Biological signals最新文献

The subcellular distributions of the endogenous eukaryotic translation initiation factor, eIF-5A, and Rev, a protein of the human immunodeficiency virus proposed to interact with eIF-5A, were studied in COS-7 cells treated with inhibitors of RNA or protein synthesis. We have previously shown that transiently expressed Rev is localized in the nucleolus, whereas eIF-5A is primarily in the cytoplasm. The subcellular localization of Rev was not affected by treatment with protein synthesis inhibitors (cycloheximide, CHX, 10 micrograms/ml; puromycin, 10 micrograms/ml), although its location changed from predominantly the nucleolus to the cytoplasm after treatment with RNA synthesis inhibitors (actinomycin D, 4 micrograms/ml, and 5,6-dichloro-1 beta-D-ribofuranosylbenzimidazole, DRB; 0.1 mM), as previously reported. In contrast, none of the RNA synthesis inhibitors (alpha-amanitin, 10 micrograms/ml; actinomycin D, 4 micrograms/ml, and DRB, 0.1 mM) caused any significant changes in the subcellular distribution pattern of eIF-5A. However, treatment with puromycin, a protein synthesis inhibitor known to dissociate ribosomes, dramatically altered the subcellular distribution pattern of eIF-5A in 30% of the cell population. In these cells, the staining of eIF-5A was changed from an endoplasmic reticulum (ER) net work-like perinuclear structure to a patched dotted pattern dispersed throughout the cytoplasm. This change was not observed in the same cells stained for calnexin, an ER resident protein, nor in cells treated with CHX, which freezes the ribosomes to block protein synthesis. Our data suggest that eIF-5A does not shuttle between the nucleus and cytoplasm in the same way as Rev. Our findings are consistent with our previous conclusion that eIF-5A is associated with the ER through ribosomes and support a role for eIF-5A in protein synthesis.

Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor represents a unique posttranslational modification that is ubiquitously present in eukaryotic cells and archaebacteria. Specific inhibition of deoxyhypusine synthase leads to growth arrest and cell death. The precise cellular function of eIF-5A and the physiological significance of hypusine modification are not clear. Although the methionyl-puromycin synthesis has been suggested to be the functional assay for eIF-5A activity in vitro, the role of eIF-5A in protein synthesis has not been established. Recent studies have suggested that eIF-5A may be the cellular target of the human immunodeficiency virus type 1 Rev and human T cell leukemia virus type 1 Rex proteins. Motif analysis suggested that eIF-5A resembles a bimodular RNA-binding protein in that it contains a stretch of basic amino acids clustered at the N-terminal region and a leucine-rich stretch at the C-terminal region. Using Rev target RNA, RRE, as a model, we tested the hypothesis that eIF-5A may be an RNA-binding protein. We found that both deoxyhypusine and hypusine-containing eIF-5A can bind to the 252-nt RRE RNA, as determined by a gel mobility shift assay. In contrast, the unmodified eIF-5A precursor cannot. Deoxyhypusine-containing eIF-5A, but not its precursor, could also cause supershift of the Rev stem-loop IIB RRE complex. Preliminary studies also indicated that eIF-5A can bind to RNA such as U6 snRNA and that deoxyhypusine modification appears to be required for the binding. The ability of eIF-5A to directly interact with RNA suggests that deoxyhypusine formation of eIF-5A may be related to its role in RNA processing and protein synthesis. Our study also suggests the possibility of using a gel mobility shift assay for eIF-5A-RNA binding as a functional assay for deoxyhypusine and hypusine formation.

Hypusine [N epsilon-(4-amino-2-hydroxybutyl)lysine] occurs in all eukaryotes at one residue in a highly conserved protein, the putative eukaryotic translation initiation factor 5A (eIF-5A, old terminology eIF-4D). This unusual amino acid is produced in a unique posttranslational modification reaction that involves the conjugation of the 4-aminobutyl moiety of the polyamine spermidine to the epsilon-amino group of a specific lysine residue of the eIF-5A precursor protein to form the deoxyhypusine [N epsilon-(4-aminobutyl)lysine] residue and its subsequent hydroxylation. The strict specificity of hypusine synthesis, its derivation from spermidine and its requirement for the activity of eIF-5A and for eukaryotic cell proliferation have raised keen interest in the physiological function of the hypusine-containing protein, eIF-5A.

The polyamines are essential for eukaryotic cell growth. One of the most critical effects of polyamines on cell growth is the availability of spermidine for the post-translational modification of eIF-5A. Because hypusine-containing eIF-5A is necessary for cell proliferation, depletion of cellular polyamines suppresses growth by depleting cellular modified eIF-5A content. Excess putrescine accumulations in DH23A/b cells induces apoptosis and suppresses the formation of hypusine-containing eIF-5A. Treatment of DH23A/b cells with diaminoheptane also suppresses modified eIF-5A formation and induces apoptosis. These data suggest that suppression of modified eIF-5A formation may play a role in putrescine-induced apoptosis as well.

A unique amino acid, hypusine, is formed posttranslationally in the precursor of eukaryotic translation initiation factor 5A (eIF-5A). Deoxyhypusine synthase catalyzes the first of two steps in the biosynthesis of hypusine. We reported earlier that the DYS1 gene encoding deoxyhypusine synthase is essential for cell viability and proliferation in yeast. Here, we show by deletion studies that both N- and C-terminal regions, which are not so well conserved, are necessary for the activity of the yeast enzyme. Of the seven cysteine residues present in the yeast enzyme, only one cysteine (position 252; C252) appeared to be essential for its activity. Moderate overexpression of DYS1 showed very little effects on cell growth and no obvious effects on the intracellular level of eIF-5A. However, repression of the expression of DYS1 resulted in near-complete depletion of eIF-5A 24 h after the initiation of repression and was followed by cell growth arrest after another 24 h. This novel finding suggests that the major role of deoxyhypusine synthase in cell proliferation is mediated not only through its modification of the eIF-5A precursor, but also through its regulation of intracellular eIF-5A levels.

Eukaryotic initiation factor 5A (eIF-5A) is the only cellular protein known to contain the unusual amino acid hypusine, a modification that appears to be required for cell proliferation. This hypusine-modified protein stimulates synthesis of methionyl-puromycin in an in vitro assay which mimics the formation of the first peptide bond during protein synthesis, although the exact role of eIF-5A in vivo is still unknown. The unexpected finding that eIF-5A is a cellular cofactor of the HIV-1 Rev trans-activator protein may, however, provide a novel opportunity to reveal precisely what function eIF-5A performs in eukaryotic cells. In this review article, we first present a brief description of HIV-1 Rev function, followed by an overview of the data that identified eIF-5A as a Rev cofactor and, finally, discuss novel findings with respect to cellular eIF-5A activities.

Ovarian follicular kinetics and gravimetric changes in the ovary and oviducts were studied in the skipper frog, Rana cyanophlyctis, following exposure to continuous light and melatonin treatment during the breeding season. Daily late-afternoon injections of melatonin (15 micrograms subcutaneous) for 30 days decreased the gonadosomatic index (GSI), whereas continuously available melatonin from subcutaneous implants did not influence the GSI compared to those of controls. Exposure to continuous light for 30 days stimulated the GSI, and melatonin given as daily injections prevented the continuous-light induced increase in GSI. Oviductal weights decreased only in the melatonin-injected groups. Data on follicular kinetics revealed a decrease in first-growth-phase (FGP) oocytes and an increase in medium-sized second-growth-phase (MSGP) and large-sized second-growth-phase (LSGP) oocytes following continuous-light exposure. Melatonin administered to continuous-light-exposed frogs did not change the FGP oocyte number; however, it reduced both the MSGP and LSGP oocytes. Melatonin administration to frogs held in a light and dark cycle increased FGP oocytes and decreased MSGP and LSGP oocytes. Atretic follicles increased in all melatonin-treated groups. The results suggest that continuous light stimulates and melatonin inhibits reproductive function in this species.

Effects of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) and cations on 2-[125I]iodomelatonin binding were investigated in membrane preparations of the chicken spinal cord. At concentrations of 10 and 50 mumol/l, GTP gamma S dose-dependently increased (p < 0.05) the equilibrium dissociation constant (Kd) and depressed (p < 0.05) the maximum number of binding sites (Bmax). Na+ at a concentration of 125 mmol/l significantly increased (p < 0.05) the Kd and decreased (p < 0.05) the Bmax, and Mg2+ (2.5 mmol/l) significantly increased (p < 0.05) the Bmax without changes in Kd. In addition, Na+ and Mg2+ affected the interactions of GTP gamma S with melatonin receptors. In the spinal cord explants, melatonin (10 nmol/l) attenuated forskolin-stimulated cyclic AMP production by 53.1%, and preincubation with pertussis toxin abolished this effect of melatonin. These results suggest that the melatonin receptors in the chicken spinal cord are linked to its second messenger via a pertussis-toxin-sensitive guanine-nucleotide-binding protein, and that cations modulate these receptors. Our studies further support a previous hypothesis that melatonin exerts a direct action on spinal cord functions.

In an attempt to define the role of the pineal hormone melatonin and two analogues (5-methoxytryptamine, 5MT, and 6-hydroxymelatonin, 6HM) in limiting oxidative stress, the present study investigated the changes in glutathione, lipid peroxidation, and the activity of the antioxidant enzyme glutathione peroxidase after exercise (swimming for 60 min) with or without treatment with the indolamines mentioned. Lipid peroxidation was measured by estimating tissue levels of malondialdehyde and 4-hydroxyalkenals; the experimental animals in these studies were male Sprague-Dawley rats. In the liver, swimming exercise increased the levels of reduced glutathione (GSH) and also significantly increasing oxidized glutathione (GSSG), while decreasing the GSH/GSSG ratio, an index directly related to oxidative stress. When the animals were treated with melatonin, the concentrations of GSH and GSSG were also increased after swimming; however, no reduction in the GSH/GSSG ratio appeared. In the animals treated with 6HM the changes were the same as in those treated with melatonin. In muscle as well, the concentration of GSH and the GSH/GSSG ratio were decreased following 60 min of swimming. Pretreatment of the rats with melatonin prevented these effects. Pretreatment of the rats with both 5MT and 6HM also prevented the changes. Brain GSH/GSSG ratio was not affected by either exercise or indolamine administration. Swimming enhanced lipid peroxidation in the liver, muscle and brain; however, this was prevented in animals treated with melatonin or 6HM before swimming. Glutathione peroxidase was significantly elevated after exercise in the brain but not in the liver and muscle. It is concluded that swimming imposes a severe oxidative stress and suggests that melatonin and, to a lesser degree, 5MT and 6HM confer protection against the oxidative damage associated with swimming for 60 min. This mechanism may be reasonably attributed to their indole structure, which possibly allows these molecules to act as free-radical scavengers.