Analysis of protein synthesis dynamic model in eukaryotic cells: Input control

This paper presents the analysis of initiation control model of protein synthesis via eukaryotic initiation factor (eIF)-2 unit, introduced by [N.S. Bar, D.R. Morris, Dynamic model of the process of protein synthesis in eukaryoric cells, Bulletin of Mathematical Biology 69 (2007) 361-393, doi:10.1007/s11538-006-9128-2.] and propose methods to control it.Linearization of the model is presented as a measure to simplify the analysis and control application. The properties of the linear model were investigated and compared to the non-linear model using simulations. It was shown that the linear model is (marginally) stable and the states converge to a finite value. Linear optimal control theory can then be applied to the model under the value range where the linearized model is accurate. The effect of the input signals GCN2·tRNA and eIF-2 on the non-linear system was investigated. A few characteristics known from in vitro experiments of the initiation process were proven from a mathematical aspect and some conclusions about the function of the initiation complexes such as eIF2B and the ternary complex were derived. Consistent with published experiments, it was shown that overexpression of eIF-2 increases the concentration of 48S initiation complex and promote initiation rate. A state feedback control was applied in order to manipulate the initiation rate and it was proven that the 48S initiation complex can be driven to a desired value by calculating an input control law using measurement techniques available today. If this strategy can be implemented de facto, then a genuine control on protein synthesis process can be obtained.     

Translation in micrococcal nuclease-treated cell-free extracts from Ehrlich ascites tumor cells: Stimulation by initiation factor eIF-2B

Translation of exogenous mRNAs in micrococcal nuclease-treated extracts from Ehrlich ascites tumor cells is greatly stimulated by the addition of crude initiation factors or initiation factors eIF-2B and eIF-2 containing eIF-2B. The requirement for exogenous eIF-2B in micrococcal nuclease-treated extracts does not result from either loss or enhanced phosphorylation of eIF-2 during incubation.     

Purification of protein synthesis initiation factor eIF-3 from rat liver microsomes by affinity chromatography on rRNA-cellulose

An efficient four-step procedure is described for preparing highly purified polypeptide chain initiation factor eIF-3 from rat liver microsomal saltwash. The method involves fractionation with ammonium sulfate between 25–40% saturation (0°C) followed by affinity chromatography on rRNA-cellulose, DEAE-cellulose chromatography and sucrose density gradient centrifugation. eIF-3 is eluted from the affinity column at a KCl concentration of 0.18 M. The purification is 10-times and the recovery of activity better than 85%. In the sucrose gradients, eIF-3 sediments as a 15 S particle indicating a total mass of 650 000 Da. The purified eIF-3 is highly active in stimulating globin synthesis in a fractionated translation system. Factor eIF-3 contains eight subunits with molecular weights ranging from 40 000 to 110 000. Seven of the subunits are present in one copy per eIF-3, whereas the factor contains two copies of one subunit. The isoelectric points of the factor subunits range from 5.5 to 7.3 with most of the polypeptides being acidic.     

Inhibition of cell-substratum attachment of cultured rat heart cells by protein synthesis inhibitors

Addition of cycloheximide to growth medium of neonatal rat heart cell cultures prevented cell-substratum attachment. Even concentrations of cycloheximide which inhibited only 50% of normal protein synthesis prevented some cells from attaching. Cells which required the longest time to attach were most dependent on protein synthesis. The kinetics of cell-substratum adhesion in the presence of various concentrations of cycloheximide supported the hypothesis that repair of damaged cell membranes was required prior to attachment. An alternate hypothesis that protein synthesis was required for substratum attachment either to synthesize new unique proteins or higher concentrations of existing proteins not damaged by enzymes was not supported by experimentally obtained data. If the second hypothesis were true, no cells would have attached when protein synthesis was completely inhibited (greater than 95%) and all cells should have been equally affected by protein synthesis inhibition; such was not the case. Inhibition of mRNA formation by actinomycin D also should have inhibited attachement completely and this was not observed. Since attachment was minimally affected by actinomycin D, protein synthesis on long-lived mRNA was apparently sufficient for cell-substratum adhesion.     

Ultraviolet-crosslinking reveals specific affinity of eukaryotic initiation factors for Semliki Forest virus mRNA

Eukaryotic initiation factors (eIF) associate readily with ³²P-labeled Semliki Forest virus (SFV) mRNA in vitro, forming complexes which can be crosslinked by 254 nm ultraviolet irradiation. After ribonuclease digestion, the initiation factors were released and analysed by gel electrophoresis. Autoradiography revealed proteins by virtue of crosslinked ³²P-labeled mRNA fragments. eIF-4A, -4B and -4C as well as three subunits of eIF-3 could be crosslinked with SFV mRNA. None of these proteins bound to ribosomal RNAs.     

The effects of monocyte-conditioned medium and interleukin 1 on the synthesis of collagenous and non-collagenous proteins by mouse bone and human bone cells in vitro

Cultural adherent human mononuclear cells produce factor(s) which stimulate the release of calcium from new-born mouse calvaria in organ culture. This stimulation of bone resorption is accompanied by an inhibition of the incorporation of [³H]proline into collagen which is independent of increased prostaglandin production by the bone. When human osteoblast-like cells are treated with conditioned medium from human mononuclear cells, collagen accounts for a decreased proportion of the protein synthesised. This effect on matrix synthesis is not accompanied by an inhibitory action of the monocyte-conditioned medium preparations on net cell proliferation. In human osteoblast-like cell cultures, partially purified human interleukin 1 also inhibits the production of the bone-specific protein osteocalcin in a dose-dependent fashion. These observations are consistent with the hypothesis that products of human monocytes similar to, or identical with, human interleukin 1 may be important regulators of bone metabolism and may contribute to the bone loss seen in diseases such as chronic rheumatoid arthritis.     

Regulation of protein kinases activity by quercetin in Ehrlich ascites tumor cells

Cyclic AMP-independent protein kinase activities from Ehrlich ascites tumor cells, partially purified by DEAE-cellulose and phosphocellulose chromatography were inhibited by quercetin. The cyclic AMP in the tumor ascites cells and the cyclic AMP-dependent protein kinase activity from this tumor and from bovine and mouse tissues were unaffected by this drug. Since we reported that quercetin elevates cyclic AMP level in Ehrlich ascites tumor cells, this bioflavonoid may have a dual effect on the protein kinae activities in these cells, thus, increasing the cyclic AMP-dependent and decreasing the cyclic AMP-independent protein kinase activities.     

Stimulation of protein synthesis by lysine analogues in lysine-deprived Ehrlich ascites tumour cells

This paper describes experiments in which we have investigated the mechanism by which amino acid starvation regulates the initiation of protein synthesis in mammalian cells. We have examined the ability of a range of lysine analogues to stimulate protein synthesis in lysine-deprived mouse Ehrlich ascites tumour cells in culture. Of those analogues tested, only those which are cleaved to lysine intracellularly are capable of restoring protein synthesis to the level seen in fully fed cells. Lysine which is covalently linked to agarose does not stimulate translation. After 5 min incubation of lysine-deprived cells with the analogue lysine p-nitroanilide, the lysine concentration in cell extracts is restored to that found in extracts from fed cells, and protein synthesis is maximally stimulated within 5–10 min. During this period of time there is no increase in the concentration of lysine in the medium. These data indicate that it is the size of the intracellular rather than the extracellular amino acid pool which regulates the rate of protein synthesis during amino acid deprivation.     

Erythromycin inhibition of cell proliferation and in vitro mitochondrial protein synthesis in human HeLa cells is pH dependent

The growth of HeLa cells in Hepes-buffered medium was significantly more sensitive to the inhibitory effects of erythromycin than in medium buffered by the more conventional bicarbonate-CO₂ system. Since growth inhibition by erythromycin became more pronounced as the pH of the medium was increased the difference in erythromycin sensitivity between the Hepes-buffered medium vs. the bicarbonate-CO₂-buffered medium is most likely due to pH effects. The relative growth sensitivity to erythromycin of ERY2301, an erythromycin-resistant mutant of HeLa, was also affected by elevated pH of the growth medium. However, ERY2301 cells were able to proliferate to a greater extent in the presence of erythromycin than HeLa cells grown under the same conditions. The selective growth advantage of ERY2301 (in the presence of erythromycin) is best seen in medium of pH 7.4, or in the Hepes-buffered medium. In vitro protein synthesis by intact mitochondria isolated from HeLa cells was relatively insensitive to erythromycin inhibition at pH 7.4 and 7.6, but at high pH values was inhibited approx. 50%. Although the erythromycin sensitivity of ERY2301 mitochondrial protein synthesis was also affected by increasing the pH, the incorporation of [³H]leucine was more resistant to erythromycin than that observed for HeLa mitochondria over the pH range tested. Increasing the concentration of erythromycin at a given pH did not result in a further increase in the inhibition of either HeLa or ERY2301 mitochondrial protein synthesis. When the mitochondrial membranes were disrupted by Triton X-100, erythromycin inhibition of HeLa mitochondrial protein synthesis was pH dependent and, at the lower pH values tested, greater inhibition was observed as the erythromycin concentration was increased. ERY2301 mitochondrial protein synthesis under the same conditions displayed a high level of erythromycin-resistant activity independent of both pH and erythromycin concentration. It is suggested that, as has been proposed for bacterial systems, only the non-protonated molecule of erythromycin is effective in inhibiting mitochondrial protein synthesis. The ability of erythromycin to permeate the mitochondrial membranes and the plasma membres may also be facilitated by a higher pH.     

Translocation of nucleolar phosphoprotein B23 (37kDapI 5.1) induced by selective inhibitors of ribosome synthesis

To elucidate the possible role of nucleolar phosphoprotein B23 in ribosome synthesis, drugs which inhibit the processing of ribosomal RNA were employed. After treatment with actinomycin D, toyocamycin or high doses of α-amanitin, a uniform nucleoplasmic fluorescence was observed. Low doses of α-amanitin and the protein synthesis inhibitor puromycin and cycloheximide had no effect on protein B23 translocation. By ELISA immunoassay, there was a 60% decrease in the amount of protein B23 in the nucleoli of the actinomycin D-treated cells as compared with the control nucleoli. Conversely, the amount of protein B23 in the nucleoplasm (excluding nucleoli) was 3-fold higher in the actinomycin D-treated cells. Preribosomal ribunucleoprotein particles (pre-rRNPs) were extracted from isolated nucleoli of Novikoff hepatoma ascites cells and fractionated on sucrose density gradients. Protein B23 was found co-localized with the pre-rRNPs as determined by ELISA assays which agrees with previous studies. The proteins in these 80 S and 55 S pre-ribosomal ribonucleoprotein particles were fractionated by 10% gel electrophoresis. Immunoblots showed protein B23 was present in both pre-rRNPs.     

Inhibition of protein synthesis by the cordycepin analog of (2'-5')ppp(Ap)nA, (2'-5')ppp(3'dAp)n3'dA, in intact mammalian cells

The introduction of the cordycepin analog of (2'-5')An, (2'-5')ppp(3'dAp)n3'dA [referred to as (2'-5')p33'dAn], into mouse L929 cells and cultured human fibroblasts resulted in a dose-dependent inhibition of protein synthesis which was comparable to the inhibition observed by (2'-5')ppp(Ap)nA [referred to as (2'-5')p3An]. The inhibition of protein synthesis by (2'-5')p33'dAn was much more persistent than that of the naturally occurring (2'-5')p3An following prolonged incubation of cells. Furthermore, the (2'-5')p3An was cytotoxic to mammalian cells in culture, whereas the (2'-5')p33'dAn was not.     

Requirements for antiribosomal activity of pokeweed antiviral protein

It has been known for some time that pokeweed antiviral protein acts by enzymatically inhibiting protein synthesis on eucaryotic ribosome systems. The site of this action is known to be the ribosome itself. In this paper we show that the pokeweed antiviral protein reaction against ribosomes is a strong function of salt concentrations, where 160 mM K⁺ and 3 mM Mg²⁺ retards the reaction, while 20 mM K⁺ and 2 mM Mg²⁺ allows maximum reaction rate. It is also shown, however, that an unidentified protein in the postribosomal supernatant solution, together with ATP, allows the ribosome to be attacked even in the presence of high salt. Kinetic analysis of the antiviral protein reaction has been carried out under both sets of conditions, and reveals that the turnover number for the enzyme is about 300–400 mol/mol per min. in each case. The K_m for ribosomes is 1 μM in the presence of low salt and 0.2 μM at higher salt in the presence of postribosomal supernatant factors plus ATP. The antiviral protein reaction is also shown to be pH dependent and is controlled by a residue with pK_a value of approx. 7.0, apparently a histidine. Stoichiometric reaction of the enzyme with iodoacetamide results in a significant loss of antiribosomal activity.     

Battling for Ribosomes: Translational Control at the Forefront of the Antiviral Response

In the early stages of infection, gaining control of the cellular protein synthesis machinery including its ribosomes is the ultimate combat objective for a virus. To successfully replicate, viruses unequivocally need to usurp and redeploy this machinery for translation of their own mRNA. In response, the host triggers global shutdown of translation while paradoxically allowing swift synthesis of antiviral proteins as a strategy to limit collateral damage. This fundamental conflict at the level of translational control defines the outcome of infection. As part of this special issue on molecular mechanisms of early virus–host cell interactions, we review the current state of knowledge regarding translational control during viral infection with specific emphasis on protein kinase RNA-activated and mammalian target of rapamycin-mediated mechanisms. We also describe recent technological advances that will allow unprecedented insight into how viruses and host cells battle for ribosomes.