Translational control Interferon

Escherichia coli Easy to grow in large-scale volumes Transcriptional and translational control well known Successfully used in the manufacture of insulin, interferon and human somatotropin Difficult to achieve export of some proteins into growth medium Degradation of small proteins by proteases Unable to undertake most post-translational modifications, e.g. glycosylation Many proteins retained in the cytoplasm as insoluble aggregates... 

Several models, yet relatively little direct evidence has been presented to explain the genetic regulation of interferon production. No matter what model may be built to accomodate all known facts, it should be composed, in its simplest form, of an operon (interferon gene), a repressor protein, and a mRNA. The repressor protein may combine with the operator of the operon (referred to as transcriptional control in Fig. 7) in this state, the interferon gene would be repressed. It would be derepressed if the repressor is inactivated, e.g. following contact of the cell with the interferon inducer. The derepressed interferon gene would then be transcribed to mRNA, which in turn is translated to interferon. Hypothetically, the repressor protein may also combine with the mRNA for interferon (referred to as translational control), and even with the interferon molecule itself (referred to as posttranslational control in Fig. 7). 

Cell sap contains a specific phosphoprotein phosphatase (P) which dephosphoiylates protein 67, eIF-2 and several other dsRNA-dependent phosphorylated proteins (5). The protein phosphatase P is separated from PK-i on DEAE-cellulose and elutes at 25O1DM KCl. As shown in Figure 5> P stimulates mengo RNA translation in interferon-treated cell extracts (SlO-int) supplemented with dsRNA, but not in control cell extracts (SIO-cont). This suggests a regulatory mechanism which controls the level of PK-i dependent inhibition of protein synthesis. 

Le Dinh T, Freneaux E, Labbe G, Letteron P, Degott C, Geneve J, Berson A, Larrey D, Pessayre D (1988) Amineptine, a tricyclic antidepressant, inhibits the mitochondrial oxidation of fatty acids and produces microvesicular steatosis of the liver in mice. J Pharmacol Exp Ther 247 745-750 Le Roy F, Bisbal C, Silhol M, Martinand C, Lebleu B, Salehzada T (2001) The 2-5A/RNase L/RNase inhibitor (RLl) pathway regulates mitochondrial mRNAs stability in interferon-a-treated H9 cells. J Biol Chem 276 48473 8482 Le Roy F, Silhol M, Salehzada T, Bisbal C (2007) Regulation of mitochondrial mRNA stability by RNase L is translation-dependent and controls IFNalpha-induced apoptosis. Cell Death Differ 14 1406-1413... 

Figure 3 Separation of various translation regulatory activities on DEAE cellulose. Cell sap from interferon-treated cultures was fractionated and assayed on mengo RNA translation (as in Table 2) in S10 from control (c) and interferon-treated cells (int) with O.O4 M g/ml dsENA. Activity without fraction = 1. Oligo-isoadenylate synthetase E (IO), factor F (18). Protein phosphatase (P) and protein kinase activator A, are discussed in the text.

Factor P was first detected as an activity which inhibits translation only when added to extracts from interferon-treated cells in the presence of dsRNA, but not in control cell extracts or without dsRNA. Figure 5 shows that P elutes from DEAE-cellulose at 120mM KCl. It is- not well separated at this stage from a stimulator of protein synthesis, but could be purified on phosphocellulose pH 7 9 from which P eluted at 200-500mM KCl. Overall purification was 120-fold (I8). 

Most of the cell-free extracts used in these studies had been pre-incubated to reduce the translation of endogenous mRNA. Since different preparations of lysates exhibited varying degrees of interferon-induced inhibition, we studied the influence of the length of time of pre-incubation on the interferon-induced inhibition of vitro protein synthesis. e could establish that the ability of cell-free extracts to translate mengo virus RNA was a function of the time of pre-incubation. The ability of an extract from interferon-treated (int. S-10) cells to translate mengo virus RNA was not impaired when compared with an extract from control cells until between 60 and 90 of pre-incubation. At this point, although the activity of the control extract declined somewhat, the activity of the Int. S-10 declined much more rapidly. 

Table 2. Effect of tENA(pj Q) on the translation of poly (U,C) by extracts from control and interferon-treated cells pre-incubated for different times. Protein synthesis directed by poly (H,G) was assayed in the absence or presence (30 ig/ml) of tENA y...

Addition of dsENA to extracts prepared from interferon-treated cells caused 80-90% inhibition of mengo virus EEA translation, regardless of the time of pre-incubation (Table 5) The same dose of dsEEA caused an inhibition of only 8-15% in control extracts. 

Table 4 Effect of Added dsRRA and tRRA on poly(lJ,C) translation by extracts of control and interferon-treated cells pre-incubated for different times>...

Translation is more sensitive to inhibition by dsRNA in extracts from interferon-treated cells than in those from control cells (Kerr et al., 1974). As in reticulocyte lysates, dsRNA causes eIF-2 to be phos-phorylated to a significant extent in interferon-treated cell extracts, but much less in extracts of uninfected cells (Lebleu et al., 1976 Roberts et al., 1976 Zilberstein et al., 1978). The translational block observed in the presence of dsRNA in extracts of interferon-treated mouse L cells can be overcome completely by the addition of eIF-2 (Kaempfer et al., 1979b)... 

One question that deserves attention is the fact that, at least in the case of some virus-cell systems, viral RNA translation is inhibited preferentially over host mRNA translation, for example, in reovirus-infected L cells (Gupta et al., 1974). An interesting possibility was raised by Nilsen and Baglioni (1979). They showed that, in extracts of interferon-treated cells, VSV mRNA hybridized with poly (U) at its poly (A) tail or EMC RNA hybridized with poly (I) at its poly (C) tract are more rapidly degraded than the corresponding control mRNAs. They proposed that, in infected, interferon-treated cells, activation of the endoribonuclease takes place near the replicative intermediate of RNA viruses, because the dsRNA moiety therein promotes the formation of (2 -5 )oligo (A) in its vicinity. As a result, the viral mRNA portion in the replicative intermediate may be more sensitive to degradation than host mRNA. 

Lengyel, P., 1982Z , Interferon action Control of RNA processing, translation and degradation, in Protein Biosynthesis in Eukaryotes (R. Perez-Bercoff, ed.), pp. 459-483, Plenum Publishing, New York. 

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