scholarly journals Cancer‐secreted miRNAs regulate amino‐acid‐induced mTORC1 signaling and fibroblast protein synthesis

EMBO Reports ◽  
2020 ◽  
Author(s):  
Miranda Y Fong ◽  
Wei Yan ◽  
Majid Ghassemian ◽  
Xiwei Wu ◽  
Xin Zhou ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Mtorc1 Signaling

Leucine and mTORC1: a complex relationship

2012 ◽  
Vol 302 (11) ◽  
pp. E1329-E1342 ◽  
Author(s):  
Kayleigh M. Dodd ◽  
Andrew R. Tee
Keyword(s):  
Signal Transduction ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Turnover ◽  
Muscle Growth ◽  
Protein Translation ◽  
Amino Acid Availability ◽  
Intracellular Amino Acid ◽  
System L ◽  
Mtorc1 Signaling

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.

scholarly journals Intracellular Staphylococcus aureus Infection Decreases Milk Protein Synthesis by Preventing Amino Acid Uptake in Bovine Mammary Epithelial Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Yuhao Chen ◽  
Yuze Ma ◽  
Qiang Ji ◽  
Xiaoru Yang ◽  
Xue Feng ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Milk Protein ◽  
Mammary Epithelial Cells ◽  
Protein Composition ◽  
Mammary Epithelial ◽  
Bovine Mammary Epithelial Cells ◽  
Mtorc1 Signaling

Staphylococcus aureus (S. aureus) is one of the main pathogens in cow mastitis, colonizing mammary tissues and being internalized into mammary epithelial cells, causing intracellular infection in the udder. Milk that is produced by cows that suffer from mastitis due to S. aureus is associated with decreased production and changes in protein composition. However, there is limited information on how mastitis-inducing bacteria affect raw milk, particularly with regard to protein content and protein composition. The main purpose of this work was to examine how S. aureus infection affects milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were infected with S. aureus, and milk protein and amino acid levels were determined by ELISA after S. aureus invasion. The activity of mTORC1 signaling and the transcription factors NF-κB and STAT5 and the expression of the amino acid transporters SLC1A3 and SLC7A5 were measured by western blot or immunofluorescence and RT-qPCR. S. aureus was internalized by BMECs in vitro, and the internalized bacteria underwent intracellular proliferation. Eight hours after S. aureus invasion, milk proteins were downregulated, and the level of BMECs that absorbed Glu, Asp, and Leu from the culture medium and the exogenous amino acids induced β-casein synthesis declined. Further, the activity of mTORC1 signaling, NF-κB, and STAT5 was impaired, and SLC1A3 and SLC7A5 were downregulated. Eight hours of treatment with 100 nM rapamycin inhibited NF-κB and STAT5 activity, SLC1A3 and SLC7A5 expression, and milk protein synthesis in BMECs. Thus mTORC1 regulates the expression of SLC1A3 and SLC7A5 through NF-κB and STAT5. These findings constitute a model by which S. aureus infection suppresses milk protein synthesis by decreasing amino acids uptake in BMECs.

ASCT2 silencing regulates mammalian target-of-rapamycin growth and survival signaling in human hepatoma cells

2007 ◽  
Vol 293 (1) ◽  
pp. C55-C63 ◽  
Author(s):  
Bryan C. Fuchs ◽  
Richard E. Finger ◽  
Marie C. Onan ◽  
Barrie P. Bode
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Ribosomal Protein ◽  
Binding Protein ◽  
Mammalian Target Of Rapamycin ◽  
Amino Acid Transporter ◽  
Human Hepatoma ◽  
Growth And Survival ◽  
Mtorc1 Signaling ◽  
Survival Signaling

System ASC amino acid transporter-2 (ASCT2) was previously demonstrated to be essential for human hepatoma cell growth and survival, as its silencing via inducible antisense RNA expression results in complete apoptosis within 48 h by a mechanism that transcends its role in amino acid delivery. To gain mechanistic insights into the reliance of cancerous liver cells on ASCT2, the aim of this study was to determine the early consequences of its silencing on the growth and survival signaling that presage apoptosis. Induced antisense ASCT2 RNA in SK-Hep1 cells led to >90% suppression of ASCT2 mRNA by 6 h and inhibition of mammalian target-of-rapamycin (mTOR)/raptor (mTOR complex-1; mTORC1) signaling by 8 h, as manifested by diminished p70 ribosomal protein S6 kinase-1 and eukaryotic initiation factor-4E (eIF4E) binding protein-1 phosphorylation, while protein synthesis rates declined by nearly 50% despite no measurable decreases in the cap binding protein eIF4G or cellular ribosomal protein content. Depressed mTORC1 signaling occurred before detectable reduction in ASCT2 activity but coincided with a 30% decline in total cellular ASCT2 protein. By 12 h after ASCT2 silencing, further decrements were observed in protein synthesis rates and ASCT2 protein and activity, each by ∼50%, while signaling from mTOR/rictor (mTOR complex-2; mTORC2) was stimulated as indexed by enhanced phosphorylation of the Akt/PKB kinase on serine-473 and of its proapoptotic substrate Bad on serine-136. These results suggest that ASCT2 silencing inhibits mTORC1 signaling to the translational machinery followed by an mTORC2-initiated survival response, establishing a link between amino acid transporter expression and mTOR function.

scholarly journals The Late Endosome is Essential for mTORC1 Signaling

2010 ◽  
Vol 21 (5) ◽  
pp. 833-841 ◽  
Author(s):  
Rory J. Flinn ◽  
Ying Yan ◽  
Sumanta Goswami ◽  
Peter J. Parker ◽  
Jonathan M. Backer
Keyword(s):  
Protein Synthesis ◽  
Growth Factors ◽  
Amino Acid ◽  
Cell Growth ◽  
Endocytic Trafficking ◽  
Nutrient Regulation ◽  
Late Endosomes ◽  
Mtorc1 Signaling ◽  
Regulate Protein ◽  
Early Late

The multisubunit mTORC1 complex integrates signals from growth factors and nutrients to regulate protein synthesis, cell growth, and autophagy. To examine how endocytic trafficking might be involved in nutrient regulation of mTORC1, we perturbed specific endocytic trafficking pathways and measured mTORC1 activity using S6K1 as a readout. When early/late endosomal conversion was blocked by either overexpression of constitutively active Rab5 (Rab5CA) or knockdown of the Rab7 GEF hVps39, insulin- and amino acid–stimulated mTORC1/S6K1 activation were inhibited, and mTOR localized to hybrid early/late endosomes. Inhibition of other stages of endocytic trafficking had no effect on mTORC1. Overexpression of Rheb, which activates mTOR independently of mTOR localization, rescued mTORC1 signaling in cells expressing Rab5CA, whereas hyperactivation of endogenous Rheb in TSC2−/− MEFs did not. These data suggest that integrity of late endosomes is essential for amino acid– and insulin-stimulated mTORC1 signaling and that blocking the early/late endosomal conversion prevents mTOR from interacting with Rheb in the late endosomal compartment.

scholarly journals An increase in essential amino acid availability upregulates amino acid transporter expression in human skeletal muscle

2010 ◽  
Vol 298 (5) ◽  
pp. E1011-E1018 ◽  
Author(s):  
Micah J. Drummond ◽  
Erin L. Glynn ◽  
Christopher S. Fry ◽  
Kyle L. Timmerman ◽  
Elena Volpi ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Protein Expression ◽  
Human Skeletal Muscle ◽  
Amino Acid Transporter ◽  
Dependent Manner ◽  
Mtorc1 Signaling ◽  
Acid Transporter ◽  
Transporter Expression

Essential amino acids (EAA) stimulate skeletal muscle mammalian target of rapamycin complex 1 (mTORC1) signaling and protein synthesis. It has recently been reported that an increase in amino acid (AA) transporter expression during anabolic conditions is rapamycin-sensitive. The purpose of this study was to determine whether an increase in EAA availability increases AA transporter expression in human skeletal muscle. Muscle biopsies were obtained from the vastus lateralis of seven young adult subjects (3 male, 4 female) before and 1–3 h after EAA ingestion (10 g). Blood and muscle samples were analyzed for leucine kinetics using stable isotopic techniques. Quantitative RT-PCR, and immunoblotting were used to determine the mRNA and protein expression, respectively, of AA transporters and members of the general AA control pathway [general control nonrepressed (GCN2), activating transcription factor (ATF4), and eukaryotic initiation factor (eIF2) α-subunit (Ser52)]. EAA ingestion increased blood leucine concentration, delivery of leucine to muscle, transport of leucine from blood into muscle, intracellular muscle leucine concentration, ribosomal protein S6 (Ser240/244) phosphorylation, and muscle protein synthesis. This was followed with increased L-type AA transporter (LAT1), CD98, sodium-coupled neutral AA transporter (SNAT2), and proton-coupled amino acid transporter (PAT1) mRNA expression at 1 h ( P < 0.05) and modest increases in LAT1 protein expression (3 h post-EAA) and SNAT2 protein expression (2 and 3 h post-EAA, P < 0.05). Although there were no changes in GCN2 expression and eIF2α phosphorylation, ATF4 protein expression reached significance by 2 h post-EAA ( P < 0.05). We conclude that an increase in EAA availability upregulates human skeletal muscle AA transporter expression, perhaps in an mTORC1-dependent manner, which may be an adaptive response necessary for improved AA intracellular delivery.

scholarly journals Effect of the lysosomotropic agent chloroquine on mTORC1 activation and protein synthesis in human skeletal muscle

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Michael S. Borack ◽  
Jared M. Dickinson ◽  
Christopher S. Fry ◽  
Paul T. Reidy ◽  
Melissa M. Markofski ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Protein Synthesis ◽  
Amino Acid ◽  
Human Skeletal Muscle ◽  
Muscle Protein ◽  
C2c12 Cells ◽  
Mtorc1 Signaling ◽  
Lysosomotropic Agent ◽  
Amino Acid Sensing

Abstract Background Previous work in HEK-293 cells demonstrated the importance of amino acid-induced mTORC1 translocation to the lysosomal surface for stimulating mTORC1 kinase activity and protein synthesis. This study tested the conservation of this amino acid sensing mechanism in human skeletal muscle by treating subjects with chloroquine—a lysosomotropic agent that induces in vitro and in vivo lysosome dysfunction. Methods mTORC1 signaling and muscle protein synthesis (MPS) were determined in vivo in a randomized controlled trial of 14 subjects (10 M, 4 F; 26 ± 4 year) that ingested 10 g of essential amino acids (EAA) after receiving 750 mg of chloroquine (CHQ, n = 7) or serving as controls (CON, n = 7; no chloroquine). Additionally, differentiated C2C12 cells were used to assess mTORC1 signaling and myotube protein synthesis (MyPS) in the presence and absence of leucine and the lysosomotropic agent chloroquine. Results mTORC1, S6K1, 4E-BP1 and rpS6 phosphorylation increased in both CON and CHQ 1 h post EAA ingestion (P < 0.05). MPS increased similarly in both groups (CON, P = 0.06; CHQ, P < 0.05). In contrast, in C2C12 cells, 1 mM leucine increased mTORC1 and S6K1 phosphorylation (P < 0.05), which was inhibited by 2 mg/ml chloroquine. Chloroquine (2 mg/ml) was sufficient to disrupt mTORC1 signaling, and MyPS. Conclusions Chloroquine did not inhibit amino acid-induced activation of mTORC1 signaling and skeletal MPS in humans as it does in C2C12 muscle cells. Therefore, different in vivo experimental approaches are required for confirming the precise role of the lysosome and amino acid sensing in human skeletal muscle. Trial registration NCT00891696. Registered 29 April 2009.

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