scholarly journals Differences in degradation processes for insulin and its receptor in cultured foetal hepatocytes

1983 ◽  
Vol 212 (3) ◽  
pp. 529-537 ◽  
Author(s):  
E Pringault ◽  
C Plas
Keyword(s):  
Protein Synthesis ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Inhibitory Effect ◽  
Insulin Degradation ◽  
Protein Synthesis Inhibitors ◽  
Degradation Processes ◽  
Degradation Rates ◽  
Number Of Binding Sites ◽  
The Stability

Binding and degradation of 125I-labelled insulin were studied in cultured foetal hepatocytes after exposure to the protein-synthesis inhibitors tunicamycin and cycloheximide. Tunicamycin (1 microgram/ml) induced a steady decrease of insulin binding, which was decreased by 50% after 13 h. As the total number of binding sites per hepatocyte was 20000, the rate of the receptor degradation could not exceed 13 sites/min per hepatocyte. Cycloheximide (2.8 micrograms/ml) increased insulin binding by 30% within 6 h, an effect that persisted for up to 25 h. This drug had a specific inhibitory effect on the degradation of proteins prelabelled for 10 h with [14C]glucosamine, without affecting the degradation of total proteins. Chronic exposure to 10 nM-insulin neither decreased insulin binding nor modified the effect of the drugs. The absence of down-regulation of insulin receptors cannot be attributed to rapid receptor biosynthesis in foetal hepatocytes. Cellular insulin degradation, which is exclusively receptor-mediated, was determined by two different parameters. First, the rate of release of degraded insulin into the medium was 600 molecules/min per hepatocyte with 1 nM labelled hormone, and increased (preincubation with cycloheximide) or decreased (tunicamycin) as a function of the amount of cell-bound insulin. Secondly, the percentage of cell-bound insulin degraded was not changed by the presence of protein-synthesis inhibitors (25-30%). The stability of insulin degradation suggested that this process was dependent on long-life proteinase systems. Such differences in degradation rates and cycloheximide sensitivity imply that hormone- and receptor-degradation processes utilize distinct pathways.

Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi

1986 ◽  
Vol 6 (1) ◽  
pp. 54-61
Author(s):  
E J Baker ◽  
L R Keller ◽  
J A Schloss ◽  
J L Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Control ◽  
Protein S ◽  
Protein Synthesis Inhibitors ◽  
Control Factors ◽  
Flagellar Proteins ◽  
The Stability ◽  
And Control

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha- and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081, 1984). Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.

scholarly journals Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability.

1987 ◽  
Vol 7 (12) ◽  
pp. 4357-4368 ◽  
Author(s):  
N G Theodorakis ◽  
R I Morimoto
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Posttranscriptional Regulation ◽  
High Efficiency ◽  
Hsp70 Expression ◽  
Mrna Levels ◽  
Protein Synthesis Inhibitors ◽  
Hsp70 Mrna ◽  
Infected Cells ◽  
The Stability

We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated.

scholarly journals Protein synthesis is required for rapid degradation of tubulin mRNA and other deflagellation-induced RNAs in Chlamydomonas reinhardi.

1986 ◽  
Vol 6 (1) ◽  
pp. 54-61 ◽  
Author(s):  
E J Baker ◽  
L R Keller ◽  
J A Schloss ◽  
J L Rosenbaum
Keyword(s):  
Protein Synthesis ◽  
Transcriptional Control ◽  
Protein S ◽  
Protein Synthesis Inhibitors ◽  
Control Factors ◽  
Flagellar Proteins ◽  
The Stability ◽  
And Control

After flagellar detachment in Chlamydomonas reinhardi, there is a rapid synthesis and accumulation of mRNAs for tubulin and other flagellar proteins. Maximum levels of these mRNAs (flagellar RNAs) are reached within 1 h after deflagellation, after which they are rapidly degraded to their predeflagellation levels. The degradation of alpha- and beta-tubulin RNAs was shown to be due to the shortening of their half-lives after accumulation (Baker et al., J. Cell Biol. 99:2074-2081, 1984). Deflagellation in the presence of protein synthesis inhibitors results in the accumulation of tubulin and other flagellar mRNAs by kinetics similar to those of controls. However, unlike controls, in which the accumulated mRNAs are rapidly degraded, these mRNAs are stabilized in cycloheximide. The stabilization by cycloheximide is specific for the flagellar mRNAs accumulated after deflagellation, since there is no change in the levels of flagellar mRNAs in nondeflagellated (uninduced) cells in the presence of cycloheximide. The kinetics of flagellar mRNA synthesis after deflagellation are shown to be the same in cycloheximide-treated and control cells by in vivo labeling and in vitro nuclear runoff experiments. These results show that protein synthesis is not required for the induced synthesis of flagellar mRNAs, and that all necessary transcriptional control factors are present in the cell before deflagellation, but that protein synthesis is required for the accelerated degradation of the accumulated flagellar mRNAs. Since cycloheximide prevents the induced synthesis and accumulation of flagellar proteins, it is possible that the product(s) of protein synthesis required for the accelerated decay of these mRNAs is a flagellar protein(s). The possibility that one or more flagellar proteins autoregulate the stability of the flagellar mRNAs is discussed.

Insulin binding, internalization, and receptor regulation in cultured human fibroblasts

1981 ◽  
Vol 241 (3) ◽  
pp. E251-E260
Author(s):  
D. Baldwin ◽  
M. Prince ◽  
P. Tsai ◽  
C. Johnson ◽  
R. Lotan ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Degradation Products ◽  
Human Fibroblasts ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Insulin Concentration ◽  
Target Cells ◽  
Kinetic Properties ◽  
Insulin Degradation ◽  
Normal Human

Insulin binding to receptors was studied using monolayers of cultured normal human fibroblasts. Binding was rapid and inversely related to the incubation temperature; prolonged periods of steady-state binding were achieved at all temperatures studied and the amount of degradation of extracellular insulin was minimal. Competition curves demonstrated half-maximal inhibition of 125I-insulin binding at an unlabeled insulin concentration of 125I-insulin binding at an unlabeled insulin concentration of 7 ng/ml. Scatchard plots of the binding data were curvilinear and revealed that fibroblasts contained about 7,000 receptor sites per cell. Bound 125I-insulin dissociated from fibroblasts with a t 1/2 of 10 min at 30 degrees C and 35 min at 16 degrees C. After 60 min dissociation at 30 degrees C, 45% of the dissociated radioactivity consisted of 125I-insulin degradation products, whereas only 8% of the dissociated material was in the form of degraded products after 60 min of dissociation at 16 degrees C. This indicates that fibroblasts possess a temperature-sensitive receptor-mediated process for insulin degradation. Preincubation of the monolayers with insulin led to a hormone-induced loss of insulin receptors. Thus, incubating cells with 25 ng/ml insulin for 6 h at 37 degrees C caused a 50% reduction in subsequently measured 125I-insulin binding. This hormone-induced receptor loss was sensitive to physiologic insulin levels, with approximately 5 ng/ml causing a half-maximal receptor loss. When monolayers were treated with the lysosomotropic agent chloroquine and subsequently incubated with 5 X 10(-11) M 125I-insulin, a 130% increase in cell-associated radioactivity was observed after 120 min at 30 degrees C. In summary, 1) cultured normal human fibroblasts possess insulin receptors that exhibit kinetic properties and specificity identical to that of other insulin target cells; 2) incubation of fibroblasts with physiologic concentrations of insulin causes a marked loss of cell-surface insulin receptors; and 3) receptor-bound 125I-insulin is internalized through an energy-dependent endocytotic pathway and subsequently degraded by a chloroquine-sensitive reaction.

Phosphate-Transport Appearance in the Sea-Urchin Egg. I. Effects of Protein-Synthesis Inhibitors on Fertilized Eggs and Embryos

1974 ◽  
Vol 52 (6) ◽  
pp. 1178-1185 ◽  
Author(s):  
Joël de la Noüe
Keyword(s):  
Protein Synthesis ◽  
Active Transport ◽  
Sea Urchin ◽  
Developmental Stages ◽  
Sea Water ◽  
Mitochondrial Protein ◽  
Phosphate Transport ◽  
Inhibitory Effect ◽  
Protein Synthesis Inhibitors ◽  
Transport Carrier

When fertilized eggs of Strongylocentrotus purpuratus, the purple sea urchin, are incubated in sea water containing 32PO43− and L-14C-valine, valine incorporation is inhibited in the presence of puromycin, cycloheximide, or chloramphenicol, but only the last inhibits the active transport of phosphate. Since chloramphenicol does not depress the egg respiration, it is likely that this drug acts specifically. This is in line with a lack of inhibitory effect of chloramphenicol on phosphate uptake at later developmental stages, except at the time of gastrulation, when the appearance of new phosphate carriers might well occur. It is suggested that the active transport carrier for phosphate, or some element required for its operation, is synthesized after fertilization, with the likely participation of the mitochondrial protein-synthesis machinery. A similar proposal holds for valine uptake.

scholarly journals Incompatibility of chemical protein synthesis inhibitors with accurate measurement of extended protein degradation rates

2017 ◽  
Vol 5 (5) ◽  
pp. e00359 ◽  
Author(s):  
Christina Chan ◽  
Philip Martin ◽  
Neill J. Liptrott ◽  
Marco Siccardi ◽  
Lisa Almond ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Protein Degradation ◽  
Protein Synthesis Inhibitors ◽  
Degradation Rates ◽  
Chemical Protein Synthesis

Interleukin-1 alpha stimulates KC synthesis in rat mesangial cells: glucocorticoids inhibit KC induction by IL-1

1994 ◽  
Vol 266 (5) ◽  
pp. F713-F722 ◽  
Author(s):  
L. Feng ◽  
Y. Xia ◽  
J. I. Kreisberg ◽  
C. B. Wilson
Keyword(s):  
Protein Synthesis ◽  
Mesangial Cells ◽  
Interleukin 1 ◽  
Inhibitory Effect ◽  
Protein Synthesis Inhibitor ◽  
Synthesis Inhibitor ◽  
The Stability ◽  
New Protein ◽  
Neutrophil Chemotactic Activity

To assess the possible role of the production of chemokines by intrinsic glomerular cells in the generation of inflammation in glomerulonephritis, the chemokine, KC, was cloned from a rat macrophage cDNA library. Transfection of rat KC into COS-7 cells resulted in increased neutrophil chemotactic activity. The KC cDNA was expressed as a fusion protein in Escherichia coli for generation of an antibody. By using a riboprobe derived from the cDNA and the antibody, interleukin-1 (IL-1) was found to induce the expression of KC in rat mesangial cells. The induction of KC by IL-1 could be inhibited by dexamethasone (DEX). The protein synthesis inhibitor cycloheximide reversed the DEX-mediated inhibition, which suggested that new protein synthesis was necessary for the inhibitory effect. A nuclear runoff analysis indicated that DEX inhibited the transcription of KC induced by IL-1. The stability of KC mRNA was not decreased in the presence of DEX. Furthermore, immunoblots showed that DEX also inhibited KC expression at the level of translation. Together the inhibition of transcription and translation of the KC gene by DEX contribute to decreased KC expression in mesangial cells. The finding that mesangial cells express KC in response to proinflammatory cytokines, such as IL-1, points to a central role for the mesangial cell as a chemotactic source in glomerular inflammation.

Posttranscriptional regulation of hsp70 expression in human cells: effects of heat shock, inhibition of protein synthesis, and adenovirus infection on translation and mRNA stability

1987 ◽  
Vol 7 (12) ◽  
pp. 4357-4368
Author(s):  
N G Theodorakis ◽  
R I Morimoto
Keyword(s):  
Protein Synthesis ◽  
Heat Shock ◽  
Posttranscriptional Regulation ◽  
High Efficiency ◽  
Hsp70 Expression ◽  
Mrna Levels ◽  
Protein Synthesis Inhibitors ◽  
Hsp70 Mrna ◽  
Infected Cells ◽  
The Stability

We have examined the posttranscriptional regulation of hsp70 gene expression in two human cell lines, HeLa and 293 cells, which constitutively express high levels of HSP70. HSP70 mRNA translates with high efficiency in both control and heat-shocked cells. Therefore, heat shock is not required for the efficient translation of HSP70 mRNA. Rather, the main effect of heat shock on translation is to suppress the translatability of non-heat shock mRNAs. Heat shock, however, has a marked effect on the stability of HSP70 mRNA; in non-heat-shocked cells the half-life of HSP70 mRNA is approximately 50 min, and its stability increases at least 10-fold upon heat shock. Moreover, HSP70 mRNA is more stable in cells treated with protein synthesis inhibitors, suggesting that a heat shock-sensitive labile protein regulates its turnover. An additional effect on posttranscriptional regulation of hsp70 expression can be found in adenovirus-infected cells, in which HSP70 mRNA levels decline precipititously late during infection although hsp70 transcription continues unabated.

Effects of serine on protein synthesis and insulin receptors

1981 ◽  
Vol 241 (3) ◽  
pp. C167-C171 ◽  
Author(s):  
R. A. Galbraith ◽  
M. G. Buse
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Actinomycin D ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Essential Amino Acids ◽  
Leukemic Cells ◽  
Posttranscriptional Control ◽  
Receptor Number ◽  
Nonessential Amino Acids

Erythroblastic leukemic cells incubated in media containing essential amino acids, glutamine, and serine subsequently bound approximately 30% more [125I]insulin than those incubated without serine. The effect was due to an increase in receptor number, without change in affinity. Other nonessential amino acids had no effect. Increased insulin binding was observed with serine concentrations of 5.5 microM and maximum effects were seen at 22 microM. Serine-induced increases in insulin binding were detectable after 15 min of incubation and were abolished by the addition of cycloheximide (1 micrograms/ml) but not by actinomycin D (1 microgram/ml). Incorporation of [3H]leucine into protein was increased fourfold within 1 h by incubation with serine. The effect was detectable with 5.5 microM serine but, in contrast to insulin binding, reached a maximum at 88 microM serine. This differential dose responsiveness may represent selective posttranscriptional control of receptor synthesis, processing, insertion into the membrane, or recycling.

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