Inhibition Of Peptide Chain Initiation Research Articles

Insulin-like growth factor I accelerates protein synthesis in skeletal muscle during sepsis.

Sepsis causes an inhibition of protein synthesis in gastrocnemius that is resistant to the anabolic effects of insulin. The purpose of the present studies was to investigate the effect of recombinant human insulin-like growth factor I (IGF-I) on protein synthesis during a 30-min perfusion of the isolated rat hindlimb from septic rats. Inclusion of IGF-I (1 or 10 nM) in the perfusate stimulated protein synthesis in gastrocnemius of septic rats 2.5-fold and restored rates of protein synthesis to those observed in control rats. The stimulation of protein synthesis did not result from an increase in the RNA content but was correlated with a 2.5-fold increase in the translational efficiency. The enhanced translational efficiency was accompanied by a 33 and 55% decrease in the abundance of free 40S and 60S ribosomal subunits, respectively, indicating that IGF-I accelerated peptide-chain initiation relative to elongation/termination. These studies provide evidence that IGF-I can accelerate protein synthesis in gastrocnemius during chronic sepsis by reversing the sepsis-induced inhibition of peptide-chain initiation.

Sepsis-induced changes in protein synthesis: differential effects on fast- and slow-twitch muscles.

Sepsis is associated with severe muscle wasting. Mechanisms responsible for sepsis-induced alterations in muscle protein metabolism were investigated in vivo and compared with changes induced by nonseptic inflammation. The rate of protein synthesis in mixed hindlimb muscles was not altered in inflammation but was inhibited 50% in sepsis. This inhibition did not result from a decreased RNA content. Instead, the translational efficiency was significantly reduced by 50% in skeletal muscle of septic animals compared with control. The effect of sepsis to lower the rate of protein synthesis was further examined using individual muscles containing different fiber types. Both the protein concentration and protein synthetic rate in fast-twitch muscles were reduced by sepsis, whereas neither of these parameters was affected in slow-twitch muscles or heart. The decreased translational efficiency did not result from a change in the rate of peptide-chain elongation. Instead, the sepsis-induced inhibition of protein synthesis resulted from a restraint in peptide-chain initiation because sepsis caused a 1.6-fold increase in free ribosomal subunits. Overall, sepsis, but not inflammation, caused an inhibition of protein synthesis primarily in muscles composed of fast-twitch fibers. The mechanism involved in the reduced rates of protein synthesis in muscles resulted from an inhibition of peptide-chain initiation, with no change in peptide-chain elongation.

Phosphorylation of only serine-51 in protein synthesis initiation factor-2 is associated with inhibition of peptide-chain initiation in reticulocyte lysates

We have examined the phosphorylation of the α-subunit of initiation factor-2 (eIF-2α) in reticulocyte lysates in which translational shut-off was induced by haem-deficiency or by double-stranded RNA. To maximise the phosphorylation of eIF-2α, lysates were supplemented with the broad spectrum phosphatase inhibitor microcystin. Under all conditions tested, serine-51 was the only residue to become labelled. This is consistent with the observation of only two species of eIF-2α in isoelectric focusing/immunoblotting analyses of lysates treated as described above.

Mechanism of inhibition of peptide chain initiation by amino acid deprivation in perfused rat liver. Regulation involving inhibition of eukaryotic initiation factor 2 alpha phosphatase activity

In previous studies, initiation of protein synthesis was shown to be inhibited in perfused rat livers deprived of single essential amino acids. In the present study, histidinol, a competitive inhibitor of histidinyl-tRNA synthetase, was used to amplify the effects of histidine deprivation on protein synthesis in perfused liver to facilitate investigation of mechanisms involved in the inhibition of peptide chain initiation. Protein synthesis was reduced to 77% of the control rate in livers deprived of histidine and to 13% of the control rate in livers deprived of histidine and exposed to 2.0 mM histidinol. The inhibition of protein synthesis caused by histidine deprivation alone was accompanied by a 2-fold increase in the number of free ribosomal particles, a 29% decrease in Met-tRNA(i) binding to 43 S preinitiation complexes, and a 31% reduction in activity of eukaryotic initiation factor 2B (eIF-2B). By comparison, histidine deprivation combined with histidinol addition resulted in a 3-fold increase in free ribosomal particles, a 66% decrease in Met-tRNAi binding, and a 78% reduction in eIF-2B activity. The proportion of the alpha-subunit of eukaryotic initiation factor two (eIF-2) in the phosphorylated form increased from 8.9 +/- 0.8% in control livers to 52.4 +/- 5.5% in response to histidinol. The increase in the amount of eIF-2 alpha in the phosphorylated form apparently was not due to an increase in kinase activity, because there was no change in eIF-2 alpha kinase activity in extracts of liver perfused with medium containing histidinol compared to controls. Instead, the increased phosphorylation of eIF-2 alpha was associated with an inhibition of eIF-2 alpha phosphatase activity. Thus, in contrast to other systems that have been examined, the mechanism involved in the increase in the phosphorylation state of eIF-2 alpha appears to involve an inhibition of eIF-2 alpha phosphatase activity rather than activation of an eIF-2 alpha kinase.

Regulation of initiation of protein synthesis by insulin in skeletal muscle

Protein synthesis is impaired in skeletal muscle and heart from diabetic rats. In muscles composed primarily of slow-twitch fibres (e.g. heart or soleus), the inhibition of protein synthesis can be accounted for entirely by a decrease in the amount of RNA. In contrast, in muscles of mixed fibre composition (e.g. gastrocnemius or psoas), the inhibition of protein synthesis is associated with an impairment in peptide-chain initiation. We have found that the inhibition of peptide-chain initiation that occurs in muscles composed of mixed fast-twitch fibres involves eukaryotic initiation factor 2B (eIF-2B). Thus, eIF-2B activity is inhibited in gastrocnemius and psoas but not heart or soleus from diabetic rats. In other systems eIF-2B activity is regulated by phosphorylation of the alpha-subunit of a second initiation factor, eIF-2. However, we have found no change in the phosphorylation state of eIF-2 alpha in either fast- or slow-twitch muscles from diabetic compared to control animals. Instead, the available evidence suggests that eIF-2B activity may be modulated by an alternate mechanism such as a change in the extent of phosphorylation of the 82,000 Mr subunit of the factor or a change in the NADPH/NADP+ ratio.

Effect of starvation on initiation of protein synthesis in skeletal muscle and heart.

Psoas muscle of rats starved for 2 or 4 days contained increased levels of ribosomal subunits and exhibited reduced rates of protein synthesis in vitro, demonstrating a starvation-induced inhibition of peptide-chain initiation. The activity of an eIF-2-like initiation factor, assayed in postribosomal supernatants, decreased in psoas during starvation, parallel to a 25% reduction in the RNA level. Reduced eIF-2 activity did not result from nucleotide depletion or increased deacylation of initiator tRNA, nor was it abolished by extensive dialysis. Perfusion of psoas muscle in the presence of insulin reversed the starvation-induced block in peptide-chain initiation, but did not alter the activity of eIF-2 or level of RNA. Furthermore, heart muscle did not manifest a starvation-induced block in peptide-chain initiation even though the activity of eIF-2 and the level of RNA decreased as a result of food deprivation. Thus loss of eIF 2 activity in psoas and heart did not parallel changes in peptide-chain initiation but was associated with a reduction in tissue RNA. These results indicate that the level of eIF-2 is not rate-limiting for peptide-chain initiation under the conditions tested in this study.

Protein turnover in rat skeletal muscle: effects of hypophysectomy and growth hormone.

The role of growth hormone in regulating protein turnover was examined in a perfused preparation of rat skeletal muscle. The perfused muscle maintained in vivo levels of ATP and creatine phosphate and exhibited constant rates of oxygen consumption and protein synthesis. Hypophysectomy reduced the rate of protein synthesis, the concentration of RNA, and the efficiency of protein synthesis in gastrocnemius muscle to 30, 46, and 66 percent of normal, respectively. In vivo treatment of hypophysectomized (hypox) rats with bovine growth hormone (250 microgram/day for 5 days) resulted in small increases in protein synthesis and RNA, whereas synthesis/RNA was returned to near normal. Elevation of ribosomal subunits in psoas muscle indicated an inhibition of peptide-chain initiation in hypox rats that was reversed by in vivo growth hormone treatment. Thus, hypox rats exhibited both a decreased capacity and a decreased efficiency of protein synthesis. Growth hormone replacement primarily increased efficiency of protein synthesis. The rate of protein degradation and the activity of cathepsin D in gastrocnemius muscle were decreased by hypophysectomy. Growth hormone treatment had no significant effect on degradation.

The stringent response to inhibition of peptide chain initiation in Escherichia coli

In most strains of E. coli synthesis of the stable RNA species is coordinately depressed if protein synthesis is lowered either by lack of any aminoacid or by inactivation of any aminoacyl-tRNA synthetase [ 1 ] . This stringent dependence of RNA accumulation on aminoacid availability or activation was termed the ‘stringent response’ [2]. Mutant relaxed strains have been obtained [ 1 ] in which however this dependence does not occur i.e. stable RNA synthesis is not inhibited in the absence of aminoacids (‘relaxed response’). In addition to RNA metabolism, several other cellular activities respond to aminoacid deprivation but only in stringent (rer*) strains and not in relaxed (rel-) strains (for review see [3]). Namely, in the absence of protein synthesis, unusual guanosine polyphosphates ppGpp and pppGpp accumulate in the nucleotide pool; also the level of polysomes is considerably lowered even though the persisting polysomes are maintained in a state of turnover [4-T]. Protein synthesis in intact bacteria can obviously be arrested by other means than withdrawing an essential aminoacid from the culture medium or inactivating a particular aminoacyl-tRNA synthetase. Thus it can be specifically blocked in the initiation phase by treating the cells with hydroxylamine or trimethoprim which both inhibit the formylation of the initiator N-formylMet-tRNA, [8-l 31. It has been previously shown that the response of re2’ and relstrains of E. coli to treatment by either inhibitor is identical to that to aminoacid deprivation as far as RNA synthesis and guanosine polyphosphate accumulation are concerned [ 10,11,14] . However, to our knowledge, polysome behaviour has not been

Individual translational efficiencies of SV 40 and cellular mRNAs

The technique of selective inhibition of peptide chain initiation by growth medium hypertonicity was adapted for cell monolayer cultures and applied to a study of protein synthesis in SV40 infected BSC-1 cells. The translational efficiences for individual peptide chain initiation sites on SV40 mRNAs were determined and compared to those for cellular mRNA species under conditions of a reduced rate of peptide chain initiation. The SV40 mRNAs show different translational efficiences. The results indicate that the synthesis of the SV40 proteins VP1, 2 and 3 are initiated independently and with different rates on viral mRNA(s). It is proposed, therefore, that VP2 is not a precursor for VP3.

Inhibition of peptide chain initiation in lysates from ATP-depleted cells I. Stages in the evolution of the lesion and its reversal by thiol compounds, cyclic AMP or purine derivatives and phosphorylated sugars

Anaerobic incubation of rabbit reticulocytes at 37°C in Krebs-Ringer solution supplemented with hemin but devoid of glucose resulted at the end of 1–2 h in a drastic decline of their ATP content and an attendant arrest of protein synthesis. Subsequent provision of glucose and reoxygenation of the cells was followed by a rapid replenishment of the ATP pool, while resumption of protein synthesis was markedly delayed. This lag period could be considerably reduced by addition of 5–10 mM adenine or 2,6-diaminopurine to the incubation medium. Lysates prepared from ATP-depleted cells exhibited disaggregation of the polysomes and an inhibition of the endogenously coded protein synthesis, when tested in a cell-free system supplied with an adequate ATP generator. Both alterations increased in severity with the progressive decay of the intracellular ATP pool. The early phase of partial inhibition following a 40–70% decrease of the cellular ATP level was fully reversible by fortifying the cell-free preparation with dithiothreitol or a suitable NADPH-generating system. Alternatively, the inhibition could be also overcome by millimolar amounts of adenine, 2,6-diaminopurine and a variety of other purine derivatives or cyclic AMP. The effect of these compounds was unrelated to the endogenous cyclic AMP pool. Joint addition of both dithiothreitol and cyclic AMP or adenine was necessary for relieving the initiation block in lysates derived from cells depleted of 80–90% of their ATP content. On further aggravating the conditions of energy starvation, an additional requirement for phosphorylated sugars, e.g. glucose 6-phosphate or fructose 1,6-diphosphate, became apparent. ATP depletion brought about by exposing the cells to Antimycin A or 2,4-dinitrophenol resulted in a lesion which was indistinguishable from that induced by anaerobic incubation. On the other hand, energy deprivation in cell-free lysates from untreated reticulocytes, preincubated in the absence of an ATP-generating system failed to duplicate the deleterious effect of intracellular ATP depletion. Some aspects bearing on the biochemical mechanism of the lesion and its reversal are discussed in the light of the available data.

Inhibition of peptide chain initiation in lysates from ATP-depleted cells II. Studies on the mechanism of the lesion and its relation to similar alterations caused by oxidized glutathione and hemin deprivation

The impairment of peptide chain initiation in lysates from ATP-depleted rabbit reticulocytes is accompanied by a loss of their ability to form the 40 S methionyl-tRNA Met f complex with a resultant failure to promote the AUG codon-dependent combination of the complex with the 60 S ribosomal subunit. These partial initiation reactions, as well as the overall protein-synthetic activity of the defective lysates could be restored by addition of a 0.5 M KCl ribosomal extract or normal postribosomal supernatant or a 40–70% (NH 4) 2- SO 4 fraction derived from it. Alternatively, reactivation of the impaired lysates could be achieved by supplementation with millimolar amounts of cyclic AMP or certain purine derivatives. The same subcellular fractions, as well as cyclic AMP or purine derivatives were also capable of overcoming the inhibition caused by incubating reticulocyte lysates in the presence of oxidized glutathione or in the absence of hemin. Severe intracellular ATP deprivation resulted in accumulation of a soluble translational inhibitor in the postribosomal fraction, thus resembling the parallel phenomenon described in hemin-deprived lysates. The striking similarities between the three kinds of inhibition studied by us point to an identical site of the underlying biochemical lesion, despite the different mechanisms mediating their induction.

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from friend leukemia cells

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA fMet to the ribosomal initiation complex and the NaF-sensitive reaction.

Resuscitation of the monkey brain after one hour complete ischemia. III. Indications of metabolic recovery

Adult rhesus monkeys were subjected to complete cerebral ischemia for one hour and subsequent recirculation for up to 24 h. Animals with signs of functional recovery ( e.g. spontaneous EEG activity) exhibited a partial replenishment of cellular energy sources (ATP, phosphocreatine) and a progressive normalization of cerebral lactate levels. Glucose and pyruvate concentrations showed a transient increase over control values during the early stages of postischemic recirculation. Monkeys without functional recovery lacked a significant resynthesis of energy-rich compounds; adenine nucleotides continued to decrease and lactate concentrations were higher than in animals subjected to ischemia without recirculation. Cerebral polysome profiles remained unaltered during the ischemic period but in all animals a marked disaggregation of polyribosomes with a concomitant increase in ribosomal subunits occurred after the onset of recirculation. In monkeys with indications of functional recovery these changes were reversible but a normal polysome profile was only observed after 24 h of recirculation. The results obtained indicate a postischemic depression of protein synthesis due to an inhibition of peptide chain initiation. After recirculation of the brain for 3–6 h there was evidence for an induction of enzymes involved in polyamine synthesis (ornithine decar☐ylase and S-adenosyl-methionine decar☐ylase). No changes in the activity of these enzymes were observed at the end of the ischemic period, indicating that during complete cerebral ischemia not only the synthesis but also the catabolism of proteins is inhibited.

Mechanism of inhibition of protein synthesis initiation by diacetoxyscirpenol and fusarenon X in the reticulocyte lysate system

The mechanism of inhibition of peptide chain initiation by diacetoxyscirpenol and fusarenon X, (trichotecene mycotoxins), was studied in the reticulocyte lysate protein-synthesizing system. The mycotoxins did not inhibit formation of the complex between Met-tRNA f and the 40S ribosomal subunit nor the initiation codon AUG-promoted combination of the complex with 60S ribosomal subunit. Formyl-methionyl-valine accumulated when the system was incubated with the mycotoxins and fMet-tRNA f. This suggests that the inhibition occurs after the formation of the first peptide bond.

Inhibition of peptide chain initiation in Escherichia coli by thermorubin

The antibiotic thermorubin specifically inhibits protein synthesis when added to growing cultures of Escherichia coli, while RNA and DNA synthesis are not affected. In vitro studies show that thermorubin is a specific inhibitor of initiation of protein synthesis directed by natural mRNA. The drug inhibits protein synthesis directed by MS2 RNA but not poly(U)-dependent polyphenylalanine synthesis. Furthermore, during protein synthesis directed by endogenous messenger RNA, only the fraction of polypeptide synthesis dependent on ex novo-synthesized mRNA is sensitive to the drug. Thermorubin inhibits the binding of fMet—tRNA to ribosomes in the presence of AUG, GTP and initiation factors whereas the synthesis of the first peptide bond, assayed as fMet-puromycin synthesis, is not impaired by the antibiotic. The implications of these findings, with respect to the hypothesis that RNA synthesis in E. coli is controlled by initiation of protein synthesis, are discussed.

Inhibition of peptide chain initiation in Escherichia coli by hydroxylamine. Reaction of hydroxylamine with folate coenzymes.

Inhibition of peptide chain initiation in Escherichia coli by hydroxylamine. Reaction of hydroxylamine with folate coenzymes.

Inhibition of peptide chain initiation in Escherichia coli by fusidic acid

Fusidic acid, a steroidal antibiotic, has been shown to inhibit the activity of transfer factor G, a factor required for peptide chain elongation, but not for peptide chain initiation. The present investigation shows that the antibiotic also inhibits the reactions for peptide chain initiation, possibly by interfering with the initiation factor F 1. However, at low concentrations (< 1 · 10 −3 M) fusidic acid inhibits only chain elongation, while higher concentrations (> 5 · 10 −3 M) are required to inhibit the reactions for peptide chain initation.

Inhibition of peptide-chain initiation in Escherichia coli by hydroxylamine and effects on ribonucleic acid synthesis.

Inhibition of peptide-chain initiation in Escherichia coli by hydroxylamine and effects on ribonucleic acid synthesis.

Stimulation of the differential rate of β-galactosidase synthesis in E. coli by trimethoprim

It was found that partial inhibition of peptide chain initiation by trimethoprim causes a significant increase in the differential rate of β-galactosidase in cultures of E. coli growing in the presence of suboptimal concentrations of a galactoside inducer. This stimulation is not produced by inhibitors of peptide chain growth, such as chloramphenicol and puromycin. Trimethoprim, furthermore, does not cause a significant increase in the differential rate of β-galactosidase synthesis in E. coli cultures (a) of i+ genotype which are fully induced, (b) of fully constitutive i- genotype, (c) of partially constitutive oc genotype, or (d) of noninducible is genotype. These findings are compatible with the idea that the differential rate of β-galactosidase synthesis is regulated by means of an inducer-dependent growth period of a “regulatory” polypeptide.