Effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in rabbit reticulocyte lysates

Nilsson, Anders; Nygård, Odd

doi:10.1016/0167-4781(94)00198-c

Cited by 4 publications

(2 citation statements)

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“…6). GSSG-induced inhibition of elongation may be mediated through changes in eEF-2 phosphorylation, which has recently been reported to be stimulated by oxidizing agents, such as GSSG and NAD ϩ , in RRL [47]. Similarly, the inability of Hsc70 to protect protein synthesis completely from heat-induced inhibi-tion appears to be due to its inability to protect eIF-4E from dephosphorylation in heat-shocked RRL (Fig.…”

Section: Model For Hsc70-mediated Suppression Of Hri Activationmentioning

confidence: 99%

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Thulasiraman

Uma

et al. 1998

European Journal of Biochemistry

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The role of the heat-shock cognate protein, Hsc70, in regulating the activity of the heme-regulated eIF-2A kinase (HRI) in hemin-supplemented rabbit reticulocyte lysate (RRL) in response to heat and oxidative stress was examined and compared with the effect of Hsc70 on HRI activation in response to heme deficiency. Hsc70 suppressed eIF-2A phosphorylation and maintained the guanine nucleotide exchange activity of eIF-2B in heme-deficient RRL and in hemin-supplemented RRL exposed to elevated temperatures (42°C), denatured protein (reduced carboxymethylated bovine serum albumin, RCM-BSA), oxidized glutathione or Hg 2ϩ . The ability of Hsc70 to inhibit HRI activation was mediated through its ability to inhibit the hyper-autophosphorylation of transformed HRI, which causes the hyperactivation of HRI. Maintenance of protein-synthesis rate was observed to be an unreliable indicator of the ability of Hsc70 to suppress HRI activation in response to stress. While Hsc70 completely reversed protein synthesis inhibition caused by Hg 2ϩ , Hsc70 only partially reversed translational inhibition caused by oxidized glutathione (GSSG) or heat shock. The inability of Hsc70 to fully protect protein synthesis from inhibition induced by heat shock or GSSG was due to inability of Hsc70 to protect eIF-4 E from heat-induced dephosphorylation, and its inability to protect translational elongation from GSSG-induced inhibition, respectively. Activation of HRI in heat-shocked hemin-supplemented lysate correlated with a marked decrease in the pool of Hsc70 that was available to bind RCM-BSA and the loss of the interaction of Hsc70 with HRI. These observations indicate that heat shock induced the accumulation of a sufficient quantity of Hsc70 binding substrates (e.g., denatured protein) to sequester Hsc70 and inhibit the ability of Hsc70 to suppress HRI activation. Our results indicate that Hsc70 not only negatively modulates the activation of HRI in heme-deficienct RRL, but also in hemin-supplemented RRL in response to heat and oxidative stress.

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Section: Model For Hsc70-mediated Suppression Of Hri Activationmentioning

confidence: 99%

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Thulasiraman

Uma

et al. 1998

European Journal of Biochemistry

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“…Elongation factor 2 (EF-2), the eukaryotic counterpart of EF-G, was also identified as a protein that is susceptible to oxidation. Treatment of rabbit reticulocytes with the oxidant cumene hydroperoxide resulted in the inhibition of the phosphorylation of EF-2 (2). Treatment of rat liver with cumene hydroperoxide stimulated the carbonylation of amino acids and subsequent ADP-ribosylation of EF-2 (6).…”

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Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

Nagano

Kojima

Hisabori

et al. 2012

Journal of Biological Chemistry

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Effect of Oxidative Stress, Produced by Cumene Hydroperoxide, on the Various Steps of Protein Synthesis

Ayala

Parrado

Bougria

et al. 1996

Journal of Biological Chemistry

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We have studied the effect of oxidative stress on protein synthesis in rat liver. Cumene hydroperoxide (CH) was used as an oxidant agent. The approach used was to determine the ribosomal state of aggregation and the time for assembly and release of polypeptide chains in the process of protein synthesis in rat liver in vivo. The results suggest that the elongation step is the most sensitive to CH treatment. The measurement of both carbonyl groups content and ADP-ribosylatable elongation factor 2 (EF-2), the main protein involved in the elongation step, indicates that under CH treatment EF-2 is oxidatively modified and a lower amount of active EF-2 is present. These results are corroborated by in vitro oxidation of EF-2 and could explain for the decline in the elongation step.The effect of oxidative stress on protein metabolism have been reported by various laboratories (1-13). Most of these studies have focused on protein degradation, the major finding being that oxidatively modified proteins become susceptible to proteolytic digestion (2, 4 -7, 9 -13). With respect to protein synthesis, the effect of active oxygen species has been less studied and has been addressed by using oxidizing agents (14 -16). One of such compounds is cumene hydroperoxide (CH), 1 which has been used as an intracellular source of reactive oxygen intermediates (17)(18)(19)(20)(21)(22). Besides its damaging effects such as membrane damage, cell lysis, organ necrosis, tumor promotion (20), and certain aspects of aging (23), it has been reported that this compound and/or cytotoxic aldehydes derived from it inhibit protein synthesis in human skin fibroblasts (14). These in vitro results were obtained by measuring the incorporation of radioactive amino acid precursors into proteins, a methodology which is not suitable when using whole animal systems because of the difficulty in measuring the specific activity of the amino acid precursor pool (23). Furthermore, a detailed molecular mechanism of the decline of protein synthesis caused by the oxidant is not available.Because of the potential importance of decreased protein synthesis in structural and functional deterioration of the cell, the molecular basis for decreased rate of protein synthesis by CH, and hence by oxidative stress, is of interest, especially since this decrease may be an important contributor to certain processes in which free radicals seem to be involved, such as aging (25).In order to understand the mechanism underlying the inhibition of protein synthesis by CH, we have studied: 1) which of the individual steps in polypeptide synthesis is most affected, and 2) the possible mechanism of how active oxygen species may interact with the protein synthetic machinery.The effect of CH on the stages of protein synthesis has been studied by investigating the ribosome half-transit time (elongation time) required for the synthesis of an average halflength of a nascent polypeptide chain (26), and the ribosomal state of aggregation (27, 28), which is reflected by the polyribosomal profiles....

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Effect of oxidizing agents and haemin on the phosphorylation of eukaryotic elongation factor 2 in rabbit reticulocyte lysates

Cited by 4 publications

References 48 publications

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Evidence that Hsc70 negatively modulates the activation of the heme‐regulated eIF‐2α kinase in rabbit reticulocyte lysate

Elongation Factor G Is a Critical Target during Oxidative Damage to the Translation System of Escherichia coli

Effect of Oxidative Stress, Produced by Cumene Hydroperoxide, on the Various Steps of Protein Synthesis

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