Evidence supporting a physiological role for the hemin-controlled translational repressor of globin synthesis in reticulocytes.

The synthesis of heme and globin in rabbit reticulocytes was compared at 35 and 25 degrees C. The lower temperature decreased heme synthesis significantly more than globin synthesis and resulted in a much greater accumulation of globin dimers. After 16 h of incubation in the absence of iron, globin synthesis in reticulocytes which were at 35 degrees C could not be stimulated by iron, whereas cells which were at 25 degrees C responded with nearly control levels of globin synthesis. Since the formation of the hemin-controlled translational repressor in reticulocyte lysates is also decreased much more than protein synthesis at reduced temperature the results provide evidence for a physiological role for the translational repressor in controlling globin synthesis in reticulocytes.     

Partial reaction of peptide initiation inhibited by the reticulocyte hemin-controlled repressor

The hemin-controlled repressor from rabbit reticulocytes inhibits binding of Met-tRNA_f to reticulocyte 40S ribosomal subunits in a partial reaction containing these components, two initiation factor fractions and GTP. The inhibitor does not interfere with the formation of the Met-tRNA_f· initiation factor IF-E₂·GTP complex.     

Effect of antibody to the hemin-controlled translational repressor in rabbit reticulocyte lysate

We have examined the effect of the purified IgG from the serum of guinea pigs immunized with a highly purified preparation of rabbit reticulocyte, hemin-controlled translational repressor (HCR) on protein synthesis in the reticulocyte lysate. We have found that the anti-HCR (but not non-immune) IgG completely prevents or reverses the suppression of protein synthesis that occurs in hemin-deficient lysate, providing a direct and definitive demonstration that the inhibitory effect of hemin-deficiency is mediated solely by the activation of HCR. The anti-HCR IgG also prevents or reverses the phosphorylation of eIF-2 alpha and the reduced binding of Met-tRNAf to 40 S ribosomal subunits that accompanies the inhibition of protein synthesis in hemin-deficient lysate. In contrast, the anti-HCR IgG has no effect on the inhibition produced by low levels of double-stranded RNA (that is due to the activation of a separate protein kinase), but it does partly reverse inhibition due to oxidized glutathione, ethanol, and phosphatidylserine, indicating that the effect of these components is mediated, at least in part, by the activation of HCR. Finally, we have confirmed our earlier observation that an excess of proHCR, the inactive precursor of HCR, has little effect on the neutralization of HCR by limiting anti-HCR IgG, suggesting that the antigenic determinants on HCR are not exposed on ProHCR.     

Reversal of the inhibitory action of the hemin-controlled translational repressor by a post-ribosomal supernatant factor from rabbit reticulocyte lysate.

The inhibitory effect of the hemin-controlled translational repressor (HCR) on protein synthesis by rabbit reticulocyte lysates can be overcome by a factor in the post-ribosomal supernatant fraction. When chromatographed on Sepharose 6B, this supernatant factor migrates as a high molecular weight component that is distinct from the precursor of HCR (prorepressor). The supernatant factor does not appear to act by enzymatically degrading the repressor or by forming a stoichiometric complex with it, but may, rather, replace what has become limiting for protein synthesis due to repressor action.     

Structural requirements for porphyrin inhibition of the hemin-controlled protein kinase and maintenance of protein synthesis in reticulocytes

The role of hemin in the maintenance of protein synthesis in reticulocyte lysates was examined by comparing the effects of various porphyrins and metalloporphyrins on the protein kinase activity of the hemin-controlled repressor and on protein synthesis. The porphyrin requirements for maintenance of protein synthesis were relatively specific. Iron and cobalt metalloporphyrins sustained protein synthesis whereas other metalloporphyrins, metal-deficient porphyrins, and non-porphyrin precursor and degradation products of protoporphyrin IX were ineffective. These same compounds were examined for their effectiveness in inhibiting the protein kinase activity of the hemin-controlled repressor with initiation factor 2 (eIF-2). Most of the metalloporphyrins and porphyrins tested were inhibitory. The presence of the iron atom in the porphyrin was not essential for inhibition, but the maintenance of the integrity of the porphyrin ring was imperative. The porphyrins which inhibited the hemin-regulated protein kinase contained vinyl groups or ethyl groups, or were protonated in the 2- and 4-positions of the porphyrin ring, whereas those with bulky or acidic groups in these positions were ineffective. Precursor and degradation products of protoporphyrin IX and synthetic porphyrins modified at other positions had no effect on the enzyme. Both hemin and protoporphyrin IX inhibited phosphorylation of eIF-2 exogenously added to a reticulocyte lysate; however, hemin sustained protein synthesis in the lysate, whereas protoporphyrin IX did not. These results suggest that regulation of the protein kinase phosphorylating the alpha subunit of eIF-2 is not the only point at which hemin modulates protein synthesis in reticulocytes and reticulocyte lysates, since a correlation between inhibition of protein synthesis, inhibition of protein kinase activity, and phosphorylation of eIF-2 is not observed with all porphyrins.     

Additional evidence that the hemin-controlled translational repressor from rabbit reticulocytes is a protein kinase

Recent reports have suggested that the hemin-controlled translational repressor (HCR) which mediates the hemin control of protein synthesis in reticulocyte lysates, acts as a specific protein kinase, phosphorylating a subunit of the Met-tRNA_f binding factor (IF-1). We have found that crude and highly purified HCR can phosphorylate a 38,000 molecular weight component of IF-1, but that crude prorepressor (the precursor of HCR), which is not inhibitory, does not phosphorylate this component. Prolonged warming of the prorepressor induces the formation of the inhibitor and the protein kinase that phosphorylates the 38,000 molecular weight protein, and the formation of both is blocked by hemin. In addition, a brief incubation of the prorepressor with N-ethylmaleimide, which produces maximal inhibitory activity within 5 minutes, also induces formation of the protein kinase. These findings suggest that HCR and the protein kinase are the same protein and provide additional support for the concept that HCR controls protein synthesis by phosphorylating the Met-tRNA_f binding factor.     

Inhibition of an initiation codon function by hemin deficiency and the hemin-controlled translational repressor in the reticulocyte cell-free system

The trinucleotide codon, AUG, promotes the combination of reticulocyte 40S and 60S native ribosomal subunits as well as recombination of those derived by dissociation factor. This interaction is inhibited by hemin deficiency or the hemin-controlled repressor and results in the loss of methionine from ribosomal bound met-tRNA_f. The locus of inhibition among the partial reactions of peptide chain initiation is between met-tRNA_f and initiation codon binding to the 40S ribosomal subunit and peptide bond formation.     

Fluoride inhibition of the initiation of protein synthesis in the reticulocyte lysate cell-free system.

KF (30 mM) strongly inhibits polypeptide chain initiation in the reticulocyte lysate cell-free system.. Chain elongation is partially inhibited but proceeds to a significant extent with little initiation of new chains. Polysome breakdown is incomplete after incubations as long as 20 min. Under these conditions deacylated tRNA-Met accumulates in a fraction sedimenting faster than 120 S and thus may be associated with ribosomes bound to mRNA. Incubation of the system with KF results in the accumulation of a complex which can initiate synthesis of polypeptide chains in the presence of aurintricarboxylate; KF thus inhibits a step in initiation after that inhibited by aurintricarboxylate. The accumulation of deacylated tRNA-Met is correlated with the accumulation of the aurintricarboxylate-resistant complex. Both phenomema are dependent on KF and both are inhibited by aurintricarboxylate in the same range of concentrations which inhibit initiation of protein synthesis.     

Control of protein synthesis in reticulocyte lysates: the effect of nucleotide triphosphates on formation of the translational repressor.

High concentrations (1–2mM) of GTP completely prevented the decline in protein synthesis which occurs in a reticulocyte lysate incubated in the absence of haemin. The effect of GTP was diminished by raising the concentration of ATP. When formation of the haemin-controlled translational repressor was studied as a partial reaction using post-ribosomal supernatant, it was found that GTP acted by blocking repressor formation, whilst ATP was found to stimulate this process.     

Highly potent dUTPase inhibition by a bacterial repressor protein reveals a novel mechanism for gene expression control

Transfer of phage-related pathogenicity islands of Staphylococcus aureus (SaPI-s) was recently reported to be activated by helper phage dUTPases. This is a novel function for dUTPases otherwise involved in preservation of genomic integrity by sanitizing the dNTP pool. Here we investigated the molecular mechanism of the dUTPase-induced gene expression control using direct techniques. The expression of SaPI transfer initiating proteins is repressed by proteins called Stl. We found that Φ11 helper phage dUTPase eliminates SaPIbov1 Stl binding to its cognate DNA by binding tightly to Stl protein. We also show that dUTPase enzymatic activity is strongly inhibited in the dUTPase:Stl complex and that the dUTPase:dUTP complex is inaccessible to the Stl repressor. Our results disprove the previously proposed G-protein-like mechanism of SaPI transfer activation. We propose that the transfer only occurs if dUTP is cleared from the nucleotide pool, a condition promoting genomic stability of the virulence elements.     

p53-induced inhibition of protein synthesis is independent of apoptosis.

Activation of a temperature-sensitive form of p53 in murine erythroleukaemia cells results in a rapid impairment of protein synthesis that precedes inhibition of cell proliferation and loss of cell viability by several hours. The inhibition of translation is associated with specific cleavages of polypeptide chain initiation factors eIF4GI and eIF4B, a phenomenon previously observed in cells induced to undergo apoptosis in response to other stimuli. Although caspase activity is enhanced in the cells in which p53 is activated, both the effects on translation and the cleavages of the initiation factors are completely resistant to inhibition of caspase activity. Moreover, exposure of the cells to a combination of the caspase inhibitor z-VAD.FMK and the survival factor erythropoietin prevents p53-induced cell death but does not reverse the inhibition of protein synthesis. We conclude that the p53-regulated cleavages of eIF4GI and eIF4B, as well as the overall inhibition of protein synthesis, are caspase-independent events that can be dissociated from the induction of apoptosis per se.     

On the mechanism of the glucagon-induced inhibition of hepatic protein synthesis.

The intraperitoneal administration of glucagon (200 μg) to rats produced a transient increase of the hepatic polypeptide chain completion time, the increase being maximum at 5 min returning to control values at 20 min. This inhibitory effect was sustained when glucagon was constantly supplied by continuous infusion. Postmitochondrial supernatants from livers of the control group or rats treated with glucagon for 5 min showed no difference in their protein synthetic activity. After 20 min of intraperitoneal administration of the hormone, that is, when the effect on protein synthesis had vanished, the levels of cAMP were still 40% above those of the control group, and the ribosomal proteins were 110% more phosphorylated. These results suggest that the observed effect of glucagon is not due to its direct action on the protein synthesis machinery. On the other hand, the variations in the hepatic amino acid content brought about by glucagon do not appear to be quantitatively significant to account for the observed inhibition of protein synthesis. The effect of glucagon was always paralleled by a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio which may be responsible for the observed decrease in the rates of elongation and/or termination steps of protein synthesis. Glucagon also produced a rise in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio in both cellular compartments, cytosol and mitochondria, as reflected by the rise in the lactate to pyruvate and the β-hydroxybutyrate to acetoacetate ratios. This shift of the NAD⁺ couple to a more reduced state seems to be the result of an increased mobilization and oxidation of fatty acids brought about by the hormone. It is postulated then that the primary effect of glucagon leading to a decrease in protein synthesis is probably to increase the state of reduction of the hepatic nicotinamide nucleotide system. This point of view is supported by the fact that the nicotinamide and adenine nucleotide systems in rat liver are in equilibrium through cytosolic equilibrium reactions, so that a decrease in the $\text{[ATP]}\text{[ADP]}$ ratio brought about by glucagon may be secondary to the increase in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ ratio. This hypothesis is supported by the fact that glucagon was not effective in inhibiting hepatic protein synthesis in rats pretreated with a drug, 2-benzene-sulfonamido-5-(β-methoxy-ethoxy)pyrimidine, that prevents fatty acid mobilization and the subsequent changes in the $\text{[NADH]}\text{[NAD}{}^{\mathrm{+}}\text{]}$ and $\text{[ATP]}\text{[ADP]}$ ratios. Furthermore, the administration of exogenous fatty acid brings about an inhibition of the rate of hepatic protein synthesis accompanied by a decrease in the ATP levels and an increase in the state of reduction of the NAD⁺ system.     

Differential inhibition of protein synthesis in reticulocyte lysates and wheat germ extracts by a protein isolated from wheat germ extracts.

A protein with a molecular mass of 35-37 kDa has been isolated and partially purified from the postribosomal supernatant of wheat germ by ammonium sulfate precipitation (60-90%), Sephadex G-75, and DEAE-cellulose chromatography. It inhibited endogenous protein synthesis in rabbit reticulocyte lysates but had no effect on translation in wheat germ extracts. At low concentrations (0.34-1.36 ng/15 microliter assay), inhibition was limited to initiation of peptide synthesis. At higher concentrations (13.6 ng/15 microliter assay), elongation was also suppressed.     

Purification and characterization of a protein factor that reverses the inhibition of protein synthesis by the heme-regulated translational inhibitor in rabbit reticulocyte lysates.

We have purified and partially characterized a supernatant factor which reverses the effect of the heme-regulated translational inhibitor on protein synthesis in rabbit reticulocyte lysates. The anti-inhibitor restores protein synthesis activity in heme deficient lysates (and in lysates to which the inhibitor has been added) to the level observed in the presence of heme. The factor has no effect on the phosphorylation of eIF-2 by the inhibitor nor on any reaction carried out with purified initiation factors. The anti-inhibitor probably consists of three subunits with molecular weights of 81000, 60000 and 41000. The factor is isolated from the postribosomal supernatant of rabbit reticulocytes both free and complexed to eIF-2. A possible mechanism of action is discussed.