n(1)-guanyl-1-7-diaminoheptane and hypusine

Inflammation is part of an important mechanism triggered by the innate immune response that rapidly responds to invading microorganisms and tissue injury. One important elicitor of the inflammatory response is the Gram-negative bacteria component lipopolysaccharide (LPS), which induces the activation of innate immune response cells, the release of proinflammatory cytokines, such as interleukin 1 and tumor necrosis factor α(TNF-α), and the cellular generation of nitric oxide (NO) by the inducible nitric oxide synthase (iNOS). Although essential to the immune response, uncontrolled inflammatory responses can lead to pathological conditions, such as sepsis and rheumatoid arthritis. Therefore, identifying cellular targets for new anti-inflammatory treatments is crucial to improving therapeutic control of inflammation-related diseases. More recently, the translation factor eIF5A has been demonstrated to have a proinflammatory role in the release of cytokines and the production of NO. As eIF5A requires and essential and unique modification of a specific residue of lysine, changing it to hypusine, eIF5A is an interesting cellular target for anti-inflammatory treatment. The present study reviews the literature concerning the anti-inflammatory effects of inhibiting eIF5A function. We also present new data showing that the inhibition of eIF5A function by the small molecule GC7 significantly decreases TNF-α release without affecting TNF-α mRNA levels. We discuss the mechanisms by which eIF5A may interfere with TNF-α mRNA translation by binding to and regulating the function of ribosomes during protein synthesis.

Topics: Animals; Anti-Inflammatory Agents; Cytokines; Eukaryotic Translation Initiation Factor 5A; Guanine; Humans; Inflammation; Lysine; Macrophages; Mice; Molecular Targeted Therapy; Nitric Oxide; Peptide Initiation Factors; RNA-Binding Proteins

In eukaryotes, the polyamine pathway generates spermidine that activates the hypusination of the translation factor eukaryotic initiation factor 5A (eIF5A). Hypusinated-eIF5A modulates translation, elongation, termination and mitochondrial function. Evidence in model organisms like drosophila suggests that targeting polyamines synthesis might be of interest against ischemia. However, the potential of targeting eIF5A hypusination in stroke, the major therapeutic challenge specific to ischemia, is currently unknown. Using in vitro models of ischemic-related stress, we documented that GC7, a specific inhibitor of a key enzyme in the eIF5A activation pathway, affords neuronal protection. We identified the preservation of mitochondrial function and thereby the prevention of toxic ROS generation as major processes of GC7 protection. To represent a thoughtful opportunity of clinical translation, we explored whether GC7 administration reduces the infarct volume and functional deficits in an in vivo transient focal cerebral ischemia (tFCI) model in mice. A single GC7 pre- or post-treatment significantly reduces the infarct volume post-stroke. Moreover, GC7-post-treatment significantly improves mouse performance in the rotarod and Morris water-maze, highlighting beneficial effects on motor and cognitive post-stroke deficits. Our results identify the targeting of the polyamine-eIF5A-hypusine axis as a new therapeutic opportunity and new paradigm of research in stroke and ischemic diseases.

Topics: Animals; Behavior, Animal; Cognition; Eukaryotic Translation Initiation Factor 5A; Guanine; Injections, Intraperitoneal; Ischemic Attack, Transient; Lysine; Male; Mice; Mice, Inbred C57BL; Mitochondria; Models, Animal; Neuroprotection; Oxidative Stress; Oxidoreductases Acting on CH-NH Group Donors; Peptide Initiation Factors; Polyamines; Reactive Oxygen Species; RNA-Binding Proteins; Stroke

Glioblastomas are highly aggressive brain tumors of adults with poor clinical outcome. Despite a broad range of new and more specific treatment strategies, therapy of glioblastomas remains challenging and tumors relapse in all cases. Recent work demonstrated that the posttranslational hypusine modification of the eukaryotic initiation factor 5A (eIF-5A) is a crucial regulator of cell proliferation, differentiation and an important factor in tumor formation, progression and maintenance. Here we report that eIF-5A as well as the hypusine-forming enzymes deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH) are highly overexpressed in glioblastoma patient samples. Importantly, targeting eIF-5A and its hypusine modification with GC7, a specific DHS-inhibitor, showed a strong antiproliferative effect in glioblastoma cell lines in vitro, while normal human astrocytes were not affected. Furthermore, we identified p53 dependent premature senescence, a permanent cell cycle arrest, as the primary outcome in U87-MG cells after treatment with GC7. Strikingly, combined treatment with clinically relevant alkylating agents and GC7 had an additive antiproliferative effect in glioblastoma cell lines. In addition, stable knockdown of eIF-5A and DHS by short hairpin RNA (shRNA) could mimic the antiproliferative effects of GC7. These findings suggest that pharmacological inhibition of eIF-5A may represent a novel concept to treat glioblastomas and may help to substantially improve the clinical course of this tumor entity.

Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Agents, Alkylating; Apoptosis; Carmustine; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cellular Senescence; Dacarbazine; Eukaryotic Translation Initiation Factor 5A; Female; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glioblastoma; Guanine; Humans; Lysine; Male; Mixed Function Oxygenases; Molecular Targeted Therapy; Neoplasm Grading; Oxidoreductases Acting on CH-NH Group Donors; Peptide Initiation Factors; RNA-Binding Proteins; Temozolomide

The eukaryotic translation initiation factor 5A (eIF5A) contains a special amino acid residue named hypusine that is required for its activity, being produced by a post-translational modification using spermidine as substrate. Stem cells from rat skeletal muscles (satellite cells) were submitted to differentiation and an increase of eIF5A gene expression was observed. Higher content of eIF5A protein was found in satellite cells on differentiation in comparison to non-differentiated satellite cells and skeletal muscle. The treatment with N1-guanyl-1,7-diaminoheptane (GC7), a hypusination inhibitor, reversibly abolished the differentiation process. In association with the differentiation blockage, an increase of glucose consumption and lactate production and a decrease of glucose and palmitic acid oxidation were observed. A reduction in cell proliferation and protein synthesis was also observed. L-Arginine, a spermidine precursor and partial suppressor of muscle dystrophic phenotype, partially abolished the GC7 inhibitory effect on satellite cell differentiation. These results reveal a new physiological role for eIF5A and contribute to elucidate the molecular mechanisms involved in muscle regeneration.

Topics: Animals; Arginine; Cell Differentiation; Cell Proliferation; Chromatin; Connectin; Eukaryotic Translation Initiation Factor 5A; Gene Expression Regulation; Guanine; Lysine; Male; Muscle Proteins; Muscle, Skeletal; MyoD Protein; Oxidation-Reduction; Peptide Initiation Factors; Protein Biosynthesis; Rats; Rats, Wistar; Reverse Transcriptase Polymerase Chain Reaction; RNA-Binding Proteins; RNA, Messenger; Satellite Cells, Skeletal Muscle; Stem Cells

Eukaryotic translation initiation factor 5A (eIF5A) is the only protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)-lysine). We isolated a 1385-bp eIF5A cDNA containing an open reading frame (ORF) of 468 bp, which encodes a protein of 155 amino acids with a conserved hypusine modification site, from the olive flounder Paralichthys olivaceus. Pairwise alignments revealed that flounder eIF5A had a high sequence identity with those of other known species including mammals. Real-time RT-PCR analysis showed the expression of eIF5A mRNA was constitutively detected in various tissues of healthy flounder. In HINAE cells or flounder kidney infected with the viral hemorrhagic septicemia virus (VHSV), the expression of eIF5A mRNA was slightly increased before cells showed cytopathic effects and then decreased when cells showed cytopathic effects. Treatment of N-guanyl-1,7-diaminoheptane (GC-7), a potent inhibitor of eIF5A hypusination, inhibited the expression of VHSV G protein in a dose-dependent manner suggesting a potential role for eIF5A and its hypusination in viral protein expression.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Cloning, Molecular; Eukaryotic Translation Initiation Factor 5A; Flounder; Glycoproteins; Guanine; Kidney; Lysine; Molecular Sequence Data; Novirhabdovirus; Peptide Initiation Factors; Phylogeny; RNA-Binding Proteins; Viral Proteins

Interferon-alpha (IFNalpha) can induce apoptosis, a process regulated by a complex network of cell factors. Among these, eukaryotic initiation factor-5A (eIF-5A) is peculiar because its activity is modulated by the post-translational formation of the amino acid hypusine. Here we report the effects of IFNalpha and epidermal growth factor (EGF) on apoptosis and eIF-5A activity in human epidermoid oropharyngeal KB and lung H1355 cancer cells. We found that 48-h exposure to 1000 and 2000 IU/ml IFNalpha induced about 50% growth inhibition and apoptosis in H1355 and KB cells, respectively, and the addition of EGF completely antagonized this effect. When IFNalpha induced apoptosis, a hyperactivation of MEK-1 and ERK signalling and a decrease of the hypusine-containing form and, thus, of eIF-5A activity were recorded. The latter effect was again antagonized by the addition of EGF to IFNalpha-pretreated cells, probably through the activation of the EGF-->ERK-dependent pathway, since the addition of the specific MEK-1 inhibitor PD098059 abrogated the recovery of intracellular hypusine content induced by EGF in IFNalpha-pretreated cancer cells. Subsequently, we evaluated if the hypusine synthesis inhibitor (and eIF-5A inactivator) N1-guanyl-1,7-diaminoheptane (GC7) synergized with IFNalpha in the induction of cell growth inhibition and apoptosis. The analysis of the isobologram of IFNalpha and GC7 demonstrated a strong synergism between the two drugs in inducing cell growth inhibition. We also found that GC7 and IFNalpha had a synergistic effect on apoptosis. These data suggest that the apoptosis induced by IFNalpha could be regulated by eIF-5A that, therefore, could represent a useful target for the potentiation of IFNalpha antitumor activity.

Topics: Antineoplastic Agents; Apoptosis; Cell Division; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Combinations; Epidermal Growth Factor; Eukaryotic Translation Initiation Factor 5A; Guanine; Humans; Interferon-alpha; KB Cells; Lysine; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Peptide Initiation Factors; Phosphorylation; Protein Processing, Post-Translational; RNA-Binding Proteins

Hypusination is an essential posttranslational modification unique to archaeal and eukaryotic protein synthesis initiation factor 5A (aIF5A and eIF5A, respectively). We have investigated the effect of the efficient hypusination inhibitor N(1)-guanyl-1,7-diaminoheptane (GC(7)) on four archaeal and one bacterial species. We found that (i) archaea are sensitive to GC(7), whereas the bacterium Escherichia coli is not, (ii) GC(7) causes rapid and reversible arrest of growth of the archaeon Sulfolobus acidocaldarius, and (iii) the growth arrest is accompanied by a specific reversible arrest of the cell cycle prior to cell division. Our findings establish a link between hypusination and sustained growth of archaea and thereby provide the framework to study molecular details of archaeal cell cycle in connection with in vivo functions of hypusine and of aIF5A and eIF5A.

Topics: Cell Cycle; Enzyme Inhibitors; Escherichia coli; Flow Cytometry; Guanine; Lysine; Sulfolobus acidocaldarius

Dendritic cells (DCs), nature's adjuvant, must mature to sensitize T cells. However, although the maturation process is essential, it is not yet fully understood at the molecular level. In this study, we investigated the course of expression of the unique hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A), which is part of a particular RNA nuclear export pathway, during in vitro generation of human DCs. We show that eIF-5A expression is significantly upregulated during DC maturation. Furthermore, an inhibitor of the hypusine modification, GC7 (N(1)-guanyl-1, 7-diaminoheptane), prevents CD83 surface expression by apparently interfering with nucleocytoplasmic translocation of the CD83 mRNA and, importantly, significantly inhibits DC-mediated T lymphocyte activation. The data presented suggest that CD83 mRNA is transported from the nucleus to the cytoplasm via a specific nuclear export pathway and that hypusine formation appears to be essential for the maturation of functional DCs. Therefore, pharmacological interference with hypusine formation may provide a new possibility to modulate DC function.

Topics: Antigen Presentation; Antigens, CD; Biological Transport; CD83 Antigen; Cell Compartmentation; Cell Differentiation; Cell Nucleus; Dendritic Cells; Eukaryotic Translation Initiation Factor 5A; Guanine; Humans; Immunoglobulins; Lymphocyte Activation; Lysine; Membrane Glycoproteins; Peptide Initiation Factors; Protein Processing, Post-Translational; RNA-Binding Proteins; RNA, Messenger; T-Lymphocytes; Up-Regulation

Deoxyhypusine synthase catalyzes the initial step in the posttranslational formation of the amino acid hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] in eukaryotic initiation factor 5A (eIF-5A). eIF-5A and its hypusine modification are believed to be essential for cell growth. A number of compounds related to diamines and triamines were synthesized and tested as inhibitors of this enzyme. The findings indicate that the long chain triamines 2a and 2b and their guanyl derivatives 3a, 3b, 4a, and 4b exert inhibition by binding to enzyme through only a portion of their structures at any one time. The inhibition exhibited by N-ethyl-1,7-diaminoheptane 20 and its guanyl derivative 21 supports this notion and is evidence for participation of the secondary amino group in binding to enzyme. There is preliminary evidence that amidino and isothiuronium groups may also serve as basic centers for binding to enzyme. Few of the compounds tested here were comparable in inhibitory potency to 1-guanidino-7-aminoheptane (GC7) the most effective known inhibitor of deoxhypusine synthase, and none proved nearly as efficient as GC7 in inhibiting the enzyme in Chinese hamster ovary cells. Hence, unlike the antiproliferative effect of GC7, for which there is evidence of cause by interference with deoxhypusine synthase catalysis (Park, M. H.; Wolff, E. C.; Lee, Y. B.; Folk, J. E. J. Biol. Chem. 269, 1994, 27827-27832), the effective growth arrest exerted by several of the newly synthesized compounds cannot be attributed to inhibition of hypusine synthesis.

Topics: Animals; Binding, Competitive; Cell Division; CHO Cells; Cricetinae; Cricetulus; Diamines; Eukaryotic Translation Initiation Factor 5A; Humans; Lysine; Male; Oxidoreductases Acting on CH-NH Group Donors; Peptide Initiation Factors; Polyamines; Rats; RNA-Binding Proteins; Spermidine; Structure-Activity Relationship; Testis