scholarly journals Total 4EBP1 Is Elevated in Liver of Rats in Response to Low Sulfur Amino Acid Intake

2013 ◽  
Vol 2013 ◽  
pp. 1-11 ◽  
Author(s):  
Angelos K. Sikalidis ◽  
Kevin M. Mazor ◽  
Minji Kang ◽  
Hongyun Liu ◽  
Martha H. Stipanuk
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Dietary Treatment ◽  
Binding Activity ◽  
Initiation Factor ◽  
Sulfur Amino Acid ◽  
Sulfur Amino Acids ◽  
Protein Levels ◽  
Cellular Stresses

Translation initiation is known to be regulated by the binding of eukaryotic initiation factor 4E (eIF4E) by binding proteins (4EBPs), and there is evidence that amino acid deprivation and other cellular stresses upregulate 4EBP1 expression. To pursue the question of whether diets limited in an essential amino acid lead to induction of 4EBP1 expression in vivo, diets that varied in methionine and cystine content were fed to rats for 7 days, and 4EBP1 mRNA and protein levels and 4EBP1 phosphorylation state were determined. Total 4EBP1 mRNA and protein abundance increased in liver of rats with severely deficient intakes of sulfur amino acids (0.23% or 0.11% methionine without cystine) but not in animals with a less restricted intake of sulfur amino acids (0.11% methionine plus 0.35% cystine) but a similarly restricted intake of total diet (53 to 62% of control). The amount of 4EBP1 binding activity (α + β forms) was elevated in liver of rats fed sulfur amino acid-deficient diets, whereas the hyperphosphorylation of 4EBP1 was not affected by dietary treatment. Results suggest that changes in total 4EBP1 expression should be considered when examining mechanisms that attenuate protein synthesis during amino acid deficiency states.

Wool growth and sulfur amino acid entry rate in sheep fed roughage based diets supplemented with bentonite and sulfur amino acids

1989 ◽  
Vol 40 (4) ◽  
pp. 889 ◽  
Author(s):  
PD Fenn ◽  
RA Leng
Keyword(s):  
Amino Acids ◽  
Drinking Water ◽  
Amino Acid ◽  
Continuous Infusion ◽  
Basal Diet ◽  
Sulfur Amino Acid ◽  
Sulfur Amino Acids ◽  
Entry Rate ◽  
Wool Growth ◽  
Water Supplementation

In two experiments, sheep were offered a roughage-based diet supplemented with either cysteine or bentonite as a solid, or bentonite, cysteine or methionine added to their drinking water. Supplementation with cysteine as a solid had no effect on wool growth, while supplementation via drinking water had no effect on wool growth or cysteine entry rate into the blood. Supplementation with methionine via drinking water increased the entry rate of methionine into blood by 69% (P< 0.05) as measured by a continuous infusion of [35S]-methionine. This coincided with subsequent increases in wool growth of 16% (P< 0.05) compared to sheep fed a basal diet alone. Compared with the basal diet alone, supplementation with 30 g/day bentonite as a dry powder or 60 g/day as a suspension in drinking water increased wool growth by 19 and 20% respectively. Bentonite given as sole supplement did not increase the entry rate of either cysteine or methionine into the blood of sheep. When bentonite and sulfur amino acids were complexed or mixed, wool growth was not increased above that for bentonite or the amino acid alone.

A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins

1993 ◽  
Vol 13 (12) ◽  
pp. 7295-7302
Author(s):  
J E Fajardo ◽  
R B Birge ◽  
H Hanafusa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Cell Transformation ◽  
Binding Activity ◽  
Sh2 Domain ◽  
Translational Initiation ◽  
Phosphorylated Proteins

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.

scholarly journals Mechanisms involved in the nutritional regulation of mRNA translation: features of the avian model

2006 ◽  
Vol 19 (1) ◽  
pp. 104-116 ◽  
Author(s):  
Sophie Tesseraud ◽  
Mourad Abbas ◽  
Sophie Duchene ◽  
Karine Bigot ◽  
Pascal Vaudin ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Muscle Growth ◽  
Mammalian Target Of Rapamycin ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Nutritional Regulation ◽  
Translational Repressor

Abstract:Insulin and amino acids are key factors in regulating protein synthesis. The mechanisms of their action have been widely studied for several years. The insulin signal is mediated by the activation of intracellular kinases such as phosphatidylinositol–3'kinase and the mammalian target of rapamycin (mTOR), affecting the phosphorylation of some major effectors involved in the regulation of translation initiation, i.e. p70 S6 kinase (p70S6K) and the translational repressor eukaryotic initiation factor 4E binding protein (4E-BP1). The amino acid–induced signalling cascade also originates from mTOR and promotes p70S6K and 4E–BP1 activation. However, the mechanisms of regulation are complex and little understood, especially in vivo. Elucidating these mechanisms is important for both fundamental physiology and nutritional applications, i.e. better control of the use of nutrients and optimisation of dietary amino acid supplies in various physiological and physiopathological situations. In comparative physiology, the chicken is an interesting model to gain better understanding of the nutritional regulation of mRNA translation because of the very high rates of muscle growth and protein synthesis, and the unusual features compared with mammals. In the present review we provide an overview of the roles of insulin and amino acids as regulators of protein synthesis in both mammals and avian species.

scholarly journals A 31-amino-acid N-terminal extension regulates c-Crk binding to tyrosine-phosphorylated proteins.

1993 ◽  
Vol 13 (12) ◽  
pp. 7295-7302 ◽  
Author(s):  
J E Fajardo ◽  
R B Birge ◽  
H Hanafusa
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Binding Affinity ◽  
Egf Receptor ◽  
Cell Transformation ◽  
Binding Activity ◽  
Sh2 Domain ◽  
Translational Initiation ◽  
Phosphorylated Proteins

Overproduction of v-Crk, but not of c-Crk, in chicken embryo fibroblasts results in cell transformation. The transforming activity of v-Crk mutants correlates with their ability to cause increased tyrosine phosphorylation of specific cellular proteins, a property that depends on the binding of v-Crk to phosphotyrosine residues via its SH2 domain. In this study, proteins translated in rabbit reticulocyte lysates were used to analyze interactions between Crk derivatives and tyrosine-phosphorylated proteins, particularly the epidermal growth factor (EGF) receptor. The results demonstrate that the binding affinity of c-Crk is much lower than that of v-Crk, despite the fact that both proteins contain identical SH2 domains. Moreover, a 31-amino-acid N-terminal extension of c-Crk, resulting from upstream translational initiation at a CUG codon, significantly increases the ability of the resulting protein to bind to phosphotyrosine-containing proteins. Of those 31 amino acids, 24 can be found in the 27-amino-acid region between Gag and Crk sequences in v-Crk, and removal of this region results in a protein with lower affinity toward the EGF receptor. In addition, fusion of Gag to the amino terminus of c-Crk yields a protein with a binding activity that is lower than that of v-Crk but significantly higher than that of c-Crk without the fusion. These data suggest that sequences N terminal to the Crk SH2 regulate binding activity to tyrosine-phosphorylated proteins and that the amino acids encoded immediately 5' to the c-Crk initiator AUG specifically increase binding affinity. In contrast, deletion of one or two SH3 domains of c-Crk proteins did not change their affinity for the EGF receptor. These results were confirmed in vivo by using A431-derived cell lines overproducing either the chicken c-Crk protein or c-Crk with the 31-amino-acid N-terminal extension. Furthermore, the in vivo experiments suggest that binding of Crk proteins to the stimulated EGF receptor results in Crk phosphorylation and subsequent loss of binding affinity.

TOTAL SULFUR AS A SCREENING CRITERION FOR SULFUR AMINO ACIDS IN LEGUMES

1977 ◽  
Vol 57 (1) ◽  
pp. 177-183 ◽  
Author(s):  
R. S. BHATTY ◽  
A. J. FINLAYSON ◽  
S. L. MACKENZIE
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Content ◽  
Sulfur Amino Acid ◽  
Total Sulfur ◽  
Sulfur Amino Acids ◽  
Meal Protein ◽  
Faba Beans ◽  
Sulfur Determination ◽  
The Mean

The relationship between total sulfur, determined by two methods, and sulfur-containing amino acids methionine and cystine was investigated in 33 genotypes of beans (Phaseolus vulgaris L.), faba beans (Vicia faba L.), peas (Pisum sativum L.), and lentils (Lens culinaris Medik.). The Leco Sulfur Analyzer (method 1) gave values for total sulfur in the legumes 29.2–47.6% lower than the gravimetric procedure (method 2). The mean coefficient of variability of method 1 was 36% higher than for method 2. The correlation coefficient between the two methods for the 33 genotypes was +0.42*. The correlation between percent meal protein and percent total sulfur in the legumes was +0.56**. The correlation between percent meal protein and total sulfur expressed as percent of meal protein or g/16 g N was negative (−0.75**). Faba beans, peas and lentils contained, on the average, 48% more cystine than methionine. Except for lentils, the mean total sulfur amino acid content of the legumes was generally similar. The correlation between percent total sulfur and total sulfur amino acid (mmoles/g meal) content of the legumes was +0.69** for peas and +0.46** for all the legumes. There was little or no change in the correlation when total sulfur and total sulfur amino acid data were expressed as percentages of the meal protein. The sulfur in the sulfur amino acids formed between 44.8 and 66.5% of the total meal sulfur. The rest of the sulfur was present in the meal as non-methionine and non-cystine sulfur. Total sulfur determination in the legume species used in the study is a poor indicator of methionine and cystine contents.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals BIOSYNTHESIS IN ISOLATED ACETABULARIA CHLOROPLASTS

1972 ◽  
Vol 54 (2) ◽  
pp. 279-294 ◽  
Author(s):  
David C. Shephard ◽  
Wendy B. Levin
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Technical Problems ◽  
Hydrolyzed Protein ◽  
Protein Amino Acids ◽  
Amino Acid Labeling ◽  
Labeling Pattern

The ability of chloroplasts isolated from Acetabulana mediterranea to synthesize the protein amino acids has been investigated. When this chloroplast isolate was presented with 14CO2 for periods of 6–8 hr, tracer was found in essentially all amino acid species of their hydrolyzed protein Phenylalanine labeling was not detected, probably due to technical problems, and hydroxyproline labeling was not tested for The incorporation of 14CO2 into the amino acids is driven by light and, as indicated by the amount of radioactivity lost during ninhydrin decarboxylation on the chromatograms, the amino acids appear to be uniformly labeled. The amino acid labeling pattern of the isolate is similar to that found in plastids labeled with 14CO2 in vivo. The chloroplast isolate did not utilize detectable amounts of externally supplied amino acids in light or, with added adenosine triphosphate (ATP), in darkness. It is concluded that these chloroplasts are a tight cytoplasmic compartment that is independent in supplying the amino acids used for its own protein synthesis. These results are discussed in terms of the role of contaminants in the observed synthesis, the "normalcy" of Acetabularia chloroplasts, the synthetic pathways for amino acids in plastids, and the implications of these observations for cell compartmentation and chloroplast autonomy.

scholarly journals Hsp90 Binds and Regulates the Ligand-Inducible α Subunit of Eukaryotic Translation Initiation Factor Kinase Gcn2

1999 ◽  
Vol 19 (12) ◽  
pp. 8422-8432 ◽  
Author(s):  
Olivier Donzé ◽  
Didier Picard
Keyword(s):  
Amino Acid ◽  
Constitutive Expression ◽  
Amino Acid Starvation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Restrictive Temperature ◽  
Α Subunit ◽  
Macbecin I

ABSTRACT The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

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