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V M Pain

Publications and source records attributed to V M Pain.

At least 37 records · Page 2Linked to original sources
PubMed1995

Proteolytic cleavage of initiation factor eIF-4 gamma in the reticulocyte lysate inhibits translation of capped mRNAs but enhances that of uncapped mRNAs.

Infection of cells with the foot-and-mouth-disease virus, a member of the picornavirus family, results in the shut-off of host protein synthesis. A major contributory mechanism is the proteolytic destruction of the gamma subunit of the complex eIF-4, which functions in translation to promote the binding of the 43S ribosomal preinitiation complex to the 5' end of the cellular mRNA molecules bearing a 5' terminal cap structure. Picornavirus RNA molecules, which are uncapped, use a distinct mechanism for translational initiation, which can operate in the absence, or at low levels, of eIF-4. The proteolysis of eIF-4 gamma in cells infected by foot-and-mouth-disease virus results from expression of a virus-encoded cysteine proteinase known as Leader (or L) protease. We have used a transcription plasmid encoding this protease as a tool to deplete in vitro translation systems of eIF-4 gamma in order to elucidate in more detail the role of this polypeptide in the control of translation. Using in vitro transcribed mRNAs we have observed a marked contrast between capped and uncapped transcripts in the response of their translation to the proteolysis of eIF-4 gamma. Translation of capped mRNAs is, as expected, severely impaired, and is restored by addition of eIF-4 complex containing the intact gamma-subunit. On the other hand, translation of uncapped transcripts, normally inefficient, is substantially enhanced. The data suggest that the translation of uncapped mRNAs may be stimulated in this system by one or more of the proteolytic degradation products of eIF-4 gamma.

3C Viral Proteases↗
PubMed1995

Hormone-induced meiotic maturation in Xenopus oocytes occurs independently of p70s6k activation and is associated with enhanced initiation factor (eIF)-4F phosphorylation and complex formation.

Hormone-induced meiotic maturation of the Xenopus oocyte is regulated by complex changes in protein phosphorylation. It is accompanied by a stimulation in the rate of translation, manifest at the level of polypeptide chain initiation. At laser times in the maturation process, this reflects an increased ability for mRNA to interact with the 40 S ribosomal subunit. In mammalian cells there is growing evidence for the regulation of translation by phosphorylation of ribosomal protein S6 and of initiation factors responsible for the binding of mRNA to ribosomes. In this report, we show that although the 70 kDa form of S6 kinase is activated within 1.5 hours in response to progesterone or insulin, a time critical for protein synthesis, its activation is not required for hormone-induced stimulation of translation rates or maturation. In response to progesterone, activation of translation occurs in parallel with enhanced phosphate labelling of eIF-4 alpha and eIF-4 gamma and eIF-4F complex formation, events which are thought to facilitate the interaction of eIF-4F with the mRNA cap structure. However, with insulin, activation of translation occurs prior to detectable de novo phosphorylation of eIF-4F, although a small enhancement of turnover of phosphate on eIF-4 alpha may occur at this early time. With either hormone, enhanced phosphate labelling of eIF-4 alpha is shown to reflect activation of eIF-4 alpha kinase(s), which coincides temporally with activation of p42 MAP and p90rsk kinases. The possible role of initiation factor modification on increased translation rates during meiotic maturation is discussed.

Animals↗
PubMed1994

Translational control during amino acid starvation.

Amino acid starvation of mammalian cells results in a pronounced fall in the overall rate of protein synthesis. This is associated with increased phosphorylation of the alpha-subunit of the initiation factor eIF-2, which in turn impairs the activity of the guanine nucleotide exchange factor, eIF-2B. Similar mechanisms have now been found to operate in the yeast, Saccharomyces cerevisiae, where the major physiological result is to circumvent the lack of external amino acids by promoting the translation of a transcription factor, GCN4, that facilitates the expression of a number of enzymes required for amino acid biosynthesis. This article reviews current knowledge of these mechanisms in both mammalian and yeast cells and identifies questions still requiring elucidation.

Amino Acids↗
PubMed1993

Increased phosphorylation of eukaryotic initiation factor 4 alpha during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation.

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low translation rates consistent with their non-proliferative state. When cultured in the presence of the appropriate mitogen, there is a 2-4-fold increase in the rate of protein synthesis per ribosome within 4 h of stimulation [Kay, J. E., Ahern, T. and Atkins, M. (1971) Biochim. Biophys. Acta 247, 322-334]. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahern, T., Sampson, J. and Kay, J. E. (1974) Nature 248, 519-521]. In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol ester phorbol 12-myristate 13-acetate or concanavalin A leads to a rapid 2-4-fold increase in the rate of protein synthesis within 1 h or 4 h, respectively, which is insensitive to the transcriptional inhibitor, 5,6-dichlorobenzimidazole riboside. Relative to control cells, both phorbol ester and concanavalin A induce a 2-4-fold increase in labelling of the eukaryotic initiation factor eIF-4 alpha with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF-4 alpha. Similarly, with the human leukaemic T cell line JURKAT, stimulation of the T cell receptor with the monoclonal antibody, OKT-3, or treatment with phorbol ester induces a 2-3-fold increase in eIF-4 alpha phosphorylation within 30 min. Analysis of phosphorylation by two-dimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF-4 alpha kinase activity rather than phosphate turnover on eIF-4 alpha. Of central importance is the finding that, concomitant with increased rates of protein synthesis following stimulation of PPBMCs with either phorbol ester or concanavalin A, there is a significant increase in the level of eIF-4 alpha recovered in high-molecular-mass complexes. These data suggest that, in quiescent PPBMCs, eIF-4F may be limiting and that the association of eIF-4 alpha and eIF-4 gamma into high-molecular-mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.

Amino Acid Sequence↗
PubMed1993

Stimulation by insulin of protein synthesis in cultured chick embryo fibroblasts.

Insulin stimulates protein synthesis in skeletal muscle of young animals and has been reported to exert similar effects on a variety of mammalian cell types in culture. However, with chick embryo fibroblasts we found that the extent of this effect was very sensitive to the culture conditions of the cells prior to the insulin treatment. The most reproducible results were obtained with cells that had been sub-cultured into medium in which fetal calf serum was replaced with 2% horse serum. Insulin only stimulated protein synthesis when added at supra-physiological concentrations. Insulin-like growth factor 1 was effective at much lower concentrations. Since chick embryo fibroblasts can be obtained in good yield, they offer, if treated under appropriate conditions, a suitable system for study of the mechanisms by which insulin and IGF-1 promote the initiation of protein synthesis in eukaryotic cells.

Animals↗
PubMed1990

Effect of starvation and diabetes on the activity of the eukaryotic initiation factor eIF-2 in rat skeletal muscle.

The ability of the initiation factor eIF-2 in skeletal muscle extracts to form ternary initiation complexes ([Met-tRNA(f).eIF-2.GDP]) is decreased by either starvation or diabetes. These conditions also impair the ability of muscle extracts to dissociate [eIF-2.GDP], suggesting inhibition of the guanine nucleotide exchange reaction essential for eIF-2 recycling. We could not, however, detect any change in the phosphorylation state of the alpha subunit of eIF-2. This suggests that eIF-2 activity may be regulated in this system by a mechanism not involving its phosphorylation.

Animals↗
PubMed1989

Protein metabolism in the tumour-bearing mouse. Rates of protein synthesis in host tissues and in an Ehrlich ascites tumour at different stages in tumour growth.

We have investigated the time course of the changes in protein metabolism in skeletal muscle and liver in mice during the progression of growth of an Ehrlich ascites tumour. The rate of protein synthesis in muscle begins to fall very rapidly, and the decrease is clearly established by the time the tumour first becomes visible at 4 days after implantation of the cells. Liver protein synthesis increases substantially, and protein breakdown in muscle increases, but the onset of both these changes occurs later than the fall in muscle protein synthesis. A decrease in food intake in these animals occurs very rapidly after introduction of the cells. The fractional rate of protein synthesis in the tumour cells falls from 73%/day at 5 days to 26%/day at 12 days after injection, but on an absolute basis the rate of protein synthesis in the tumour at 5 days of growth is very small compared with that in muscle and liver. These results are consistent with the notion that the initial effects on muscle protein synthesis and food intake are brought about by humoral factors rather than as direct consequences of the metabolic demands of the growing tumour.

Animals↗
PubMed1989

Preparation and characterization of cell-free protein synthesis systems from oocytes and eggs of Xenopus laevis.

During the maturation of the oocytes of the frog Xenopus laevis, the rate of protein synthesis shows a twofold increase. Studies of the mechanisms involved in this stimulation have been seriously limited by the lack of an active cell-free translation system. We have now prepared such systems from oocytes, progesterone-matured oocytes and eggs of Xenopus laevis by induction of lysis by centrifugation of whole cells. The extracts are highly active in incorporation of labelled amino acids and, in the progesterone-matured and egg extracts, a substantial proportion of this is due to reinitiation on endogenous mRNA, as shown by the use of inhibitors. The increased rate of protein synthesis previously observed in intact oocytes following progesterone-induced maturation is reflected in the relative activities of the extracts. The difference in activity is not due to the presence of a dominant inhibitor of translation in the extracts from unstimulated oocytes. Labelling studies with initiator tRNA ([35S]Met-tRNAf) indicate a higher concentration of 43S preinitiation complexes in the extracts from unstimulated oocytes, suggesting an impairment of initiation of translation at or after the mRNA-binding step. Extracts from both oocytes and progesterone-matured oocytes translated endogenous mRNAs to give products ranging over a wide spectrum of molecular weight. However, significant translation of exogenous (globin) mRNA required the presence of reticulocyte postribosomal supernatant, suggesting that one or more factors required for mRNA recruitment is limiting in these extracts.

Animals↗
PubMed1987

Protein metabolism in the mouse during pregnancy and lactation.

Protein synthesis was measured in vivo in the whole body and in a number of individual tissues in mice at various stages of pregnancy and lactation. The absolute rate of protein synthesis in the whole body increased from 640 mg/day in virgin mice to 1590 mg/day by day 18 of pregnancy, and to 2100 mg/day by day 15 of lactation. Large proportions of these increments were contributed by the rapidly growing foetuses and placentae in the pregnant animals and by protein synthesis in the mammary glands during lactation. In addition, a substantial stimulation of growth and protein synthesis was also observed in the liver and the gastrointestinal tract. Gastrocnemius muscle showed no changes in protein metabolism, indicating that in the well-fed mouse this tissue is not required to play a role as a protein reserve during pregnancy and lactation.

Animals↗
PubMed1986

Effect of diabetes on the rates of synthesis and degradation of ribosomes in rat muscle and liver in vivo.

An important component of the decrease in protein synthesis in muscle of diabetic animals is a fall in the ribosome content. Therefore, we have investigated the turnover of ribosomes in skeletal muscle, heart, and liver of rats during the onset of diabetes. Synthesis rates were measured by incorporation of label into the protein moieties of the ribosomes, and a dual isotope technique was used to relate ribosome synthesis to that of total tissue protein. Degradation rates were calculated as the difference between the rates of synthesis and accumulation. The loss of ribosomes from gastrocnemius muscle and heart took place mainly between the 2nd and 4th days of insulin deficiency and was brought about largely by a very pronounced increase in the degradation rate, though synthesis also fell by a substantial amount. Rates of total tissue protein synthesis decreased markedly, but the degradation rates were only slightly elevated, if at all. Thus, the effect of diabetes on muscle ribosome breakdown was quite distinct from that on degradation of total tissue protein. In liver the response of protein synthesis to diabetes was much less pronounced than in muscle, and ribosome synthesis was not affected.

Animals↗
PubMed1986

Insulin stimulation of growth in diabetic rats. Synthesis and degradation of ribosomes and total tissue protein in skeletal muscle and heart.

Skeletal muscle and heart of diabetic rats show a substantial decline in the rate of protein synthesis associated with decreases in both the number and activity of tissue ribosomes. We have examined the reversal of these changes during the first 3 days of resumption of insulin therapy to rats that had been diabetic for 4 days. Rates of ribosome degradation, which had been elevated in both muscle and heart of the diabetic animals, were suppressed virtually to zero after 1 day of insulin treatment. Synthesis of ribosomes was stimulated, but this change occurred more gradually. Similar, but less dramatic, changes occurred in the rates of synthesis and degradation of total protein in these tissues.

Animals↗
PubMed1984

Effects of starvation, diabetes and acute insulin treatment on the regulation of polypeptide-chain initiation in rat skeletal muscle.

The rate of protein synthesis in skeletal muscle is greatly decreased in response to diabetes and starvation. Analysis of polyribosome profiles indicates that polypeptide-chain initiation is impaired under these conditions. To identify the step in initiation that is affected, we assayed the incorporation of [35S]methionyl-tRNAfMet into [35S]methionyl-tRNAfMet . 40S-ribosomal-subunit initiation complexes in cell-free extracts based on postmitochondrial supernatants prepared from gastrocnemius muscle. Extracts from either starved or diabetic rats were 30-40% less active in forming these complexes compared with those derived from fed or insulin-maintained controls respectively. This change could be reversed by treatment of either starved or diabetic rats with insulin in vivo 30 min before death. Formation of 40S initiation complexes by extracts from either fed or starved rats could be stimulated by the addition of exogenous purified initiation factor eIF-2, but extracts from starved or diabetic rats were more sensitive than controls to stimulation by low concentrations of the factor. These results provide evidence for the acute regulation by insulin of protein synthesis in skeletal muscle at the level of polypeptide-chain initiation, and suggest that in this tissue, as in certain other eukaryotic systems, control of initiation appears to be mediated by changes in the activity of initiation factor eIF-2.

Animals↗