Essential role of eIF5A-1 and deoxyhypusine synthase in mouse embryonic development.
ABSTRACT: The eukaryotic initiation factor 5A (eIF5A) contains a polyamine-derived amino acid, hypusine [N(ε)-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed post-translationally by the addition of the 4-aminobutyl moiety from the polyamine spermidine to a specific lysine residue, catalyzed by deoxyhypusine synthase (DHPS), and subsequent hydroxylation by deoxyhypusine hydroxylase (DOHH). The eIF5A precursor protein and both of its modifying enzymes are highly conserved, suggesting a vital cellular function for eIF5A and its hypusine modification. To address the functions of eIF5A and the first modification enzyme, DHPS, in mammalian development, we knocked out the Eif5a or the Dhps gene in mice. Eif5a heterozygous knockout mice and Dhps heterozygous knockout mice were viable and fertile. However, homozygous Eif5a1 (gt/gt) embryos and Dhps (gt/gt) embryos died early in embryonic development, between E3.5 and E7.5. Upon transfer to in vitro culture, homozygous Eif5a (gt/gt) or Dhps (gt/gt) blastocysts at E3.5 showed growth defects when compared to heterozygous or wild type blastocysts. Thus, the knockout of either the eIF5A-1 gene (Eif5a) or of the deoxyhypusine synthase gene (Dhps) caused early embryonic lethality in mice, indicating the essential nature of both eIF5A-1 and deoxyhypusine synthase in mammalian development.
Project description:The natural amino acid hypusine (N ?-4-amino-2-hydroxybutyl(lysine)) is derived from the polyamine spermidine, and occurs only in a single family of cellular proteins, eukaryotic translation factor 5A (eIF5A) isoforms. Hypusine is formed by conjugation of the aminobutyl moiety of spermidine to a specific lysine residue of this protein. The posttranslational synthesis of hypusine involves two enzymatic steps, catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusine is essential for eIF5A activity. Inactivation of either the eIF5A or the DHPS gene is lethal in yeast and mouse, underscoring the vital role of eIF5A hypusination in eukaryotic cell growth and animal development. The long and basic side chain of the hypusine residue promotes eIF5A-mediated translation elongation by facilitating peptide bond formation at polyproline stretches and at many other ribosome-pausing sites. It also enhances translation termination by stimulating peptide release. By promoting translation, the hypusine modification of eIF5A provides a key link between polyamines and cell growth regulation. eIF5A has been implicated in several human pathological conditions. Recent genetic data suggest that eIF5A haploinsufficiency or impaired deoxyhypusine synthase activity is associated with neurodevelopmental disorders in humans.
Project description:The eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unique polyamine-derived amino acid, hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Hypusine is formed in eIF5A by a novel post-translational modification reaction that involves two enzymatic steps. In the first step, deoxyhypusine synthase catalyzes the cleavage of the polyamine spermidine and transfer of its 4-aminobutyl moiety to the epsilon-amino group of one specific lysine residue of the eIF5A precursor to form a deoxyhypusine intermediate. In the second step, deoxyhypusine hydroxylase converts the deoxyhypusine-containing intermediate to the hypusine-containing mature eIF5A. The structure and mechanism of deoxyhypusine synthase have been extensively characterized. Deoxyhypusine hydroxylase is a HEAT-repeat protein with a symmetrical superhelical structure consisting of 8 helical hairpins (HEAT motifs). It is a novel metalloenzyme containing tightly bound iron at the active sites. Four strictly conserved His-Glu pairs were identified as iron coordination sites. The structural fold of deoxyhypusine hydroxylase is entirely different from those of the other known protein hydroxylases such as prolyl 4-hydroxylase and lysyl hydroxylases. The eIF5A protein and deoxyhypusine/hypusine modification are essential for eukaryotic cell proliferation. Thus, hypusine synthesis represents the most specific protein modification known to date, and presents a novel target for intervention in mammalian cell proliferation.
Project description:Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.
Project description:Deoxyhypusine synthase (DHPS) uses the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that enabled the inducible, postnatal deletion of Dhps specifically in postnatal islet ? cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces ? cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation and protein secretion, reduced production of the cell cycle molecule cyclin D2, impaired ? cell proliferation, and induced overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-? and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in ? cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis.
Project description:A naturally occurring unusual amino acid, hypusine [N (epsilon)-(4-amino-2-hydroxybutyl)-lysine] is a component of a single cellular protein, eukaryotic translation initiation factor 5A (eIF5A). It is a modified lysine with structural contribution from the polyamine spermidine. Hypusine is formed in a novel posttranslational modification that involves two enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). eIF5A and deoxyhypusine/hypusine modification are essential for growth of eukaryotic cells. The hypusine synthetic pathway has evolved in eukaryotes and eIF5A, DHS and DOHH are highly conserved, suggesting maintenance of a fundamental cellular function of eIF5A through evolution. The unique feature of the hypusine modification is the strict specificity of the enzymes toward its substrate protein, eIF5A. Moreover, DHS exhibits a narrow specificity toward spermidine. In view of the extraordinary specificity and the requirement for hypusine-containing eIF5A for mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes present new potential targets for intervention in aberrant cell proliferation.
Project description:The unusual basic amino acid, hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)-lysine], is a modified lysine with the addition of the 4-aminobutyl moiety from the polyamine spermidine. This naturally occurring amino acid is a product of a unique posttranslational modification that occurs in only one cellular protein, eukaryotic translation initiation factor 5A (eIF5A, eIF-5A). Hypusine is synthesized exclusively in this protein by two sequential enzymatic steps involving deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). The deoxyhypusine/hypusine synthetic pathway has evolved in archaea and eukaryotes, and eIF5A, DHS and DOHH are highly conserved suggesting a vital cellular function of eIF5A. Gene disruption and mutation studies in yeast and higher eukaryotes have provided valuable information on the essential nature of eIF5A and the deoxyhypusine/hypusine modification in cell growth and in protein synthesis. In view of the extraordinary specificity and functional significance of hypusine-containing eIF5A in mammalian cell proliferation, eIF5A and the hypusine biosynthetic enzymes are novel potential targets for intervention in aberrant cell proliferation.
Project description:Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the polyamine-modified lysine, hypusine [N(?)-(4-amino-2-hydroxybutyl)lysine]. Hypusine occurs only in eukaryotes and certain archaea, but not in eubacteria. It is formed post-translationally by two consecutive enzymatic reactions catalyzed by deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH). Hypusine modification is essential for the activity of eIF5A and for eukaryotic cell proliferation. eIF5A binds to the ribosome and stimulates translation in a hypusine-dependent manner, but its mode of action in translation is not well understood. Since quantities of highly pure hypusine-modified eIF5A is desired for structural studies as well as for determination of its binding sites on the ribosome, we have used a polycistronic vector, pST39, to express eIF5A alone, or to co-express human eIF5A-1 with DHS or with both DHS and DOHH in Escherichia coli cells, to engineer recombinant proteins, unmodified eIF5A, deoxyhypusine- or hypusine-modified eIF5A. We have accomplished production of three different forms of recombinant eIF5A in high quantity and purity. The recombinant hypusine-modified eIF5A was as active in methionyl-puromycin synthesis as the native, eIF5A (hypusine form) purified from mammalian tissue. The recombinant eIF5A proteins will be useful tools in future structure/function and the mechanism studies in translation.
Project description: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.
Project description:The gene encoding eukaryotic initiation factor 5A (EIF5A) is found in diabetes-susceptibility loci in mouse and human. eIF5A is the only protein known to contain hypusine (hydroxyputrescine lysine), a polyamine-derived amino acid formed post-translationally in a reaction catalyzed by deoxyhypusine synthase (DHPS). Previous studies showed pharmacologic blockade of DHPS in type 1 diabetic NOD mice and type 2 diabetic db/db mice improved glucose tolerance and preserved beta cell mass, which suggests that hypusinated eIF5A (eIF5AHyp) may play a role in diabetes pathogenesis by direct action on the beta cells and/or altering the adaptive or innate immune responses. To translate these findings to human, we examined tissue from individuals with and without type 1 and type 2 diabetes to determine the expression of eIF5AHyp. We detected eIF5AHyp in beta cells, exocrine cells and immune cells; however, there was also unexpected enrichment of eIF5AHyp in pancreatic polypeptide-expressing PP cells. Interestingly, the presence of eIF5AHyp co-expressing PP cells was not enhanced with disease. These data identify new aspects of eIF5A biology and highlight the need to examine human tissue to understand disease.
Project description:Deoxyhypusine synthase (DHPS) utilizes the polyamine spermidine to catalyze the hypusine modification of the mRNA translation factor eIF5A and promotes oncogenesis through poorly-defined mechanisms. Because germline deletion of Dhps is embryonically lethal, its role in normal postnatal cellular function in vivo remains unknown. We generated a mouse model that allows for inducible, postnatal deletion of Dhps specifically in postnatal islet β cells, which function to maintain glucose homeostasis. Removal of Dhps did not have an effect under normal physiologic conditions. However, upon development of insulin resistance, which induces β-cell proliferation, Dhps deletion caused alterations in proteins required for mRNA translation, reduced production of the cell cycle molecule Cyclin D2, impaired β-cell proliferation, and overt diabetes. We found that hypusine biosynthesis was downstream of protein kinase C-ζ and was required for c-Myc-induced proliferation. Our studies reveal a requirement for DHPS in β cells to link polyamines to mRNA translation to effect facultative cellular proliferation and glucose homeostasis. Overall design: RNA sequencing data from RNA isolated from pancreatic islets from control or Dhsp β cell specific mice (C57.DhpsloxP.MIP1-CreERT)