate in stress-induced mRNA translation, in part, through the posttranslational hypusine modification of eIF5A. Rate-limiting enzymes in this circuit include the ODC, which converts Arg to polyamines, and DHPS, which controls eIF5A hypusination. Previously, we showed that inhibition of ODC or DHPS in non-obese diabetic mice using either DFMO or GC7, respectively, resulted in preserved β-cell function, enhanced β-cell mass, and significantly reduced T1D incidence. DFMO treatment subjects with recent-onset T1D was suggestive of reductions in β cell stress. We hypothesized that polyamine depletion with DFMO reduces inflammatory stress in β cells and the consequent accumulation of unhypusinated eIF5A limits stress-related protein production. To test the hypothesis, we treated the human islets with proinflammatory cytokine (PIC) cocktail to mimic T1D in presence or absence of 5 mM DFMO. We observed induction of ER stress as shown by increased expression of unfolded protein response proteins within 24 h of PIC treatment. Treatment with DFMO reduced protein levels of IRE1α and downstream XBP1. Gene expression analysis showed significant downregulation of genes in the IRE1α pathway (CHOP, TNXIP, and XBP1s) . To clarify a mechanism underlying suppression of stress protein production by unhypusinated eIF5A, we treated HEK cells with DFMO or GC7, both of which enhanced the association of unhypusinated eIF5A with GCN2, an eIF2α kinase, suggesting a non-ER stress mediated mechanism of mRNA translational blockade. Collectively, these data suggest that inhibition of the polyamines-hypusine circuit suppresses ER stress via IRE1α signaling and enhances non-ER stress mediated suppression of mRNA translation. Disclosure A. Anderson: None. A. Kulkarni: None. R. G. Mirmira: None.

Global Analyses of mRNA Expression in Human Sensory Neurons Reveals eIF5a as a Conserved Target for Inflammation-associated Pain

Sensory neurons derived from human induced pluripotent stem cells (hiPSCs) are a promising model. One limitation posed by the use of monocultures is the loss of cellular heterogeneity found in tissues. Here we make use of high-throughput RNA sequencing to quantify gene expression in hiPSC-derived mono-cultured and co-cultured sensory neurons. The following groups were compared: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. We find that co-culture of sensory neurons and astrocytes enhances expression of transcripts enriched in native DRG tissues. Numerous well-established genes linked to pain and most translation factors are consistently expressed in both hiPSC and mouse samples. Marker genes for various neuronal subtypes are also present in the hiPSC cultures. As a proof of concept for the potential use of the dataset for hypothesis generation, we validated the expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissue and hiPSC samples. eIF5A is a unique translation factor in that it requires a post-translational hypusine modification to be active. We show that inhibition of hypusine synthesis prevents hyperalgesic priming by inflammatory mediators in vivo and diminishes hiPSC neuronal firing in vitro. In total, we present the transcriptomes of hiPSC sensory neuron models and evaluate the requirement of a functional translation factor. This work was supported by NIH grants R01NS100788 (ZTC), R01NS114018 (ZTC), and 1UG3TR003149 (BJB). Sensory neurons derived from human induced pluripotent stem cells (hiPSCs) are a promising model. One limitation posed by the use of monocultures is the loss of cellular heterogeneity found in tissues. Here we make use of high-throughput RNA sequencing to quantify gene expression in hiPSC-derived mono-cultured and co-cultured sensory neurons. The following groups were compared: human induced pluripotent stem cells (hiPSCs) prior to differentiation, mature hiPSC-derived sensory neurons, mature co-cultures containing hiPSC-derived astrocytes and sensory neurons, mouse dorsal root ganglion (DRG) tissues, and mouse DRG cultures. We find that co-culture of sensory neurons and astrocytes enhances expression of transcripts enriched in native DRG tissues. Numerous well-established genes linked to pain and most translation factors are consistently expressed in both hiPSC and mouse samples. Marker genes for various neuronal subtypes are also present in the hiPSC cultures. As a proof of concept for the potential use of the dataset for hypothesis generation, we validated the expression of eukaryotic initiation factor 5A (eIF5A) in DRG tissue and hiPSC samples. eIF5A is a unique translation factor in that it requires a post-translational hypusine modification to be active. We show that inhibition of hypusine synthesis prevents hyperalgesic priming by inflammatory mediators in vivo and diminishes hiPSC neuronal firing in vitro. In total, we present the transcriptomes of hiPSC sensory neuron models and evaluate the requirement of a functional translation factor. This work was supported by NIH grants R01NS100788 (ZTC), R01NS114018 (ZTC), and 1UG3TR003149 (BJB).

Oxygen level regulates N-terminal translation elongation of selected proteins through deoxyhypusine hydroxylation.

Hypusine is a post-translational modification on eukaryotic translation initiation factor 5A (eIF5A). The last step of hypusine biosynthesis, deoxyhypusine hydroxylation, is an oxygen-dependent reaction. Here we show that deletion of the deoxyhypusine hydroxylase Lia1 compromises yeast respiration through translation downregulation of selected proteins in the respiration pathway. The translation suppression, because of the lack of deoxyhypusine hydroxylation, mainly affects translation of the N termini of the proteins, independent of the presence of proline residues but likely dependent on the interaction between the N-terminal nascent peptide and the ribosomal peptide exit tunnel. Proteomics and biochemical studies reveal that Lia1 deletion decreases N-terminal translation of proteins involved in mitochondrial respiration, oxidative stress response, and protein folding. Our work uncovers functions of the hypusine modification by considering the substrate requirement of the post-translational modification, highlights the unique challenges of translating the N termini of proteins, and reveals an oxygen-sensing mechanism in eukaryotic cells.

Post-translational formation of hypusine in eIF5A: implications in human neurodevelopment

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.

Flexible NAD+ Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A.

The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288–P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.

New Series of Potent Allosteric Inhibitors of Deoxyhypusine Synthase

Deoxyhypusine synthase (DHPS) is the primary enzyme responsible for the hypusine modification and, thereby, activation of the eukaryotic translation initiation factor 5A (eIF5A), which is key in regulating the protein translation processes associated with tumor proliferation. Although DHPS inhibitors could be a promising therapeutic option for treating cancer, only a few studies reported druglike compounds with this inhibition property. Thus, in this work, we designed and synthesized a new chemical series possessing fused ring scaffolds designed from high-throughput screening hit compounds, discovering a 5,6-dihydrothieno[2,3-c]pyridine derivative (26d) with potent inhibitory activity; furthermore, the X-ray crystallographic analysis of the DHPS complex with 26d demonstrated a distinct allosteric binding mode compared to a previously reported inhibitor. These findings could be significantly useful in the functional analysis of conformational changes in DHPS as well as the structure-based design of allosteric inhibitors.

Discovery of Novel Allosteric Inhibitors of Deoxyhypusine Synthase.

Deoxyhypusine synthase (DHPS) utilizes spermidine and NAD as cofactors to incorporate a hypusine modification into the eukaryotic translation initiation factor 5A (eIF5A). Hypusine is essential for eIF5A activation, which, in turn, plays a key role in regulating protein translation of selected mRNA that are associated with the synthesis of oncoproteins, thereby enhancing tumor cell proliferation. Therefore, inhibition of DHPS is a promising therapeutic option for the treatment of cancer. To discover novel lead compounds that target DHPS, we conducted synthetic studies with a hit obtained via high-throughput screening. Optimization of the ring structures of the amide compound (2) led to bromobenzothiophene (11g) with potent inhibitory activity against DHPS. X-ray crystallographic analysis of 11g complexed with DHPS revealed a dramatic conformational change in DHPS, which suggests the presence of a novel allosteric site. These findings provide the basis for the development of novel therapy distinct from spermidine mimetic inhibitors.

In vitro yeast reconstituted translation system reveals function of eIF5A for synthesis of long polypeptide.

We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

Trypanosomatid Deoxyhypusine Synthase Activity Is Dependent on Shared Active-Site Complementation between Pseudoenzyme Paralogs

Trypanosoma brucei is a neglected tropical disease endemic to Africa. The polyamine spermidine is essential for post-translational hypusine modification of eukaryotic initiation factor 5A (eIF5A), which is catalyzed by deoxyhypusine synthase (TbDHS). In trypanosomatids, deoxyhypusine synthase (DHS) activity is dependent on heterotetramer formation between two paralogs, DHSc and DHSp, both with minimal activity on their own due to missing catalytic residues. We determined the X-ray structure of TbDHS showing a single functional shared active site is formed at the DHSc/DHSp heterodimer interface, with deficiencies in one subunit complemented by the other. Each heterodimer contains two NAD+ binding sites, one housed in the functional catalytic site and the second bound in a remnant dead site that lacks key catalytic residues. Functional analysis of these sites by site-directed mutagenesis identified long-range contributions to the catalytic site from the dead site. Differences between trypanosomatid and human DHS that could be exploited for drug discovery were identified.

Synergistic drug combination GC7/DFMO suppresses hypusine/spermidine-dependent eIF5A activation and induces apoptotic cell death in neuroblastoma.

The eukaryotic initiation factor 5A (eIF5A), which contributes to several crucial processes during protein translation, is the only protein that requires activation by a unique post-translational hypusine modification. eIF5A hypusination controls cell proliferation and has been linked to cancer. eIF5A hypusination requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase and uniquely depends on the polyamine (PA) spermidine as the sole substrate. Ornithine decarboxylase (ODC) is the rate-limiting enzyme in PA biosynthesis. Both ODC and PAs control cell proliferation and are frequently dysregulated in cancer. Since only spermidine can activate eIF5A, we chose the hypusine-PA nexus as a rational target to identify new drug combinations with synergistic antiproliferative effects. We show that elevated mRNA levels of the two target enzymes DHPS and ODC correlate with poor prognosis in a large cohort of neuroblastoma (NB) tumors. The DHPS inhibitor GC7 (N1-guanyl-1,7-diaminoheptane) and the ODC inhibitor α-difluoromethylornithine (DFMO) are target-specific and in combination induced synergistic effects in NB at concentrations that were not individually cytotoxic. Strikingly, while each drug alone at higher concentrations is known to induce p21/Rb- or p27/Rb-mediated G1 cell cycle arrest, we found that the drug combination induced caspase 3/7/9, but not caspase 8-mediated apoptosis, in NB cells. Hypusinated eIF5A levels and intracellular spermidine levels correlated directly with drug treatments, signifying specific drug targeting effects. This two-pronged GC7/DFMO combination approach specifically inhibits both spermidine biosynthesis and post-translational, spermidine-dependent hypusine-eIF5A activation, offering an exciting clue for improved NB drug therapy.

A new non-radioactive deoxyhypusine synthase assay adaptable to high throughput screening.

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 ε -4-aminobutyl(lysine)], which is subsequently hydroxylated to hypusine [N ε -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 ε -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 [1,8-3H]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 5828: Targeting the polyamine-hypusine nexus for the treatment of neuroblastoma

Abstract The highly conserved and unique post-translational hypusine modification is essential for the activation of a single cellular protein called eukaryotic initiation factor 5A (eIF5A). Hypusination of eIF5A represents an essential mechanism in the control of cell proliferation and has been linked to cancer. The hypusine modification of eIF5A requires the enzymes deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) and, importantly, uniquely depends on the polyamine spermidine as a selective substrate. Polyamines including spermidine are frequently dysregulated in neoplastic disease and are critical for cell proliferation. Ornithine decarboxylase (ODC) is the sentinel enzyme in polyamine biosynthesis. We pioneered the repurposing of the irreversible ODC inhibitor alpha-difluoromethylornithine (DFMO) for neuroblastoma (NB), a devastating pediatric cancer of the sympathetic nervous system. Our preclinical work was confirmed by other groups and spurred two consortia (NMTRC and NANT) to test DFMO in independent phase I/II NB clinical trials. The oncogene MYCN is commonly amplified in high-risk NB patients and predicts poor prognosis. MYCN directly activates ODC (and eIF5A) and drives NB tumor progression, presumably through the activation of polyamines, but the precise molecular mechanisms are not fully understood. Since spermidine is the only molecule that can activate eI5FA through hypusine modification, we hypothesized that the polyamine-hypusine nexus provides a rational target site to identify new drug combinations that trigger synergistic anti-tumor effects. In this study we found that DFMO combined with DHPS inhibitor GC7 induced synergistic drug reactions in NB at concentrations that are not lethal on their own. While each drug alone at higher concentrations induced p27/Rb-mediated G1/S cell cycle arrest, the combination of both drugs resulted in the induction of programmed cell death (apoptosis). The intracellular free polyamine levels (putrescine, spermidine, and spermine) were quantified by C-18 RP-HPLC and responded directly to drug treatments, suggesting specific drug targeting effects. This dual-pronged drug combination approach that specifically targets the blockade of the unique polyamine/spermidine-dependent hypusine-eIF5A signaling circuit known to be involved in cell proliferation might lead to more effective DFMO treatment options by reducing high DFMO doses currently needed while achieving better therapeutic outcomes in NB. Citation Format: Andre S. Bachmann, Raid El-Khawaja, Chad Schultz. Targeting the polyamine-hypusine nexus for the treatment of neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5828. doi:10.1158/1538-7445.AM2017-5828

Amino acid substrates impose polyamine, eIF5A, or hypusine requirement for peptide synthesis.

Whereas ribosomes efficiently catalyze peptide bond synthesis by most amino acids, the imino acid proline is a poor substrate for protein synthesis. Previous studies have shown that the translation factor eIF5A and its bacterial ortholog EF-P bind in the E site of the ribosome where they contact the peptidyl-tRNA in the P site and play a critical role in promoting the synthesis of polyproline peptides. Using misacylated Pro-tRNAPhe and Phe-tRNAPro, we show that the imino acid proline and not tRNAPro imposes the primary eIF5A requirement for polyproline synthesis. Though most proline analogs require eIF5A for efficient peptide synthesis, azetidine-2-caboxylic acid, a more flexible four-membered ring derivative of proline, shows relaxed eIF5A dependency, indicating that the structural rigidity of proline might contribute to the requirement for eIF5A. Finally, we examine the interplay between eIF5A and polyamines in promoting translation elongation. We show that eIF5A can obviate the polyamine requirement for general translation elongation, and that this activity is independent of the conserved hypusine modification on eIF5A. Thus, we propose that the body of eIF5A functionally substitutes for polyamines to promote general protein synthesis and that the hypusine modification on eIF5A is critically important for poor substrates like proline.

Molecular events leading to death of Leishmania donovani under spermidine starvation after hypericin treatment.

We have previously reported that the hypericin treatment caused spermidine starvation and death of Leishmania parasite. Here, we report different molecular events under spermidine starvation and potential role of spermidine in processes other than redox homeostasis of the parasite. We have analyzed changes in expression of several genes by using quantitative gene expression analysis. Further, these changes at molecular level were also confirmed by using biochemical and cellular studies. Altered expression of several genes involved in redox metabolism, hypusine modification of eIF5A, DNA repair pathway and autophagy was observed. There was decrease in Sir2RP expression after hypericin treatment and this decrease has been found to be associated with induced ROS due to hypericin treatment as it has been rescued by either trypanothione or spermidine supplementation. Translation initiation in the parasite was decreased upon spermidine starvation. We also observed increased AMPK expression upon hypericin treatment. The increase in intracellular ATP and NAD+ levels as well as decrease in Sir2RP expression of the parasite are cytoprotective mechanism towards generated ROS due to hypericin treatment possibly by inducing autophagy as indicated by increase in autophagy related gene expression and acridine orange staining. However, the autophagy needs to be established using more rigorous methodologies.

Cyclic AMP and Polyamines Overcome Inhibition by Myelin-Associated Glycoprotein through eIF5A-Mediated Increases in p35 Expression and Activation of Cdk5.

Inhibitory molecules associated with CNS myelin, such as myelin-associated glycoprotein (MAG), represent major obstacles to axonal regeneration following CNS injury. Our laboratory has shown that elevating levels of intracellular cAMP, via application of the nonhydrolyzable analog dibutyryl cAMP (dbcAMP), can block the inhibitory effects of MAG and myelin. We have also shown that elevation of cAMP results in upregulation of arginase I and increased polyamine synthesis. Treatment with putrescine or spermidine blocks myelin-mediated inhibition of neurite outgrowth, but the mechanism underlying this effect has not yet been elucidated. Here we show that cyclin-dependent kinase 5 (Cdk5) is required for dbcAMP and putrescine to overcome MAG-mediated inhibition. The ability of dbcAMP and putrescine to overcome inhibition by MAG is abolished in the presence of roscovitine, a Cdk inhibitor that has greater selectivity for Cdk5, and expression of dominant negative Cdk5 abolishes the ability of dbcAMP or putrescine to enhance neurite outgrowth in the presence of MAG. Importantly, dbcAMP and putrescine increase expression of p35, the neuron-specific activator of Cdk5, and rat DRG neurons transduced with HSV overexpressing p35 can overcome inhibition by MAG. The upregulation of p35 by putrescine is also reflected in increased localization of p35 to neurites and growth cones. Last, we show that putrescine upregulates p35 expression by serving as a substrate for hypusine modification of eIF5A, and that this hypusination is necessary for putrescine's ability to overcome inhibition by MAG. Our findings reveal a previously unknown mechanism by which polyamines may encourage regeneration after CNS injury.

Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.

During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.