Intracellular amino acid levels as predictors of protein synthesis.

Amino acid availability is a rate-limiting factor in the regulation of protein synthesis. When amino acid supplies become restricted, mammalian cells employ homeostatic mechanisms to rapidly inhibit processes such as protein synthesis, which demands high levels of amino acids. Muscle cells in particular are subject to high protein turnover rates to maintain amino acid homeostasis. Mammalian target of rapamycin complex 1 (mTORC1) is an evolutionary conserved multiprotein complex that coordinates a network of signaling cascades and functions as a key mediator of protein translation, gene transcription, and autophagy. Signal transduction through mTORC1, which is centrally involved in muscle growth through enhanced protein translation, is governed by intracellular amino acid supply. The branched-chain amino acid leucine is critical for muscle growth and acts in part through activation of mTORC1. Recent research has revealed that mTORC1 signaling is coordinated primarily at the lysosomal membranes. This discovery has sparked a wealth of research in this field, revealing several different signaling molecules involved in transducing the amino acid signal to mTORC1, including the Rag GTPases, MAP4K3, and Vps34/ULK1. This review evaluates the current knowledge regarding cellular mechanisms that control and sense the intracellular amino acid pool. We discuss the role of leucine and mTORC1 in the regulation of amino acid transport via the system L and system A transporters such as LAT1 and SNAT2, as well as protein degradation via autophagic and proteasomal pathways. We also describe the complexities of energy homeostasis via AMPK and cell receptor-mediated growth signals that also converge on mTORC1. Leucine is a particularly potent regulator of protein turnover, to the extent where leucine stimulation alone is sufficient to stimulate mTORC1 signal transduction. The significance of leucine in this context is not yet known; however, recent advancements in this area will also be covered within this review.

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Influence of Amino-acid Levels on Protein Synthesis in vitro

Nature ◽

10.1038/2041194a0 ◽

1964 ◽

Vol 204 (4964) ◽

pp. 1194-1195 ◽

Cited By ~ 10

Author(s):

BETTY M. HANKING ◽

SIDNEY ROBERTS

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Amino Acid Levels

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Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1995.268.1.e75 ◽

1995 ◽

Vol 268 (1) ◽

pp. E75-E84 ◽

Cited By ~ 84

Author(s):

G. Biolo ◽

R. Y. Fleming ◽

S. P. Maggi ◽

R. R. Wolfe

Keyword(s):

Skeletal Muscle ◽

Amino Acids ◽

Protein Synthesis ◽

Amino Acid ◽

Human Skeletal Muscle ◽

De Novo ◽

Transmembrane Transport ◽

Acid Transport ◽

De Novo Synthesis ◽

Intracellular Amino Acid

We have used stable isotopic tracers of amino acids to measure in vivo transmembrane transport of phenylalanine, leucine, lysine, alanine, and glutamine as well as the rates of intracellular amino acid appearance from proteolysis, de novo synthesis, and disappearance to protein synthesis in human skeletal muscle. Calculations were based on data obtained by the arteriovenous catheterization of the femoral vessels and muscle biopsy. We found that the fractional contribution of transport from the bloodstream to the total intracellular amino acid appearance depends on the individual amino acid, varying between 0.63 +/- 0.02 for phenylalanine and 0.22 +/- 0.02 for alanine. Rates of alanine and glutamine de novo synthesis were approximately eight and five times their rate of appearance from protein breakdown, respectively. The model-derived rate of protein synthesis was highly correlated with the same value calculated by means of the tracer incorporation technique. Furthermore, amino acid transport rates were in the range expected from literature values. Consequently, we conclude that our new model provides a valid means of quantifying the important aspects of protein synthesis, breakdown, and amino acid transport in human subjects.

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Acidosis-Sensing Glutamine Pump SNAT2 Determines Amino Acid Levels and Mammalian Target of Rapamycin Signalling to Protein Synthesis in L6 Muscle Cells

Journal of the American Society of Nephrology ◽

10.1681/asn.2006091014 ◽

2007 ◽

Vol 18 (5) ◽

pp. 1426-1436 ◽

Cited By ~ 54

Author(s):

Kate Evans ◽

Zeerak Nasim ◽

Jeremy Brown ◽

Heather Butler ◽

Samira Kauser ◽

...

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Mammalian Target Of Rapamycin ◽

Muscle Cells ◽

Target Of Rapamycin ◽

Amino Acid Levels ◽

L6 Muscle Cells

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Cell density affects prolidase and prolinase activity and intracellular amino acid levels in cultured human cells

Clinica Chimica Acta ◽

10.1016/0009-8981(85)90305-5 ◽

1985 ◽

Vol 150 (1) ◽

pp. 1-9 ◽

Cited By ~ 16

Author(s):

Isaac Myara ◽

Christiane Charpentier ◽

Marthe Gautier ◽

Alain Lemonnier

Keyword(s):

Amino Acid ◽

Cell Density ◽

Human Cells ◽

Intracellular Amino Acid ◽

Cultured Human Cells ◽

Amino Acid Levels

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The E3 ubiquitin ligase ZNRF2 is a substrate of mTORC1 and regulates its activation by amino acids

eLife ◽

10.7554/elife.12278 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 8

Author(s):

Gerta Hoxhaj ◽

Edward Caddye ◽

Ayaz Najafov ◽

Vanessa P Houde ◽

Catherine Johnson ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Ubiquitin Ligase ◽

Protein Phosphatase ◽

E3 Ubiquitin Ligase ◽

Mechanistic Target Of Rapamycin ◽

Rag Gtpases ◽

Intracellular Amino Acid ◽

Amino Acid Levels ◽

Amino Acid Sensing

The mechanistic Target of Rapamycin complex 1 (mTORC1) senses intracellular amino acid levels through an intricate machinery, which includes the Rag GTPases, Ragulator and vacuolar ATPase (V-ATPase). The membrane-associated E3 ubiquitin ligase ZNRF2 is released into the cytosol upon its phosphorylation by Akt. In this study, we show that ZNRF2 interacts with mTOR on membranes, promoting the amino acid-stimulated translocation of mTORC1 to lysosomes and its activation in human cells. ZNRF2 also interacts with the V-ATPase and preserves lysosomal acidity. Moreover, knockdown of ZNRF2 decreases cell size and cell proliferation. Upon growth factor and amino acid stimulation, mTORC1 phosphorylates ZNRF2 on Ser145, and this phosphosite is dephosphorylated by protein phosphatase 6. Ser145 phosphorylation stimulates vesicle-to-cytosol translocation of ZNRF2 and forms a novel negative feedback on mTORC1. Our findings uncover ZNRF2 as a component of the amino acid sensing machinery that acts upstream of Rag-GTPases and the V-ATPase to activate mTORC1.

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Thermoregulation of myocardial protein synthesis

American Journal of Physiology-Legacy Content ◽

10.1152/ajplegacy.1975.228.3.884 ◽

1975 ◽

Vol 228 (3) ◽

pp. 884-889 ◽

Cited By ~ 4

Author(s):

H Taegtmeyer ◽

AG Ferguson ◽

M Lesch

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Thermal Injury ◽

Thermal Damage ◽

Specific Activity ◽

Effect Of Temperature ◽

Papillary Muscles ◽

Amino Acid Incorporation ◽

Intracellular Amino Acid ◽

Acid Incorporation

The effect of temperature on myocardial protein synthesis was evaluated using L-[14C]phenylalanine incorporation into total protein of isolated rabbit right ventricular papillary muscles. Muscles were incubated in oxygenated Krebs-Ringer buffer containing tracer amino acid at temperatures of 25-43 degrees C or incubated without tracer at varying temperatures up to 120 min and then incubated at 37 degrees C for an additional 2 h with the tracer present for the final hour of incubation. Higher as well as lower than physiological temperatures depressed tracer amino acid incorporation. Recovery of myocardial protein synthesis from thermal injury was incomplete when the experimental temperature deviated by 6 degrees C or more from the control and exposure exceeded 60 min. In addition, tracer amino acid incorporation on reoxygenation and return to 37 degrees C in muscles exposed to anoxia at 25 degrees C did not differ from that in muscles exposed to anoxia at 37 degrees C. Specific activity of the intracellular amino acid pool was directly measured in appropriate experiments and variation of this parameter could not account for the depressed tracer amino acid incorporation. Likewise methylprednisolone (10-5 M), chloroquine phosphate (10-5 M), and glucose (15 mM), if present during hyperthemia, did not ameliorate thermal damage. It is concluded that hyperthermia as well as hypothermia can cause irreversible alterations rather than reversible inhibition of myocardial protein synthesis.

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