Inhibition of mitochondrial protein synthesis influences the glucocorticoid sensitivity of lymphoid cells

Inhibition of mitochondrial protein synthesis impairs the formation of the 13 polypeptides encoded on the mitochondrial genome. These polypeptides are part of enzyme complexes involved in oxidative phosphorylation. Prolonged inhibition of mitochondrial protein synthesis thus reduces the oxidative phosphorylation capacity which ultimately results in impairment of energy-requiring processes. Via a different mechanism glucocorticoid hormones also decrease the oxidative phosphorylation capacity of, e.g., lymphoid cells. The present study shows that inhibition of mitochondrial protein synthesis influences glucocorticoid-induced responses of lymphoid cells in two opposing manners. (a) It is enhanced after induction in cells with a reduced oxidative phosphorylation capacity resulting from preceding inhibition of mitochondrial protein synthesis. This can be explained by the synergistic effects of glucocorticoids and prolonged inhibition of mitochondrial protein synthesis on energy-producing processes. (b) It is counteracted when mitochondrial protein synthesis is impaired during induction of the response. The latter observation suggests that mitochondrial protein synthesis is involved in the generation of glucocorticoid-induced effects on lymphoid cells.     

Formation of the yeast mitochondrial membrane. 3. Accumulation of mitochondrial proteins synthesized in both the cytoplasm and mitochondria in yeast undergoing glucose depression

The inhibitors of protein synthesis, chloramphenicol and cycloheximide, were added to cultures of yeast undergoing glucose derepression at different times during the growth cycle. Both inhibitors blocked the increase in activity of coenzyme QH₂-cytochrome c reductase, suggesting that the formation of complex III of the respiratory chain requires products of both mitochondrial and cytoplasmic protein synthesis.The possibility that precursor proteins synthesized by either cytoplasmic or mitochondrial ribosomes may accumulate was investigated by the sequential addition of cycloheximide and chloramphenicol (or the reverse order) to cultures of yeast undergoing glucose derepression. When yeast cells were grown for 3 hr in medium containing cycloheximide and then transferred to medium containing chloramphenicol, the activity of cytochrome oxidase increased at the same rate as the control during the first hour in chloramphenicol. These results suggest that some accumulation of precursor proteins synthesized in the mitochondria had occurred when cytoplasmic protein synthesis was blocked during the growth phase in cycloheximide. In contrast, essentially no products of mitochondrial protein synthesis accumulated as precursors for either oligomycin-sensitive ATPase or complex III of the respiratory chain during growth of the cells in cycloheximide.When yeast were grown for 3 hr in medium containing chloramphenicol followed by 1 hr in cycloheximide, the activities of cytochrome oxidase and succinate-cytochrome c reductase increased at the same rate as the control, while the activities of oligomycin-sensitive ATPase and NADH or coenzyme QH₂-cytochrome c reductase were nearly double that of the control. These data suggest that a significant accumulation of mitochondrial proteins synthesized in the cytoplasm had occurred when the yeast cells were grown in medium containing sufficient chloramphenicol to block mitochondrial protein synthesis. The possibility that proteins synthesized in the cytoplasm may act to control the synthesis of mitochondrial proteins for both oligomycin-sensitive ATPase and complex III of the respiratory chain is discussed.     

Biogenesis of mitochondrial membranes in Neurospora crassa. Mitochondrial protein synthesis during conidial germination.

The conidia of Neurospora crassa entered logarithmic growth after a 1-h lag period at 30 degrees C. Although [14C]leucine is incorporated quickly early in growth, cellular protein data indicated that no net protein synthesis occurred until after 2 h of growth. Neurospora is known to produce ethanol during germination even though respiratory enzymes are present. Also, Neurospora mitochondria isolated from cells less than 3-h old are uncoupled. Since oxygen uptake increased during germination, was largely cyanide-sensitive, and reached a maximum at 3 h, it is hypothesized that during early germination the uncoupled electron transport chain merely functions to dispose of reducing equivalents generated by substrate level ATP production. The rate of protein synthesis in vitro by mitochondria isolated from 0-8-h-old cells increased as did cell age. Mitochondrial protein synthesis in vivo, assayed in the presence of 100 mug cycloheximide/ml, increased from low levels in the cinidia to peak levels at 3-4 h of age and then slowly decreased. The rate of mitochondrial protein synthesis in vivo was linear for at least 90 min in 0-4-h-old cells, but declined after 15 min of incorporation in 6 and 8-h-old cells. The products of mitochondrial protein synthesis in vivo were analyzed with dodecylsulfate gel electrophoresis and autoradiography. Early in germination 80% of the synthesis was of two small proteins (molecular weights 7200 and 9000). At 8 h 85% of the radioactivity was in 10 larger proteins (12 200 to 80 000). Within the high-molecular-weight class, proteins of between 12 000 and 21 500 molecular weight were preferentially lavelled early in germination, whereas after 8 h of growth proteins of 27 500 to 80 000 molecular weight were preferentially labelled. It is hypothesized that the 7200 and 9000-molecular-weight products of mitochondrial protein synthesis combine with other proteins to form the larger proteins found later in growth. The availability of these other proteins in cells of different ages could affect the rate of mitochondrial protein synthesis in vivo.     

ATP浓度和缺氧暴露对大鼠脑线粒体RNA和蛋白质体外合成的影响

本文探讨介质中ATP浓度和急,慢性缺氧暴露对大鼠脑线粒体内RNA和蛋白质合成的影响。用差速离心法分离正常和低压舱模拟4000m高原急性连续缺氧暴露3d和慢性连续缺氧暴露40d大鼠脑线粒体,用体外无细胞（cell-free in vitro)^3H-UTP和^3H-Leucine掺入法分别测定线粒体RNA和蛋白质合成活性,结果显示,大鼠急性缺氧暴露后大脑皮质线粒体RNA体外合成活性降低40%,蛋白质合成活性降低60%;慢性缺氧暴露后线粒体RNA和蛋白质合成活性分别为对照的72%和76%;ATP对正常大鼠脑线粒体RNA以及蛋白质的体外合成活性的影响均呈双相性,大于或小于1mmol/L均可产生不同程度的抑制效应,结果提示,缺氧可在转录和翻译两个水平上影响脑线粒体mtDNA的表达,而慢性缺氧暴露时,线粒体半自主性功能的改善可能是机体对缺氧适应的细胞机制之一;ATP对脑线粒体内转录和释放活性的调节是一种经济有效的反馈调节方式。     

Mitochondrial RNA and protein synthesis in enucleated African green monkey cells

The behavior of extramitochondrial protein synthesis and of mitochondrial RNA and protein synthesis was examined in the cytoplasts of African green monkey kidney cells (TC-7 subline) at different times following enucleation by cytochalasin B. The rate of incorporation of [³H]isoleucine into protein of the soluble cytoplasmic fraction decreased in an approximately exponential fashion, with a half-life of about five hours, during the first 26 hours after enucleation. Discrete mitochondrial 16 S, 12 S and 4 S RNA components were identified among the products of cytoplast RNA synthesis. The rates of [³H]uridine incorporation into the 16 and 12 S RNA components as well as into total RNA declined progressively after enucleation to a barely detectable level by the 20th hour. By contrast, the rate of chloramphenicol-sensitive [³H]isoleucine incorporation into protein (due to mitochondrial protein synthesis) did not undergo a substantial decline for at least 20 hours in TC-7 cytoplasts; instead, a reproducible transient stimulation occurred in the first hours following enucleation. The products of mitochondrial protein synthesis pulse-labeled in nucleated cells and in cytoplasts 24 hours after enucleation exhibited similar electrophoretic profiles.     

Protein synthesis by hepatic mitochondria isolated from carbon tetrachloride-exposed rats

Hepatic mitochondria isolated from rats 40 h after dosage with 1.1 ml/kg CCl4 are uncoupled and display structural damage. Mitochondrial function returns during hepatic recovery. Because the products of mitochondrial protein synthesis are essential to mitochondrial structure and function, the effects of CCl4 on the rate of mitochondrial protein synthesis, and on the products, was studied using mitochondria from CCl4-exposed rats during the early, maximum development and resolution stages of CCl4-induced mitochondrial damage. Rates of mitochondrial protein synthesis (incorporation of [35S]methionine) were elevated 300% over that of mitochondria from non-exposed rats 17 h after exposure; depressed by 50% at 40 h and above control at 113 h. When the radiolabeled products of incorporation were separated and examined by autoradiography, a novel, low-molecular-weight band, of approx. 9700, was apparent 40 h after CCl4 exposure. A band of similar molecular weight appeared when control mitochondria were incubated without an exogenous supply of ATP. Mitochondria from exposed rats which displayed rates of protein synthesis greater than control consistently had a relative increase in a band that corresponded in size to that of cytochrome oxidase subunit I. It was concluded that the loss of mitochondrial function induced by CCl4 could not be attributed to inhibition of mitochondrial protein synthesis, and that the mitochondria may not always synthesize protein in constant proportions.     

β-Adrenergic receptor blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in humans

β-Adrenergic receptor (AR) signaling is a regulator of skeletal muscle protein synthesis and mitochondrial biogenesis in mice. We hypothesized that β-AR blockade blunts postexercise skeletal muscle mitochondrial protein synthesis rates in adult humans. Six healthy men (mean ± SD: 26 ± 6 yr old, 39.9 ± 4.9 ml·kg(-1)·min(-1) peak O(2) uptake, 26.7 ± 2.0 kg/m(2) body mass index) performed 1 h of stationary cycle ergometer exercise (60% peak O(2) uptake) during 1) β-AR blockade (intravenous propranolol) and 2) administration of saline (control). Skeletal muscle mitochondrial, myofibrillar, and sarcoplasmic protein synthesis rates were assessed using [(2)H(5)]phenylalanine incorporation into skeletal muscle proteins after exercise. The mRNA content of signals for mitochondrial biogenesis was determined using real-time PCR. β-AR blockade decreased mitochondrial (from 0.217 ± 0.076 to 0.135 ± 0.031%/h, P < 0.05), but not myofibrillar or sarcoplasmic, protein synthesis rates. Peroxisome proliferator-activated receptor-γ coactivator-1α mRNA was increased ～2.5-fold (P < 0.05) at 5 h compared with 1 h postexercise but was not influenced by β-AR blockade. We conclude that decreased β-AR signaling during cycling can blunt the postexercise increase in mitochondrial protein synthesis rates without affecting mRNA content.     

Sull'evoluzione Dei Mitocondri Nell'endosperma di Semi di Ricino in Germinazione

Abstract

On the behavior of mitochondria in the castor bean seed endosperm during the early phases of germination. — In the endosperm of the castor bean seed the oxidative activity and the protein nitrogen contents of the mitochondrial fraction markedly increase during the first period of germination (Beevers and coworkers). The activation of the mitochondrial system is paralleled by a similar increase of the activity of several soluble enzymes; the latter process is severely depressed by protein synthesis inhibitors (Cornaggia, Aberghina).

The present research is aimed to understand at what extent phenomena of activation and/or, respectively, of « ex novo » synthesis are responsible of the increase of mitochondrial activity. The following aspects of the mitochondrial behavior during the early period of germination were investigated:

a) Changes in the activity of cytochrome oxydase, malate dehydrogenase and of the succinate-citochrome reductase system.

b) Changes in the morphology of mitochondria and other particulated cell structures, as revealed by electron microscopy.

In the mitochondrial preparation all of the three enzymatic activities investigated were found to increase rapidly during the first days of germination. The increase during the first 24 hours was almost as large when measured as specific activity (activity per mg protein in the mitochondrial fraction) than when measured on an absolute (i.e. per seed) basis; moreover, it was not significantly inhibited by puromycin or by actinomycin. The increase of the three activities during the following period of germination (second-third day) was accompanied by an increase of the protein nitrogen (per seed) in the mitochondrial fraction, and was consistently depressed by the protein synthesis inhibitors.

In the mitochondrial preparation all of the three enzymatic activities investigated were found to increase rapidly during the first days of germination. The increase during the first 24 hours was almost as large when measured as specific activity (activity per mg protein in the mitochondrial fraction) than when measured on an absolute (i.e. per seed) basis; moreover, it was not significantly inhibited by puromycin or by actinomycin. The increase of the three activities during the following period of germination (second-third day) was accompanied by an increase of the protein nitrogen (per seed) in the mitochondrial fraction, and was consistently depressed by the protein synthesis inhibitors.

These results, integrated with those of other investigations on the same material are in agreement with the hypothesis that the activation of metabolism in the endosperm during germination depends in a very early phase mainly on the transition of enzyme systems from an inactive to an active state; while in a second phase synthesis « ex novo » of enzymes and cell structures predominates.     

Effect of mitochondrial protein synthesis inhibitors on erythroid differentiation of mouse erythroleukemia (Friend) cells.

When mouse erythroleukemia (MEL) cells were incubated in the presence of chloramphenicol (a specific inhibitor for mitochondrial protein synthesis) during the early stage of in vitro erythroid differentiation, the number of induced erythroid cells was greatly reduced. By use of cell fusion between two genetically marked MEL cells, this finding was further investigated. We found that the drug, along with other agents which inhibit mitochondrial protein synthesis, blocked the induction and turnover of the DMSO-inducible intracellular-erythroid-inducing activity (differentiation-inducing factor II) in a manner similar to that of cycloheximide, an inhibitor for nuclear protein synthesis. The inhibitory effect was confirmed by directly assaying differentiation-inducing factor II in the cell extracts. These results strongly suggest that mitochondrial protein synthesis is closely associated with in vitro erythroid differentiation of MEL cells.     

The role of mitochondrial calcium transport during inflammation and the effect of anti-inflammatory drugs

The effects of inflammation induced by the inoculation of rats with Freund's adjuvant on calcium transport by isolated rat liver mitochondria and on mitochondrial in vivo protein synthesis were investigated. Mitochondria isolated from the liver of inflamed rats exhibited (i) a reduction in 45Ca2+ uptake and, (ii) a reduction in protein synthesis. Addition of ATP to the calcium uptake medium stimulate the uptake in inflamed rat liver mitochondria. After inflammation was controlled by treatment with a mixture of Clerodendron inerme flavonoidal glycosides and indomethacin, rat liver mitochondria showed (i) an increase in 45Ca2+ uptake and, (ii) an increase in mitochondrial in vivo protein synthesis. The mechanism of mitochondrial calcium transport and the mitochondrial protein metabolism during inflammation and after treatment with anti-inflammatory drugs were discussed.     

Macromolecular synthesis in mitochondria isolated from different regions of developing rat brain

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.     

Effects of chloramphenicol isomers and erythromycin on enzyme and lipid synthesis induced by oxygen in wild-type and petite yeast

The synthesis of mitochondrial enzymes induced by exposure of anaerobically grown, lipid-depleted Saccharomyces cerevisiae to oxygen is inhibited by d(-)-threo-chloramphenicol and erythromycin. The concentration of these antibiotics required to cause 50% inhibition of this synthesis is less than 1 mm; this is also approximately the concentration required to inhibit by the same amount mitochondrial protein synthesis in situ. The synthesis of unsaturated fatty acids, ergosterol, and phospholipid induced by aeration is inhibited by d(-)-threo-chloramphenicol at high concentrations (12 mm) but is unaffected by erythromycin. l(+)-threo-Chloramphenicol affects neither enzyme nor lipid synthesis and is without effect on mitochondrial protein synthesis in situ. All three compounds inhibit the oxidative activity of isolated mitochondria; the chloramphenicol isomers also inhibit phosphorylation. In a euflavine-derived petite mutant, lacking mitochondrial protein synthesis and respiration, aeration results in the normal development of lipid in the cells, but no synthesis of mitochondrial enzymes. d(-)-threo-Chloramphenicol does not inhibit lipid synthesis in these cells. Thus inhibition of mitochondrial protein synthesis with erythromycin or genetic deletion of mitochondrial protein synthesis results in loss of the capacity to synthesize enzymes during aeration. d(-)-threo-Chloramphenicol, as well as inhibiting induced enzyme formation, inhibits lipid synthesis induced by oxygen. It is unlikely that the latter effect of chloramphenicol is due to inhibition of energy production and transformation, to direct effects on lipid synthesis, or to an inhibition of mitochondrial protein synthesis. It is, however, an effect not shared with the l isomer.     

Mitochondrial protein synthesis in chinese hamster cells (line CHO) synchronized by isoleucine deprivation

Mitochondrial protein synthesis was measured in line CHO cells after phases of the cell cycle were synchronized by isoleucine deprivation or mitotic selection. Maximum incorporation of [³H] leucine into mitochondrial polypeptides occurred within 2 hours after isoleucine was added to initiate G₁ traverse. In cells synchronized in G₁ by mitotic selection, the rate of mitochondrial protein synthesis was fairly constant throughout the cell cycle. SDS-polyacrylamide gel electrophoretic profiles of labeled mitochondrial polypeptides were similar in cells synchronized by either isoleucine deprivation or mitotic selection. Obvious changes in the distribution of polypeptides were not detected during various phases of the cell cycle. The increased rate of incorporation of [³H] leucine into mitochondrial polypeptides after reversal of G₁-arrest may indicate that mitochondrial protein synthesis and possibly mitochondrial biogenesis are synchronized in CHO cells deprived of isoleucine.     

Mitochondrial Protein Synthesis During Embryonic Development of Cricket

Protein synthetic activity in homogenate of embryos of cricket, Gryllus bimaculatus , increased transiently 25 hr after oviposition, though the activity in mitochondrial fraction increased successively thereafter. The decrease in the activity after the peak in homogenate was caused apparently by increase in the amino acid content in cytosol during development. To analyze increase in the mitochondrial protein synthetic activity 25 hr after oviposition, effect of various components was examined in the reconstituted mitochondrial system. The nucleic acids extracted from 25-hr embryos most effectively stimulated the protein synthetic activity, but those from 0-hr embryos did not. The results were discussed on the role of mitochondrial protein synthesis during development of cricket embryos.     

Effect of mitochondrial functions on synthesis of yeast cytochrome c

The effects of the mitochondrial protein synthesis inhibitor chloramphenicol and the mitochondrial F0 adenosine triphosphatase inhibitor oligomycin on the synthesis of nucleus-encoded cytochrome c protein were studied. Both inhibitors stimulated cytochrome c protein synthesis in the derepressed state (growth in media containing 2% raffinose) but had no effect on the synthesis of the cytochrome c protein in the repressed state (growth in media containing 5% glucose). Oligomycin uncoupled the synthesis of the apoprotein from its processing into the hemoprotein. Neither antibiotic had a significant effect on the rate of glucose repression of cytochrome protein synthesis. The kinetics of cytochrome c derepression and the effects of these two antibiotics on these kinetics were also studied. Cells were derepressed by transfer from glucose- to faffinose-containing media, and the rate of cytochrome c synthesis increased from the repressed to the derepressed level during the second hour of derepression. Chloramphenicol delayed this derepression, but after 5 h the rate of cytochrome c protein synthesis increased to twice the rate of synthesis in uninhibited cells. On the other hand, oligomycin inhibited derepression of cytochrome c. These results are discussed with respect to the effects of mitochondrial function in the derepressed and repressed states and during the processes of repression and derepression of cytochrome c.     

Comparison of the developmental patterns of mitochondrial malate dehydrogenase between mung bean and cucumber cotyledons during and following germination

The development of mitochondrial NAD⁺-malate dehydrogenase (EC 1.1.1.37) in mung bean and cucumber cotyledons was followed. using the antibody raised against it, during and following germination. The developmental patterns were quite different between the two. In cucumber, the content of mitochondrial malate dehydrogenase continued to increase through 3–4 days after the beginning of imbibition. This was, at least in part, due to active synthesis of the enzyme protein, and the synthesis seemed to be regulated by the availability of the translatable mRNA for the enzyme. In mung bean, on the other hand, the enzyme was present in dry cotyledons at a rather high concentration, and remained at a constant level between day 1 and day 3 after the reduction of the content to one-half its initial level during the first day. De novo synthesis of the enzyme could not be detected in mung bean cotyledons by pulse-labeling experiment.     

Protein synthesis is defended in the mitochondrial fraction of gill but not heart in cunner (Tautogolabrus adspersus) exposed to acute hypoxia and hypothermia

The cunner, Tautogolabrus adspersus, is a north-temperate teleost which relies upon metabolic depression to survive the extreme low water temperatures of its habitat during the winter. Previous study has demonstrated a decrease in protein synthesis accompanies the metabolic depression observed at the whole animal level during seasonal low temperature exposure. As such, the objective of the current study was to determine: (i) if the response of decreased protein synthesis is conserved across environmental stressors and (ii) if the response of metabolic depression is conserved across levels of cellular organization. This was accomplished through the measurement of in vivo protein synthesis rates in the whole tissue, cytosolic and mitochondrial protein pools (reflective of nuclear encoded proteins imported into mitochondria) of heart and gill in cunner exposed to either acute low temperature (8–4°C) or acute hypoxia (10% O₂ saturation). In both heart and gill, rates of protein synthesis in the whole tissue and cytosolic protein pools were substantially depressed by 80% in response to acute hypothermia. In hypoxic heart, protein synthesis was significantly decreased by 50–60% in the whole tissue, cytosolic and mitochondrial pools; however, in gill there was no significant difference in rates of protein synthesis in any cellular fraction between normoxic and hypoxic groups. Most strikingly the rate of new protein accumulation in the mitochondrial fraction of gill did not change in response to either a decrease in temperature or hypoxia. The defense of protein synthesis in the gill is most likely associated with the importance of maintaining ionic regulation and the oxidative capacity in this front line organ for gas and ion exchange.     

Problems in the estimation of mutation rates and in the detection of induced mutations in man

The contribution of nuclear-directed protein synthesis in the repair of lethal and mitochondrial genetic damage after UV-irradiation of exponential and stiationary phase haploid yeast cells was examined. This was carried out using cycloheximide (CH), a specific inhibitor of nuclear protein synthesis. It appears that nuclear protein synthesis required for the increase in survival seen after the liquid holding of cells at both stages, as well as for the “petite” recovery seen after the liquid holding of exponential phase cells. The characteristic negative liquid holding effect observed for the UV induction of “petites” in stationary phase cells (increase of the frequency of “petites” during storage) remained following all the treatments which inhibited nuclear protein synthesis. However, the application of photoreactivating light following dark holding with cycloheximide indicates that some steps of the repair of both nuclear and mitochondrial damage are performed in the absence of a synthesis of proteins.