184 results on '"Ly Q"'
Search Results
2. Chemical Chaperones Correct the Mutant Phenotype of the ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator Protein
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Brown, C. Randell, Hong-Brown, Ly Q., Biwersi, Joachim, Verkman, A. S., and Welch, William J.
- Published
- 1996
3. Integration einer zweiten Öffnungsmöglichkeit in die Rückwandklappe eines Transporters
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Frömel, C., primary and Ly, Q. T., additional
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- 2017
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4. Activation of AMPK/TSC2/PLD by Alcohol Regulates mTORC1 and mTORC2 Assembly in C2C12 Myocytes
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Hong-Brown, Ly Q., Brown, Randell C., Navaratnarajah, Maithili, and Lang, Charles H.
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- 2013
- Full Text
- View/download PDF
5. Intracellular vesicle clusters are organelles that synthesize extracellular vesicle–associated cargo proteins in yeast
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Ly Q. Hong-Brown, Chelsea M. Winters, and Hui Ling Chiang
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0301 basic medicine ,Proteomics ,Saccharomyces cerevisiae ,Intracellular Space ,Vesicular Transport Proteins ,Biochemistry ,Exocytosis ,Fungal Proteins ,03 medical and health sciences ,Extracellular Vesicles ,Protein biosynthesis ,Extracellular ,Secretion ,Molecular Biology ,030102 biochemistry & molecular biology ,biology ,Chemistry ,Intracellular vesicle ,Cell Biology ,Extracellular vesicle ,biology.organism_classification ,Cell biology ,Protein Transport ,030104 developmental biology ,Cytoplasm ,Protein Synthesis and Degradation ,Protein Biosynthesis ,Periplasm ,cardiovascular system - Abstract
Extracellular vesicles (EVs) play important roles in cell-cell communication. In budding yeast (Saccharomyces cerevisiae), EVs function as carriers to transport cargo proteins into the periplasm for storage during glucose starvation. However, intracellular organelles that synthesize these EV-associated cargo proteins have not been identified. Here, we investigated whether cytoplasmic organelles—called intracellular vesicle clusters (IVCs)—serve as sites for the synthesis of proteins targeted for secretion as EV-associated proteins. Using proteomics, we identified 377 IVC-associated proteins in yeast cells grown under steady-state low-glucose conditions, with the largest group being involved in protein translation. Isolated IVCs exhibited protein synthesis activities that required initiation and elongation factors. We have also identified 431 newly synthesized proteins on isolated IVCs. Expression of 103Q-GFP, a foreign protein with a long polyglutamine extension, resulted in distribution of this protein as large puncta that co-localized with IVC markers, including fructose-1,6-bisphosphatase (FBPase) and the vacuole import and degradation protein Vid24p. We did not observe this pattern in cycloheximide-treated cells or in cells lacking VID genes, required for IVC formation. The induction of 103Q-GFP on IVCs adversely affected total protein synthesis in intact cells and on isolated IVCs. This expression also decreased levels of EV-associated cargo proteins in the extracellular fraction without affecting the number of secreted EVs. Our results provide important insights into the functions of IVCs as sites for the synthesis of EV-associated proteins targeted for secretion to the periplasm.
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- 2020
6. The Shape of Vesicle-Containing Organelles Is Critical for Their Functions in Vesicle Endocytosis
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Ly Q. Hong-Brown, Chelsea M. Winters, and Hui Ling Chiang
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0301 basic medicine ,Cell type ,Saccharomyces cerevisiae Proteins ,Vesicle fusion ,Saccharomyces cerevisiae ,Vesicular Transport Proteins ,Biology ,Bulk endocytosis ,03 medical and health sciences ,Cytosol ,Organelle ,Genetics ,Cell Shape ,Molecular Biology ,Eisosome ,Organelles ,Vesicle ,Cell Membrane ,Biological Transport ,Cell Biology ,General Medicine ,Phosphoproteins ,biology.organism_classification ,Endocytosis ,Microvesicles ,Cell biology ,030104 developmental biology - Abstract
Exosomes are small vesicles secreted by a variety of cell types under physiological and pathological conditions. When Saccharomyces cerevisiae are grown in low glucose, small vesicles carrying more than 300 proteins with diverse biological functions are secreted. Upon glucose addition, secreted vesicles are endocytosed that requires cup-shaped organelles containing the major eisosome protein Pil1p at the rims. We aim to identify genes that regulate the function of cup-shaped organelles in vesicle endocytosis. In cells lacking either VID27 or VID21, Pil1p distribution was altered and cup-shaped organelles became elongated with narrower openings. Change in shape reduced the number of vesicles in the deeper areas and impaired vesicle endocytosis. Vid21p and Vid27p were localized to vesicle clusters and interacted with other Vid proteins. In the absence of these genes, these vesicles failed to aggregate and were secreted. Vid21p and Vid27p are required for the aggregation and retention of vesicles that contain Vid proteins in the cytoplasm. Increased vesicles near the plasma membrane in mutant strains correlate with an increased Pil1p movement resulting in the fusion of cup-shaped organelles. We conclude that the shape of vesicle-containing organelles is critical for their functions in vesicle endocytosis.
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- 2017
7. Indinavir alters regulators of protein anabolism and catabolism in skeletal muscle
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Hong-Brown, Ly Q., Pruznak, Anne M., Frost, Robert A., Vary, Thomas C., and Lang, Charles H.
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HIV (Viruses) -- Research ,Eukaryotes -- Research ,Protease inhibitors -- Research ,Biological sciences - Abstract
The HIV protease inhibitor indinavir adversely impairs carbohydrate and lipid metabolism, whereas its influence on protein metabolism under in vivo conditions remains unknown. The present study tested the hypothesis that indinavir also decreases basal protein synthesis and impairs the anabolic response to insulin in skeletal muscle. Indinavir was infused intravenously for 4 h into conscious rats, at which time the homeostasis model assessment of insulin resistance was increased. Indinavir decreased muscle protein synthesis by 30%, and this reduction was due to impaired translational efficiency. To identify potential mechanisms responsible for regulating mRNA translation, several eukaryotic initiation factors (eIFs) were examined. Under basal fasted conditions, there was a redistribution of eIF4E from the active eIF4E x eIF4G complex to the inactive eIF4E x 4E-BP1 complex, and this change was associated with a marked decrease in the phosphorylation of 4E-BP 1 in muscle. Likewise, indinavir decreased constitutive phosphorylation of eIF4G and mTOR in muscle, but not S6K1 or the ribosomal protein S6. In contrast, the ability of a maximally stimulating dose of insulin to increase the phosphorylation of PKB, 4E-BP1, S6K1, or mTOR was not altered 20 min after intravenous injection. Indinavir increased mRNA expression of the ubiquitin ligase MuRF1, but the plasma concentration of 3-methylhistidine remained unaltered. These indinavir-induced changes were associated with a marked reduction in the plasma testosterone concentration but were independent of changes in plasma levels of IGF-I, corticosterone, TNF-[alpha], or IL-6. In conclusion, indinavir acutely impairs basal protein synthesis and translation initiation in skeletal muscle but, in contrast to muscle glucose uptake, does not impair insulin-stimulated signaling of protein synthetic pathways. eukaryotic initiation factor 4E; eukaryotic initiation factor 4E-binding protein-1; eukaryotic initiation factor 4G; mammalian target of rapamycin; ribosomal protein S6 kinase; muscle RING finger 1
- Published
- 2005
8. Indinavir impairs protein synthesis and phosphorylations of MAPKs in mouse [C.sub.2][C.sub.12] myocytes
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Hong-Brown, Ly Q., Brown, C. Randell, and Lang, Charles H.
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Proteins -- Research ,Protein biosynthesis -- Research ,Biological sciences - Abstract
Indinavir impairs protein synthesis and phosphorylations of MAPKs in mouse [C.sub.2][C.sub.12] myocytes. Am J Physiol Cell Physiol 287: C1482-C1492, 2004. First published June 30, 2004; doi:10.1152/ajpcell. 00038.2004.--Anti-retroviral therapy promotes clinical, immunologic, and virologic improvement in human immunodeficiency virus-infected patients. Whereas this therapy adversely affects carbohydrate and lipid metabolism, the effects of anti-retroviral drugs on muscle protein synthesis and degradation have not been reported. To examine these processes, we treated [C.sub.2][C.sub.12] myocytes with increasing concentrations of the protease inhibitor indinavir for 1 or 2 days. Treatment of myocytes with a therapeutic concentration of indinavir (20 [micro]M) for 24 h decreased basal protein synthesis by 18%, whereas a 42% decline was observed after 48 h. A similar decrement, albeit quantitatively smaller, was detected with other protease inhibitors. Indinavir did not alter the rate of proteolysis. Likewise, indinavir did not impair the anabolic effect of insulin-like growth factor-I on protein synthesis. Mechanistically, indinavir decreased the phosphorylation of the $6 ribosomal protein (rpS6), and this reduction was associated with a decreased phosphorylation of p70S6 kinase and p90rsk as well as the upstream regulators ERK1/2 and MEK1/2. Indinavir also decreased the phosphorylation of Mnk1 and its upstream effectors, p38 MAPK and ERK1/2. Indinavir did not affect the phosphorylation of roTOR or 4E-BP1, but it did decrease the amount of the active eukaryotic initiation factor elF4G-elF4E complex. In conclusion, indinavir decreased protein synthesis in myocytes. This decrease was associated with the disruption of the ERK1/2 and p38 MAPK pathways and a reduction in both the level of functional eIF4F complex and rpS6 phosphorylation. anti-retroviral drugs; mitogen-activated protein kinases; translation initiation
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- 2004
9. Ataxia–telangiectasia mutated kinase-mediated upregulation of NKG2D ligands on leukemia cells by resveratrol results in enhanced natural killer cell susceptibility
- Author
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Luis Espinoza, J., Takami, Akiyoshi, Trung, Ly Q., and Nakao, Shinji
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- 2013
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10. Sepsis-induced muscle growth hormone resistance occurs independently of STAT5 phosphorylation
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Hong-Brown, Ly Q., Brown, C. Randell, Cooney, Robert N., Frost, Robert A., and Lang, Charles H.
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Somatotropin ,Liver -- Physiological aspects ,Bacterial infections -- Physiological aspects ,Phosphorylation -- Physiological aspects ,Biological sciences - Abstract
Growth hormone (GH) stimulates insulin-like growth factor I (IGF-I) synthesis in both liver and muscle. During sepsis, proinflammatory cytokines inhibit GH action in liver, but it is unknown whether sepsis also produces GH resistance in muscle. Sepsis was induced by cecal ligation and puncture, and 18 h later the effect of GH on signal transducer and activator of transcription (STAT) phosphorylation and IGF-I mRNA content was assessed in rat gastrocnemius and liver. The relative abundance of phosphorylated (p)STAT5a, pSTAT5b, pSTAT3, and pSTAT1 was increased in liver from control rats after GH. Sepsis alone also increased hepatic pSTAT5a, pSTAT3, and pSTAT1. Sepsis dramatically impaired the ability of GH to stimulate the phosphorylation of STAT5a and -5b, as well as to increase IGF-I mRNA in liver. In muscle from control rats, GH increased pSTAT5a and -5b, whereas content of pSTAT3 and pSTAT1 was not affected. Sepsis increased basal content of pSTAT3 but not pSTAT5a, pSTAT5b, or pSTAT1 in muscle. The GH-induced increase of pSTAT5a and -5b in muscle from septic rats was not inhibited, suggesting that muscle was not GH resistant. In contrast to these changes in pSTAT5, the ability of GH to increase IGF-I mRNA was completely absent in muscle from septic rats. Because the suppressor of cytokine signaling (SOCS) proteins may function as negative regulators of GH signaling, we examined the content of these proteins. Sepsis produced small (30-50%), albeit statistically significant, increases in SOCS-1, -2, and -3 protein in muscle. In contrast to muscle, the SOCS proteins in the liver did not change under the various experimental conditions, suggesting that these proteins are not responsible for the impaired phosphorylation of STAT5 by GH. In conclusion, sepsis produces GH resistance in both muscle and liver, with the locus of this impairment in muscle differing from that in liver and being independent of a defect in STAT5 phosphorylation. growth hormone; signal transducer and activator of transcription; suppressors of cytokine signaling; insulin-like growth factor I; liver; endotoxin
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- 2003
11. Alcohol-Induced Modulation of Rictor and mTORC2 Activity in C2C12 Myoblasts
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Hong-Brown, Ly Q., Brown, Randell C., Navaratnarajah, Maithili, Huber, Danuta S., and Lang, Charles H.
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- 2011
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12. Intracellular vesicle clusters are organelles that synthesize extracellular vesicle–associated cargo proteins in yeast
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Winters, Chelsea M., primary, Hong-Brown, Ly Q., additional, and Chiang, Hui-Ling, additional
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- 2020
- Full Text
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13. Acetaminophen (paracetamol) inhibits myeloperoxidase-catalyzed oxidant production and biological damage at therapeutically achievable concentrations
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Koelsch, Maud, Mallak, Roger, Graham, Garry G., Kajer, Tracey, Milligan, Marian K., Nguyen, Ly Q., Newsham, Dawn W., Keh, Jeremy S., Kettle, Anthony J., Scott, Kieran F., Ziegler, John B., Pattison, David I., Fu, Shanlin, Hawkins, Clare L., Rees, Martin D., and Davies, Michael J.
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- 2010
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14. Acetaminophen (Paracetamol) Inhibits the Formation of Myeloperoxidase-Derived Oxidants and Biological Damage at Therapeutically-Achievable Concentrations: 374
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Koelsch, Maud, Mallak, Roger, Graham, Garry G, Rees, Martin D, Milligan, Marian K, Newsham, Dawn W, Nguyen, Ly Q, Kettle, Antony J, Scott, Keiran, Ziegler, John B, Kajer, Tracey, Fu, Shanlin, Hawkins, Clare L, and Davies, Michael J.
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- 2009
15. Strategies for Correcting the AF508 CFTR Protein-Folding Defect
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Brown, C. Randell, Hong-Brown, Ly Q., and Welch, William J.
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- 1997
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16. Alcohol and Indinavir Adversely Affect Protein Synthesis and Phosphorylation of MAPK and mTOR Signaling Pathways in C2C12 Myocytes
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Hong-Brown, Ly Q., Brown, C. Randell, Huber, Danuta S., and Lang, Charles H.
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- 2006
17. Diagnostic specificity and prognostic value of cardiac troponins in asymptomatic chronic haemodialysis patients: a three year prospective study
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Troyanov, S, Ly, Q H, Schampaert, E, Ammann, H, Lalumière, G, Madore, F, and Quérin, S
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- 2005
18. Myocardial infarction with myocardial rupture
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Ly, Q H and Lebeau, R
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- 2003
19. Role of Laparoscopy in Hepatic Cyst Surgery
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Gloor, B., Ly, Q., and Candinas, D.
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- 2002
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20. Phase I Trial of Concurrent Stereotactic Body Radiation Therapy and Nelfinavir for Locally Advanced Borderline or Unresectable Pancreatic Cancer-Final Results
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Lin, C., primary, Verma, V., additional, Ly, Q., additional, Schwarz, J., additional, Meza, J., additional, Sasson, A., additional, Are, C., additional, Kos, B., additional, and Grem, J., additional
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- 2018
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21. Strategies for Correcting theΔF508 CFTR Protein-Folding Defect
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Brown, C. Randell, Hong-Brown, Ly Q., and Welch, William J.
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- 1997
22. Correcting Temperature-sensitive Protein Folding Defects
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Brown, C. Randell, Hong-Brown, Ly Q., and Welch, William J.
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- 1997
23. Further Characterization of IgE-binding Antigens in Horse Dander, with Particular Emphasis on Glycoprotein Allergens
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Johnsen, T. K., Thanh, D. B., Ly, Q., Paulsen, B. Smestad, and Wold, J. K.
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- 1996
24. Adamts1 Mediates Ethanol-Induced Alterations in Collagen and Elastin via a FoxO1-Sestrin3-AMPK Signaling Cascade in Myocytes
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CRandell Brown, Maithili Navaratnarajah, Ly Q. Hong-Brown, and Charles H. Lang
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endocrine system ,medicine.medical_specialty ,biology ,Chemistry ,AMPK ,FOXO1 ,Cell Biology ,Matrix metalloproteinase ,Biochemistry ,Endocrinology ,Downregulation and upregulation ,AMP-activated protein kinase ,Internal medicine ,mental disorders ,biology.protein ,medicine ,Myocyte ,Phosphorylation ,Molecular Biology ,Elastin ,reproductive and urinary physiology - Abstract
A variety of stressors including alcohol (EtOH) are known to induce collagen production and fibrotic diseases. Matrix metalloproteinases (MMP) play an important role in regulating fibrosis, but little is known regarding the relationship between EtOH and MMPs. In addition, the signaling cascades involved in this process have not been elucidated. We have identified the MMP Adamts1 as a target of EtOH regulation. To characterize the function of Adamts1, we examined EtOH-induced alterations in collagen I and elastin protein levels in C2C12 myocytes. Incubation of myocytes with 100 mM EtOH decreased elastin and increased collagen content, respectively, and these changes were associated with increased O-GLcNAc modification of Adamts1. Conversely, silencing of Adamts1 by siRNA blocked the adverse effects of EtOH on collagen and elastin levels. Similar results were obtained after treatment with a pharmacological inhibitor of MMP. Changes in collagen were due, at least in part, to a decreased interaction of Adamts1 with its endogenous inhibitor TIMP3. The AMPK inhibitor compound C blocked the EtOH-induced stimulation of collagen and O-GLcNAc Adamts1 protein. Changes in AMPK appear linked to FoxO1, since inhibition of FoxO1 blocked the effects of EtOH on AMPK phosphorylation and O-GLcNAc levels. These FoxO-dependent modifications were associated with an upregulation of the FoxO1 transcription target sestrin 3, as well as increased binding of sestrin 3 with AMPK. Collectively, these data indicate that EtOH regulates the collagen I and elastin content in an Adamts1-dependent manner in myocytes. Furthermore, Adamts1 appears to be controlled by the FoxO1-sestrin 3-AMPK signaling cascade.
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- 2014
25. FoxO1-AMPK-ULK1 Regulates Ethanol-Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1-PIK3C3 Complex
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Charles H. Lang, C. Randell Brown, Ly Q. Hong-Brown, and Maithili Navaratnarajah
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0301 basic medicine ,endocrine system ,Small interfering RNA ,Vesicular Transport Proteins ,Medicine (miscellaneous) ,Autophagy-Related Proteins ,FOXO1 ,Protein degradation ,AMP-Activated Protein Kinases ,Toxicology ,Article ,Cell Line ,Myoblasts ,03 medical and health sciences ,Mice ,Phosphatidylinositol 3-Kinases ,Autophagy ,Animals ,Autophagy-Related Protein-1 Homolog ,Autophagy-Related Protein 7 ,Ethanol ,Chemistry ,Forkhead Box Protein O1 ,AMPK ,BECN1 ,Class III Phosphatidylinositol 3-Kinases ,Cell biology ,Psychiatry and Mental health ,030104 developmental biology ,Phosphorylation ,Beclin-1 ,Protein Binding - Abstract
Background Excessive alcohol (EtOH) consumption causes an imbalance in protein metabolism. EtOH impairs protein synthesis in C2C12 myoblasts via a FoxO1-AMPK-TSC2-mTORC1 pathway and also induces protein degradation. As the underlying regulatory signaling cascades for these processes are currently poorly defined, we tested the hypothesis that alcohol-induced autophagy is mediated via activation of the PIK3C3 complex that is regulated by FoxO1-AMPK. Methods C2C12 myoblasts were incubated with EtOH for various periods of time, and autophagy pathway-related proteins were assessed by Western blotting and immunoprecipitation. Expression of targeted genes was suppressed using electroporation of specific siRNAs and chemical inhibitors. Results Incubation of C2C12 myoblasts with 100 mM EtOH increased the autophagy markers LC3B-II and ATG7, whereas levels of SQSTM1/p62 decreased. The lysosomal inhibitor bafilomycin A1 caused a similar response, although there was no additive effect when combined with EtOH. EtOH altered ULK1 S555 and S757 phosphorylation in a time- and AMPK-dependent manner. The activation of AMPK and ULK1 was associated with increased BECN1 (S93, S14) and PIK3C3/VPS34 (S164) phosphorylation as well as increased total ATG14 and PIK3C3. These changes promoted formation of the ATG14-AMBRA1-BECN1-PIK3C3 proautophagy complex that is important in autophagosome formation. EtOH-induced changes were not associated with increased production of PtdIns3P, which may be due to enhanced PIK3C3 complex binding with 14-3-3θ. Reduction of AMPK using siRNA suppressed the stimulatory effect of EtOH on BECN1 S93, BECN1 S14, and PIK3C3 S164 phosphorylation in a time-dependent manner. Likewise, knockdown of AMPK or chemical inhibition of FoxO1 attenuated phosphorylation of ULK1 at both residues. Knockdown of ULK1 or BECN1 antagonized the effect of EtOH on LC3B-II, SQSTM1, and ATG7 protein expression. Conclusions EtOH-induced autophagy is mediated through changes in phosphorylation and interaction of various PIK3C3 complex components. This, in turn, is regulated either directly via FoxO1-AMPK or indirectly via the FoxO1-AMPK-ULK1 signaling cascade in a mTORC1-independent or mTORC1-dependent manner.
- Published
- 2016
26. The Shape of Vesicle-Containing Organelles Is Critical for Their Functions in Vesicle Endocytosis
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Winters, Chelsea M., primary, Hong-Brown, Ly Q., additional, and Chiang, Hui-Ling, additional
- Published
- 2017
- Full Text
- View/download PDF
27. Anti-ageing activity of Fitchia nutans extract, a Polynesian traditional monoï skin care ingredient
- Author
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Ansel, Jean-Luc, Ly, Q., Butaud, J.-F., Nicolas, M., Herbette, G., Peno-Mazzarino, L., Lati, E., Raharivelomanana, Phila, Université de la Polynésie Française (UPF), Géoazur (GEOAZUR 7329), Institut national des sciences de l'Univers (INSU - CNRS)-Observatoire de la Côte d'Azur, Université Côte d'Azur (UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), Spectropôle - Aix Marseille Université (AMU SPEC), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Laboratoire BIO-EC, University of Copenhagen, Université Côte d'Azur (UCA)-Institut national des sciences de l'Univers (INSU - CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud])-Centre National de la Recherche Scientifique (CNRS)-Observatoire de la Côte d'Azur, Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Université Côte d'Azur (UCA)-Centre National de la Recherche Scientifique (CNRS)-Institut de Recherche pour le Développement (IRD [France-Sud]), and Benson-Rumiz, Alicia
- Subjects
[CHIM.THER] Chemical Sciences/Medicinal Chemistry ,Fitchia nutans ,Anti-ageing activity ,Activité anti-âge ,[CHIM.THER]Chemical Sciences/Medicinal Chemistry ,Monoï ,ComputingMilieux_MISCELLANEOUS - Abstract
International audience
- Published
- 2016
28. Rag GTPases and AMPK/TSC2/Rheb mediate the differential regulation of mTORC1 signaling in response to alcohol and leucine
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Ly Q. Hong-Brown, Charles H. Lang, C. Randell Brown, Abid A. Kazi, and Maithili Navaratnarajah
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endocrine system ,medicine.medical_specialty ,Physiology ,P70-S6 Kinase 1 ,mTORC1 ,AMP-Activated Protein Kinases ,Mice ,AMP-activated protein kinase ,Leucine ,Internal medicine ,Tuberous Sclerosis Complex 2 Protein ,mental disorders ,medicine ,Animals ,Cells, Cultured ,reproductive and urinary physiology ,PI3K/AKT/mTOR pathway ,Monomeric GTP-Binding Proteins ,Muscle Cells ,Ethanol ,biology ,Tumor Suppressor Proteins ,Neuropeptides ,AMPK ,Articles ,Cell Biology ,Cell biology ,Endocrinology ,biology.protein ,Phosphorylation ,Ras Homolog Enriched in Brain Protein ,Protein Multimerization ,Signal transduction ,Signal Transduction ,Transcription Factors ,RHEB - Abstract
Leucine (Leu) and insulin both stimulate muscle protein synthesis, albeit at least in part via separate signaling pathways. While alcohol (EtOH) suppresses insulin-stimulated protein synthesis in cultured myocytes, its ability to disrupt Leu signaling and Rag GTPase activity has not been determined. Likewise, little is known regarding the interaction of EtOH and Leu on the AMPK/TSC2/Rheb pathway. Treatment of myocytes with EtOH (100 mM) decreased protein synthesis, whereas Leu (2 mM) increased synthesis. In combination, EtOH suppressed the anabolic effect of Leu. The effects of EtOH and Leu were associated with coordinate changes in the phosphorylation state of mTOR, raptor, and their downstream targets 4EBP1 and S6K1. As such, EtOH suppressed the ability of Leu to activate these signaling components. The Rag signaling pathway was activated by Leu but suppressed by EtOH, as evidenced by changes in the interaction of Rag proteins with mTOR and raptor. Overexpression of constitutively active (ca)RagA and caRagC increased mTORC1 activity, as determined by increased S6K1 phosphorylation. Furthermore, the caRagA-caRagC heterodimer blocked the inhibitory effect of EtOH. EtOH and Leu produced differential effects on AMPK signaling. EtOH enhanced AMPK activity, resulting in increased TSC2 (S1387) and eEF2 phosphorylation, whereas Leu had the opposite effect. EtOH also decreased the interaction of Rheb with mTOR, and this was prevented by Leu. Collectively, our results indicate that EtOH inhibits the anabolic effects that Leu has on protein synthesis and mTORC1 activity by modulating both Rag GTPase function and AMPK/TSC2/Rheb signaling.
- Published
- 2012
29. Acetaminophen (paracetamol) inhibits myeloperoxidase-catalyzed oxidant production and biological damage at therapeutically achievable concentrations
- Author
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Jeremy S Keh, Tracey B Kajer, Roger Mallak, Anthony J. Kettle, Michael J. Davies, Garry G. Graham, David I. Pattison, Dawn W Newsham, Marian K Milligan, Kieran F. Scott, Clare L. Hawkins, Maud Koelsch, Ly Q Nguyen, John B. Ziegler, Shanlin Fu, and Martin D. Rees
- Subjects
Taurine ,Hypochlorous acid ,Neutrophils ,Biochemistry ,Catalysis ,chemistry.chemical_compound ,Superoxides ,Hypobromous acid ,medicine ,Humans ,Antipyretic ,Hydrogen peroxide ,Acetaminophen ,Peroxidase ,Pharmacology ,biology ,Bromates ,Superoxide ,digestive, oral, and skin physiology ,Analgesics, Non-Narcotic ,Oxidants ,Hypochlorous Acid ,chemistry ,Myeloperoxidase ,biology.protein ,medicine.drug - Abstract
The heme peroxidase enzyme myeloperoxidase (MPO) is released by activated neutrophils and monocytes, where it uses hydrogen peroxide (H(2)O(2)) to catalyze the production of the potent oxidants hypochlorous acid (HOCl), hypobromous acid (HOBr) and hypothiocyanous acid (HOSCN) from halide and pseudohalide (SCN(-)) ions. These oxidants have been implicated as key mediators of tissue damage in many human inflammatory diseases including atherosclerosis, asthma, rheumatoid arthritis, cystic fibrosis and some cancers. It is shown here that acetaminophen (paracetamol), a phenol-based drug with analgesic and antipyretic actions, is an efficient inhibitor of HOCl and HOBr generation by isolated MPO-H(2)O(2)-halide systems. With physiological halide concentrations, acetaminophen concentrations required for 50% inhibition of oxidant formation (IC(50)) were 77+/-6microM (100mMCl(-)) and 92+/-2microM (100mMCl(-) plus 100microMBr(-)), as measured by trapping of oxidants with taurine. The IC(50) for inhibition of HOCl generation by human neutrophils was ca. 100microM. These values are lower than the maximal therapeutic plasma concentrations of acetaminophen (< or =150microM) resulting from typical dosing regimes. Acetaminophen did not diminish superoxide generation by neutrophils, as measured by lucigenin-dependent chemiluminescence. Inhibition of HOCl production was associated with the generation of fluorescent acetaminophen oxidation products, consistent with acetaminophen acting as a competitive substrate of MPO. Inhibition by acetaminophen was maintained in the presence of heparan sulfate and extracellular matrix, materials implicated in the sequestration of MPO at sites of inflammation in vivo. Overall, these data indicate that acetaminophen may be an important modulator of MPO activity in vivo.
- Published
- 2010
30. Skeletal and cardiac myopathy in HIV-1 transgenic rats
- Author
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Robert A. Frost, Pengxiang She, Anne M. Pruznak, Rachel Lantry, Ly Q. Hong-Brown, Charles H. Lang, and Thomas C. Vary
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Male ,medicine.medical_specialty ,Physiology ,Endocrinology, Diabetes and Metabolism ,medicine.medical_treatment ,Human Immunodeficiency Virus Proteins ,Nuclease Protection Assays ,Muscle Proteins ,HIV Wasting Syndrome ,Biology ,Protein degradation ,Kidney ,Body Temperature ,Animals, Genetically Modified ,Atrophy ,Insulin resistance ,Muscular Diseases ,Physiology (medical) ,Internal medicine ,medicine ,Animals ,Insulin ,Myocytes, Cardiac ,Amino Acids ,Insulin-Like Growth Factor I ,Muscle, Skeletal ,Myopathy ,Body Weight ,Skeletal muscle ,Calorimetry, Indirect ,Organ Size ,Articles ,Blotting, Northern ,medicine.disease ,Rats, Inbred F344 ,Muscle atrophy ,Rats ,Endocrinology ,medicine.anatomical_structure ,Body Composition ,HIV-1 ,Lean body mass ,Cytokines ,medicine.symptom ,Energy Metabolism - Abstract
The mechanism by which human immunodeficiency virus (HIV)-1 infection in humans leads to the erosion of lean body mass is poorly defined. Therefore, the purpose of the present study was to determine whether transgenic (Tg) rats that constitutively overexpress HIV-1 viral proteins exhibit muscle wasting and to elucidate putative mechanisms. Over 7 mo, Tg rats gained less body weight than pair-fed controls exclusively as a result of a proportional reduction in lean, not fat, mass. Fast- and slow-twitch muscle atrophy in Tg rats did not result from a reduction in the in vivo-determined rate of protein synthesis. In contrast, urinary excretion of 3-methylhistidine, as well as the content of atrogin-1 and the 14-kDa actin fragment, was elevated in gastrocnemius of Tg rats, suggesting increased muscle proteolysis. Similarly, Tg rats had reduced cardiac mass, which was independent of a change in protein synthesis. This decreased cardiac mass was associated with a reduction in stroke volume, but cardiac output was maintained by a compensatory increase in heart rate. The HIV-induced muscle atrophy was associated with increased whole body energy expenditure, which was not due to an elevated body temperature or secondary bacterial infection. Furthermore, the atrophic response could not be attributed to the development of insulin resistance, decreased levels of circulating amino acids, or increased tissue cytokines. However, skeletal muscle and, to a lesser extent, circulating insulin-like growth factor I was reduced in Tg rats. Although hepatic injury was implicated by increased plasma levels of aspartate and alanine aminotransferases, hepatic protein synthesis was not different between control and Tg rats. Hence, HIV-1 Tg rats develop atrophy of cardiac and skeletal muscle, the latter of which results primarily from an increased protein degradation and may be related to the marked reduction in muscle insulin-like growth factor I.
- Published
- 2008
31. FoxO1-AMPK-ULK1 Regulates Ethanol-Induced Autophagy in Muscle by Enhanced ATG14 Association with the BECN1-PIK3C3 Complex
- Author
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Hong-Brown, Ly Q., primary, Brown, C. Randell, additional, Navaratnarajah, Maithili, additional, and Lang, Charles H., additional
- Published
- 2017
- Full Text
- View/download PDF
32. Alcohol Regulates Eukaryotic Elongation Factor 2 Phosphorylation via an AMP-activated Protein Kinase-dependent Mechanism in C2C12 Skeletal Myocytes
- Author
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Charles H. Lang, Ly Q. Hong-Brown, Danuta Huber, and C. Randell Brown
- Subjects
Elongation Factor 2 Kinase ,endocrine system ,p38 mitogen-activated protein kinases ,AMP-Activated Protein Kinases ,Protein Serine-Threonine Kinases ,EEF2 ,Biochemistry ,Cell Line ,Mice ,AMP-activated protein kinase ,Multienzyme Complexes ,mental disorders ,Animals ,Phosphorylation ,Muscle, Skeletal ,Protein kinase A ,Molecular Biology ,Protein Synthesis Inhibitors ,Muscle Cells ,Ethanol ,biology ,Kinase ,MEK inhibitor ,AMPK ,Cell Biology ,Cell biology ,Calcium-Calmodulin-Dependent Protein Kinases ,biology.protein ,Signal Transduction - Abstract
Ethanol decreases protein synthesis in cells, although the underlying regulatory mechanisms of this process are not fully established. In the present study incubation of C2C12 myocytes with 100 mm EtOH decreased protein synthesis while markedly increasing the phosphorylation of eukaryotic elongation factor 2 (eEF2), a key component of the translation machinery. Both mTOR and MEK pathways were found to play a role in regulating the effect of EtOH on eEF2 phosphorylation. Rapamycin, an inhibitor of mammalian target of rapamycin, and the MEK inhibitor PD98059 blocked the EtOH-induced phosphorylation of eEF2, whereas the p38 MAPK inhibitor SB202190 had no effect. Unexpectedly, EtOH decreased the phosphorylation and activity of the eEF2 upstream regulator eEF2 kinase. Likewise, treatment of cells with the inhibitor rottlerin did not block the stimulatory effect of EtOH on eEF2, suggesting that eEF2 kinase (eEF2K) does not play a role in regulating eEF2. In contrast, increased eEF2 phosphorylation was correlated with an increase in AMP-activated protein kinase (AMPK) phosphorylation and activity. Compound C, an inhibitor of AMPK, suppressed the effects of EtOH on eEF2 phosphorylation but had no effect on eEF2K, indicating that AMPK regulates eEF2 independent of eEF2K. Finally, EtOH decreased protein phosphatase 2A activity when either eEF2 or AMPK was used as the substrate. Thus, this later action may partially account for the increased phosphorylation of eEF2 in response to EtOH and the observed sensitivity of AMPK to rapamycin and PD98059 treatments. Collectively, the induction of eEF2 phosphorylation by EtOH is controlled by an increase in AMPK and a decrease in protein phosphatase 2A activity.
- Published
- 2007
33. A functional polymorphism in the NKG2D gene modulates NK-cell cytotoxicity and is associated with susceptibility to Human Papilloma Virus-related cancers
- Author
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Espinoza, J. Luis, primary, Nguyen, Viet H., additional, Ichimura, Hiroshi, additional, Pham, Trang T. T., additional, Nguyen, Cuong H., additional, Pham, Thuc V., additional, Elbadry, Mahmoud I., additional, Yoshioka, Katsuji, additional, Tanaka, Junji, additional, Trung, Ly Q., additional, Takami, Akiyoshi, additional, and Nakao, Shinji, additional
- Published
- 2016
- Full Text
- View/download PDF
34. Anti-ageing activity of Fitchia nutans extract, a Polynesian tradi-tional monoï skin care ingredient
- Author
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Ansel, JL, additional, Ly, Q, additional, Butaud, JF, additional, Nicolas, M, additional, Herbette, G, additional, Peno-Mazzarino, L, additional, Lati, E, additional, and Raharivelomanana, P, additional
- Published
- 2016
- Full Text
- View/download PDF
35. Alcohol and Indinavir Adversely Affect Protein Synthesis and Phosphorylation of MAPK and mTOR Signaling Pathways in C2C12 Myocytes
- Author
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C. Randell Brown, Danuta Huber, Ly Q. Hong-Brown, and Charles H. Lang
- Subjects
p38 mitogen-activated protein kinases ,Medicine (miscellaneous) ,Indinavir ,P70-S6 Kinase 1 ,Biology ,Toxicology ,EEF2 ,Mice ,Eukaryotic translation ,Eukaryotic initiation factor ,Animals ,Phosphorylation ,Cells, Cultured ,History, Ancient ,Muscle Cells ,Ethanol ,TOR Serine-Threonine Kinases ,EIF4E ,Molecular biology ,Cell biology ,Psychiatry and Mental health ,Protein Biosynthesis ,Mitogen-Activated Protein Kinases ,Signal transduction ,Protein Kinases - Abstract
Background: Alcohol and the antiretroviral drug indinavir (Ind) decrease protein synthesis in skeletal muscle under in vivo and in vitro conditions. The goal of the present study was to identify signaling mechanisms responsible for the inhibitory effect of ethanol (EtOH) and Ind on protein synthesis. Methods: C2C12 mouse myocytes were incubated with EtOH, Ind, or a combination of both for 24 hours. The rate of protein synthesis was determined by [35S]methionine/cysteine incorporation into cellular protein. Phosphorylation of eukaryotic initiation and elongation factors were quantitated by Western blot analysis to identify potential mechanisms for regulating translation. Results: Treatment of myocytes with Ind or EtOH for 24 hours decreased protein synthesis by 19 and 22%, respectively, while a 35% decline was observed in cells treated simultaneously with both agents. Mechanistically, treatment with EtOH or Ind decreased the phosphorylation of the S6 ribosomal protein, and this reduction was associated with decreased S6K1 and p90rsk phosphorylation. Ethanol also decreased the phosphorylation of ERK1/2, mTOR, and 4EBP1, while Ind only suppressed ERK1/2 phosphorylation. Both agents inhibited the phosphorylation of Mnk1 and its upstream regulator p38 MAPK, and they decreased the amount of the active eukaryotic initiation factor (eIF) 4G/eIF4E complex. Finally, EtOH and/or Ind increased phosphorylation of the eukaryotic elongation factor (eEF)-2 by 1.6- to 6-fold. The effects of these agents were not additive, although the combination did exert a greater effect on S6K1 and eEF2 phosphorylation. Conclusions: Ethanol and Ind decreased protein synthesis in myocytes and this response was associated with changes in the phosphorylation of proteins that regulate translation initiation and elongation.
- Published
- 2006
36. Indinavir impairs protein synthesis and phosphorylations of MAPKs in mouse C2C12myocytes
- Author
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C. Randell Brown, Ly Q. Hong-Brown, and Charles H. Lang
- Subjects
Physiology ,Ratón ,medicine.medical_treatment ,Indinavir ,Protein Serine-Threonine Kinases ,Biology ,Pharmacology ,Ribosomal Protein S6 Kinases, 90-kDa ,Mice ,Insulin-like growth factor ,medicine ,Animals ,Myocyte ,Phosphorylation ,Cells, Cultured ,Muscle Cells ,Ribosomal Protein S6 ,Dose-Response Relationship, Drug ,Intracellular Signaling Peptides and Proteins ,Ribosomal Protein S6 Kinases, 70-kDa ,HIV Protease Inhibitors ,Cell Biology ,Eukaryotic Initiation Factor-4F ,Ribosomal protein s6 ,Mitogen-activated protein kinase ,Immunology ,biology.protein ,Mitogen-Activated Protein Kinases ,C2C12 ,Cell Division ,medicine.drug - Abstract
Anti-retroviral therapy promotes clinical, immunologic, and virologic improvement in human immunodeficiency virus-infected patients. Whereas this therapy adversely affects carbohydrate and lipid metabolism, the effects of anti-retroviral drugs on muscle protein synthesis and degradation have not been reported. To examine these processes, we treated C2C12myocytes with increasing concentrations of the protease inhibitor indinavir for 1 or 2 days. Treatment of myocytes with a therapeutic concentration of indinavir (20 μM) for 24 h decreased basal protein synthesis by 18%, whereas a 42% decline was observed after 48 h. A similar decrement, albeit quantitatively smaller, was detected with other protease inhibitors. Indinavir did not alter the rate of proteolysis. Likewise, indinavir did not impair the anabolic effect of insulin-like growth factor-I on protein synthesis. Mechanistically, indinavir decreased the phosphorylation of the S6 ribosomal protein (rpS6), and this reduction was associated with a decreased phosphorylation of p70S6 kinase and p90rsk as well as the upstream regulators ERK1/2 and MEK1/2. Indinavir also decreased the phosphorylation of Mnk1 and its upstream effectors, p38 MAPK and ERK1/2. Indinavir did not affect the phosphorylation of mTOR or 4E-BP1, but it did decrease the amount of the active eukaryotic initiation factor eIF4G-eIF4E complex. In conclusion, indinavir decreased protein synthesis in myocytes. This decrease was associated with the disruption of the ERK1/2 and p38 MAPK pathways and a reduction in both the level of functional eIF4F complex and rpS6 phosphorylation.
- Published
- 2004
37. Adamts1 mediates ethanol-induced alterations in collagen and elastin via a FoxO1-sestrin3-AMPK signaling cascade in myocytes
- Author
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Ly Q, Hong-Brown, C Randell, Brown, Maithili, Navaratnarajah, and Charles H, Lang
- Subjects
endocrine system ,Muscle Cells ,Ethanol ,Forkhead Box Protein O1 ,Forkhead Transcription Factors ,AMP-Activated Protein Kinases ,Article ,Cell Line ,Elastin ,ADAM Proteins ,Mice ,ADAMTS1 Protein ,mental disorders ,Animals ,Collagen ,reproductive and urinary physiology ,Heat-Shock Proteins - Abstract
A variety of stressors including alcohol (EtOH) are known to induce collagen production and fibrotic diseases. Matrix metalloproteinases (MMP) play an important role in regulating fibrosis, but little is known regarding the relationship between EtOH and MMPs. In addition, the signaling cascades involved in this process have not been elucidated. We have identified the MMP Adamts1 as a target of EtOH regulation. To characterize the function of Adamts1, we examined EtOH-induced alterations in collagen I and elastin protein levels in C2C12 myocytes. Incubation of myocytes with 100 mM EtOH decreased elastin and increased collagen content, respectively, and these changes were associated with increased O-GLcNAc modification of Adamts1. Conversely, silencing of Adamts1 by siRNA blocked the adverse effects of EtOH on collagen and elastin levels. Similar results were obtained after treatment with a pharmacological inhibitor of MMP. Changes in collagen were due, at least in part, to a decreased interaction of Adamts1 with its endogenous inhibitor TIMP3. The AMPK inhibitor compound C blocked the EtOH-induced stimulation of collagen and O-GLcNAc Adamts1 protein. Changes in AMPK appear linked to FoxO1, since inhibition of FoxO1 blocked the effects of EtOH on AMPK phosphorylation and O-GLcNAc levels. These FoxO-dependent modifications were associated with an upregulation of the FoxO1 transcription target sestrin 3, as well as increased binding of sestrin 3 with AMPK. Collectively, these data indicate that EtOH regulates the collagen I and elastin content in an Adamts1-dependent manner in myocytes. Furthermore, Adamts1 appears to be controlled by the FoxO1-sestrin 3-AMPK signaling cascade.
- Published
- 2014
38. Alcohol Impairs Protein Synthesis and Degradation in Cultured Skeletal Muscle Cells
- Author
-
Ly Q. Hong-Brown, Charles H. Lang, and Robert A. Frost
- Subjects
endocrine system ,medicine.medical_specialty ,Basal rate ,Ethanol ,Growth factor ,medicine.medical_treatment ,Insulin ,Acetaldehyde ,Medicine (miscellaneous) ,Skeletal muscle ,Biology ,Protein degradation ,Toxicology ,Psychiatry and Mental health ,chemistry.chemical_compound ,medicine.anatomical_structure ,Endocrinology ,chemistry ,Internal medicine ,mental disorders ,medicine ,Receptor ,reproductive and urinary physiology - Abstract
Background: Acute and chronic alcohol intoxication decreases skeletal muscle protein synthesis under in vivo conditions. We investigated whether ethanol (EtOH) and its major metabolites, acetaldehyde and acetate, can directly modulate protein balance under in vitro conditions. Methods: Human myocytes were incubated with different doses of EtOH for varying periods of time (i.e., 4–72 hr). Alternatively, cells were incubated with acetaldehyde, acetate, insulin, insulin-like growth factor-I (IGF-I), or with a combination of EtOH plus insulin or IGF-I. Rates of protein synthesis or degradation were determined by 35S-methionine/cysteine incorporation into or release from cellular protein. Results: A significant, 15% to 20%, decrease in basal protein synthesis was observed after 24 hr, but not at earlier time points, in response to 80 mM EtOH. Incubation of myocytes for 72 hr decreased synthesis in cells incubated with EtOH ranging between 60 and 120 mM. The ability of IGF-I or insulin to stimulate protein synthesis was impaired by 30% and 60%, respectively, in cells incubated with 80 mM EtOH for 72 hr. Exposure of cells to 200 μM acetaldehyde or 5 mM Na-acetate also decreased basal protein synthesis. In contrast, neither EtOH, acetaldehyde, nor acetate altered the basal rate of protein degradation. However, EtOH completely impaired the ability of insulin and IGF-I to inhibit proteolysis. Finally, EtOH did not impair IGF-I receptor autophosphorylation, but inhibited the ability of insulin to phosphorylate its own receptor. EtOH also did not alter the number of insulin or IGF-I receptors or the formation of insulin/IGF-I hybrid receptors. Conclusions: We have demonstrated that EtOH can directly inhibit muscle protein synthesis under in vitro conditions. Neither EtOH nor its metabolites altered basal protein degradation, although EtOH did compromise the ability of both insulin and IGF-I to slow proteolysis. This impairment seems to be mediated by different defects in signal transduction.
- Published
- 2001
39. [Untitled]
- Author
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William J. Welch, Ly Q. Hong-Brown, and C. Randell Brown
- Subjects
Mutation ,Physiology ,Endoplasmic reticulum ,Mutant ,Cell Biology ,Biology ,medicine.disease_cause ,Biochemistry ,Mutant protein ,Chloride channel ,medicine ,Protein folding ,Chemical chaperone ,Gene - Abstract
Many human diseases arise as a result of mutations within genes encoding essential proteins. In many cases, the mutations are not so severe as to render the protein biologically inactive. Rather, the mutations oftentimes result in only subtle protein-folding abnormalities. In the case of the CFTR protein, a mutation leading to the loss of a single amino acid is responsible for the diseased state in the majority of individuals with cystic fibrosis. Here the newly synthesized mutant CFTR protein, missing a phenylalanine residue at position 508 (ΔF508 CFTR), is unable to transit from the endoplasmic reticulum to the plasma membrane, where it functions as a regulator of chloride transport. All of the available evidence indicate that the newly synthesized ΔF508 CFTR protein adopts a slightly altered conformation and therefore is retained at the level of the endoplasmic reticulum, ostensibly by the actions of the cellular quality control system. Because the mutant protein is capable of functioning as a chloride channel, developing ways to elicit its release out of the ER and to the plasma membrane has important clinical implications. Herein, we discuss our recent studies showing that the protein folding defect associated with the ΔF508 CFTR mutation, as well as a number of other temperature-sensitive mutations, can be overcome by strategies designed to influence protein folding inside the cell. Specifically we show that a number of low-molecular-weight compounds, all of which are known to stabilize proteins in their native conformation, are effective in rescuing the folding and/or processing defects associated with different mutations that oftentimes lead to human disease.
- Published
- 1997
40. Activation of AMPK/TSC2/PLD by Alcohol Regulates mTORC1 and mTORC2 Assembly in C2C12 Myocytes
- Author
-
Maithili Navaratnarajah, C. Randell Brown, Ly Q. Hong-Brown, and Charles H. Lang
- Subjects
Metabolite ,Medicine (miscellaneous) ,Phosphatidic Acids ,mTORC1 ,Mechanistic Target of Rapamycin Complex 2 ,Biology ,AMP-Activated Protein Kinases ,Mechanistic Target of Rapamycin Complex 1 ,Toxicology ,mTORC2 ,Article ,Cell Line ,chemistry.chemical_compound ,Mice ,AMP-activated protein kinase ,mental disorders ,Tuberous Sclerosis Complex 2 Protein ,Phospholipase D ,Myocyte ,Animals ,Muscle Cells ,Ethanol ,TOR Serine-Threonine Kinases ,Tumor Suppressor Proteins ,AMPK ,Central Nervous System Depressants ,Phosphatidic acid ,Cell biology ,Psychiatry and Mental health ,chemistry ,Multiprotein Complexes ,biology.protein ,lipids (amino acids, peptides, and proteins) ,biological phenomena, cell phenomena, and immunity - Abstract
Ethanol (EtOH) decreases muscle protein synthesis, and this is associated with reduced mammalian target of rapamycin complex (mTORC)1 and increased mTORC2 activities. In contrast, phospholipase D (PLD) and its metabolite phosphatidic acid (PA) positively regulate mTORC1 signaling, whereas their role in mTORC2 function is less well defined. Herein, we examine the role that PLD and PA play in EtOH-mediated mTOR signaling.C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. For PA experiments, cells were pretreated with the drug for 25 minutes followed by 50-minute incubation with PA in the presence or absence of EtOH. The phosphorylation state of various proteins was assessed by immunoblotting. Protein-protein interactions were determined by immunoprecipitation and immunoblotting. PLD activity was measured using the Amplex Red PLD assay kit. PA concentrations were determined with a total PA assay kit.PA levels and PLD activity increased in C2C12 myocytes exposed to EtOH (100 mM). Increased PLD activity was blocked by inhibitors of AMP-activated protein kinase (AMPK) (compound C) and phosphoinositide 3-kinase (PI3K) (wortmannin). Likewise, suppression of PLD activity with CAY10594 prevented EtOH-induced Akt (S473) phosphorylation. PLD inhibition also enhanced the binding of Rictor to mSin1 and the negative regulatory proteins Deptor and 14-3-3. Addition of PA to myocytes decreased Akt phosphorylation, but changes in mTORC2 activity were not associated with altered binding of complex members and 14-3-3. PA increased S6K1 phosphorylation, with the associated increase in mTORC1 activity being regulated by reduced phosphorylation of AMPKα (T172) and its target tuberous sclerosis protein complex (TSC)2 (S1387). This resulted in increased Rheb and RagA/RagC GTPase interactions with mTOR, as well as suppression of mTORC2.EtOH-induced increases in PLD activity and PA may partially counterbalance the adverse effects of this agent. EtOH and PA regulate mTORC1 via a PI3K/AMPK/TSC2/PLD signaling cascade. PA stimulates mTORC1 function and suppresses activation of mTORC2 as part of an mTORC1/2 feedback loop.
- Published
- 2013
41. Role of PLD and PA in alcohol (EtOH)‐mediated mTOR signaling in C2C12 myocytes
- Author
-
C. Randell Brown, Charles H. Lang, Maithili Navaratnarajah, and Ly Q. Hong-Brown
- Subjects
chemistry.chemical_compound ,Mtor signaling ,chemistry ,Genetics ,Myocyte ,Alcohol ,Molecular Biology ,Biochemistry ,C2C12 ,Biotechnology ,Cell biology - Published
- 2013
42. Molecular Chaperones and the Centrosome
- Author
-
C. Randell Brown, Stephen J. Doxsey, Ly Q. Hong-Brown, and William J. Welch
- Subjects
Cell Biology ,Biology ,Biochemistry ,Cell biology ,Antigen ,Microtubule ,Centrosome ,Shock (circulatory) ,Heat shock protein ,Organelle ,medicine ,medicine.symptom ,Molecular Biology ,Microinjection ,Function (biology) - Abstract
In the accompanying paper (Brown, C. R., Doxsey, S. J., Hong-Brown, L. W., Martin, R. L., and Welch, W. J. (1996) J. Biol. Chem. 271, 824-832) two molecular chaperones, hsp 73 and TCP-1, were shown to be integral components of the centrosome. Here we show that heat shock treatment adversely affects both the structure and function of the centrosome, and that hsp 73 plays a role in the repair of the organelle. After heat shock treatment, the centrosome could not be identified via indirect immunofluorescence and cells were unable to support microtubule regrowth. During recovery from heat shock, a strong correlation between the return of staining of three centrosomal antigens (hsp 73, TCP-1, and pericentrin) and the recovery of microtubule regrowth properties was found. Incubation of cells with glycerol, a protein protective agent, prevented the heat induced alterations in the structure/function of the centrosome. Likewise, the recovery of the structure and function of the centrosome after heat shock treatment was significantly accelerated in cells first made thermotolerant. We provide evidence that this process is related to the levels of hsp 73 since: 1) microinjection of hsp 73 antibody blocked centrosomal reassembly and microtubule regrowth abilities following heat shock; and 2) microinjection of purified hsp 73 protein prior to heat shock treatment accelerated both the repair and function of the organelle, similar to that observed for thermotolerant cells.
- Published
- 1996
43. Molecular Chaperones and the Centrosome
- Author
-
Stephen J. Doxsey, Ly Q. Hong-Brown, Robert L. Martin, William J. Welch, and C. Randell Brown
- Subjects
Centrosome cycle ,Microtubule organizing center ,Cell Biology ,Biology ,Biochemistry ,Spindle pole body ,Cell biology ,Nocodazole ,chemistry.chemical_compound ,Tubulin ,chemistry ,Microtubule ,Centrosome ,biology.protein ,Molecular Biology ,Microtubule nucleation - Abstract
Molecular chaperones play an important role in facilitating the proper maturation of many newly synthesized proteins. Here we provide evidence that molecular chaperones also participate in regulating the assembly of the microtubule cytoskeleton. Via indirect immunofluorescence analysis, both hsp 73 and TCP-1 localized within the centrosome in interphase and mitotic cells. These proteins, along with the centrosome-specific protein, pericentrin, were also present within an enriched preparation of centrosomes. Because the centrosome serves as an initiation site for microtubule growth, we examined the ability of cells to regrow their microtubule network in the presence of hsp 73 or TCP-1 specific antibodies. Purified tubulin and GTP were added to cells following the depolymerization and extraction of cellular microtubules. Microtubules were observed to nucleate off the centrosome using this system, even in the presence of anti-hsp 73 antibodies. Incubation with anti-TCP-1 antibodies, however, blocked microtubule regrowth off the centrosome. Similarly, anti-TCP-1 antibodies microinjected into living cells first treated with nocodazole also inhibited the regrowth of the microtubule network following removal of the microtubule poison. Our results complement earlier genetic studies in yeast implicating a role for TCP-1 in microtubule mediated processes, and may help to explain the previously reported mitotic and meiotic abnormalities associated with TCP-1 mutations.
- Published
- 1996
44. Mechanisms mediating the effects of alcohol and HIV anti-retroviral agents on mTORC1, mTORC2 and protein synthesis in myocytes
- Author
-
Ly Q. Hong-Brown, Abid A. Kazi, and Charles H. Lang
- Subjects
biology ,Mitogen-activated protein kinase ,Immunology ,biology.protein ,Protein biosynthesis ,Phosphorylation ,Translation (biology) ,mTORC1 ,Topic Highlight ,Protein degradation ,mTORC2 ,PI3K/AKT/mTOR pathway ,Cell biology - Abstract
Alcoholism and acquired immune deficiency syndrome are associated with severe muscle wasting. This impairment in nitrogen balance arises from increased protein degradation and a decreased rate of protein synthesis. The regulation of protein synthesis is a complex process involving alterations in the phosphorylation state and protein-protein interaction of various components of the translation machinery and mammalian target of rapamycin (mTOR) complexes. This review describes mechanisms that regulate protein synthesis in cultured C2C12 myocytes following exposure to either alcohol or human immunodeficiency virus antiretroviral drugs. Particular attention is given to the upstream regulators of mTOR complexes and the downstream targets which play an important role in translation. Gaining a better understanding of these molecular mechanisms could have important implications for preventing changes in lean body mass in patients with catabolic conditions or illnesses.
- Published
- 2012
45. Knockdown of Deptor, an mTOR binding protein, increases muscle protein synthesis
- Author
-
Abid A. Kazi, Ly Q. Hong-Brown, and Charles H. Lang
- Subjects
Muscle protein ,Gene knockdown ,Chemistry ,Binding protein ,Genetics ,DEPTOR ,Molecular Biology ,Biochemistry ,PI3K/AKT/mTOR pathway ,Biotechnology ,Cell biology - Published
- 2011
46. Alcohol-induced modulation of rictor and mTORC2 activity in C2C12 myoblasts
- Author
-
Ly Q, Hong-Brown, C Randell, Brown, Maithili, Navaratnarajah, Danuta S, Huber, and Charles H, Lang
- Subjects
Myoblasts ,Mice ,Rapamycin-Insensitive Companion of mTOR Protein ,Ethanol ,Trans-Activators ,Animals ,Central Nervous System Depressants ,Immunoprecipitation ,Carrier Proteins ,Transfection ,Proto-Oncogene Proteins c-akt ,Article ,Transcription Factors - Abstract
The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1.C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein-protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by (35) S-methionine/cysteine incorporation into cellular protein.EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts. EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis.
- Published
- 2011
47. Alcohol-Induced Modulation of Rictor and mTORC2 Activity in C2C12 Myoblasts
- Author
-
C. Randell Brown, Danuta Huber, Charles H. Lang, Ly Q. Hong-Brown, and Maithili Navaratnarajah
- Subjects
Psychiatry and Mental health ,Medicine (miscellaneous) ,Phosphorylation ,P70-S6 Kinase 1 ,mTORC1 ,Kinase activity ,Biology ,Toxicology ,DEPTOR ,Molecular biology ,mTORC2 ,Protein kinase B ,PI3K/AKT/mTOR pathway - Abstract
Background: The mammalian target of rapamycin (mTOR) kinase controls cell growth, proliferation, and metabolism through 2 distinct multiprotein complexes, mTORC1 and mTORC2. We reported that alcohol (EtOH) inhibits mTORC1 activity and protein synthesis in C2C12 myoblasts. However, the role that mTORC2 plays in this process has not been elucidated. In this study, we investigated whether mTORC2 functions as part of a feedback regulator in response to EtOH, acting to maintain the balance between the functions of Akt, mTORC2, and mTORC1. Methods: C2C12 myoblasts were incubated with EtOH for 18 to 24 hours. Levels of various mTORC2 proteins and mRNA were assessed by immunoblotting and real-time PCR, respectively, while protein–protein interactions were determined by immunoprecipitation and immunoblotting. An in vitro mTORC2 kinase activity assay was performed using Akt as a substrate. The rate of protein synthesis was determined by 35S-methionine/cysteine incorporation into cellular protein. Results: EtOH (100 mM) increased the protein and mRNA levels of the mTORC2 components rictor, mSin1, proline-rich repeat protein 5, and Deptor. There was also an increased association of these proteins with mTOR. EtOH increased the in vitro kinase activity of mTORC2, and this was correlated with decreased binding of rictor with 14-3-3 and Deptor. Reduced rictor phosphorylation at T1135 by EtOH was most likely due to decreased S6K1 activity. Knockdown of rictor elevated mTORC1 activity, as indicated by increased S6K1 phosphorylation and protein synthesis. Likewise, there were decreased amounts and/or phosphorylation levels of various mTORC1 and mTORC2 components including raptor, proline-rich Akt substrate 40 kDa, mSin1, Deptor, and GβL. Activated PP2A was associated with decreased Akt and eukaryotic elongation factor 2 phosphorylation. Collectively, our results provide evidence of a homeostatic balance between the 2 mTOR complexes following EtOH treatments in myoblasts. Conclusions: EtOH increased the activity of mTORC2 by elevating levels of various components and their interaction with mTOR. Decreased rictor phosphorylation at T1135 acts as mTORC1-dependent feedback mechanisms, functioning in addition to the insulin receptor substrate-I/PI3K signaling pathway to regulate protein synthesis.
- Published
- 2011
48. Deptor knockdown enhances mTOR Activity and protein synthesis in myocytes and ameliorates disuse muscle atrophy
- Author
-
Ly Q. Hong-Brown, Abid A. Kazi, Susan M. Lang, and Charles H. Lang
- Subjects
Male ,Cell Cycle Proteins ,Retinoblastoma Protein ,S Phase ,Myoblasts ,Mice ,Eukaryotic Initiation Factors ,Phosphorylation ,Genetics (clinical) ,Gene knockdown ,biology ,Chemistry ,TOR Serine-Threonine Kinases ,Retinoblastoma protein ,Intracellular Signaling Peptides and Proteins ,Organ Size ,Articles ,Cell biology ,Hindlimb Suspension ,Molecular Medicine ,Female ,RNA Interference ,medicine.medical_specialty ,Immunoblotting ,P70-S6 Kinase 1 ,DEPTOR ,Ribosomal Protein S6 Kinases, 90-kDa ,Cell Line ,Internal medicine ,Genetics ,medicine ,Animals ,Humans ,Muscle, Skeletal ,Molecular Biology ,PI3K/AKT/mTOR pathway ,Adaptor Proteins, Signal Transducing ,Cell Proliferation ,Cell Size ,Muscle Cells ,Cell growth ,Binding protein ,Muscular Dystrophy, Animal ,Phosphoproteins ,Mice, Inbred C57BL ,Endocrinology ,HEK293 Cells ,Protein Biosynthesis ,biology.protein ,Carrier Proteins - Abstract
Deptor is an mTOR binding protein that affects cell metabolism. We hypothesized that knockdown (KD) of Deptor in C2C12 myocytes will increase protein synthesis via stimulating mTOR-S6K1 signaling. Deptor KD was achieved using lentiviral particles containing short hairpin (sh)RNA targeting the mouse Deptor mRNA sequence, and control cells were transfected with a scrambled control shRNA. KD reduced Deptor mRNA and protein content by 90%, which increased phosphorylation of mTOR kinase substrates, 4E-BP1 and S6K1, and concomitantly increased protein synthesis. Deptor KD myoblasts were both larger in diameter and exhibited an increased mean cell volume. Deptor KD increased the percentage of cells in the S phase, coincident with an increased phosphorylation (S807/S811) of retinoblastoma protein (pRb) that is critical for the G(1) to S phase transition. Deptor KD did not appear to alter basal apoptosis or autophagy, as evidenced by the lack of change for cleaved caspase-3 and light chain (LC)3B, respectively. Deptor KD increased proliferation rate and enhanced myotube formation. Finally, in vivo Deptor KD (~50% reduction) by electroporation into gastrocnemius of C57/BL6 mice did not alter weight or protein synthesis in control muscle. However, Deptor KD prevented atrophy produced by 3 d of hindlimb immobilization, at least in part by increasing protein synthesis. Thus, our data support the hypothesis that Deptor is an important regulator of protein metabolism in myocytes and demonstrate that decreasing Deptor expression in vivo is sufficient to ameliorate muscle atrophy.
- Published
- 2011
49. PRAS40 regulates protein synthesis, cell cycle, proliferation and myogenic differentiation in C2C12 myocytes
- Author
-
Abid A. Kazi, Charles H. Lang, and Ly Q. Hong-Brown
- Subjects
Myogenic differentiation ,Chemistry ,Genetics ,Protein biosynthesis ,Myocyte ,Cell cycle ,Molecular Biology ,Biochemistry ,C2C12 ,Biotechnology ,Cell biology - Published
- 2010
50. Alcohol and PRAS40 knockdown decrease mTOR activity and protein synthesis via AMPK signaling and changes in mTORC1 interaction
- Author
-
Ly Q. Hong-Brown, Anne M. Pruznak, Charles H. Lang, C. Randell Brown, Danuta Huber, and Abid A. Kazi
- Subjects
Protein kinase complex ,endocrine system ,Immunoblotting ,mTORC1 ,AMP-Activated Protein Kinases ,Mechanistic Target of Rapamycin Complex 1 ,Protein Serine-Threonine Kinases ,Biochemistry ,Article ,Cell Line ,Mice ,AMP-activated protein kinase ,mental disorders ,Animals ,Immunoprecipitation ,Phosphorylation ,Kinase activity ,Molecular Biology ,Protein kinase B ,reproductive and urinary physiology ,PI3K/AKT/mTOR pathway ,Adaptor Proteins, Signal Transducing ,Muscle Cells ,Ethanol ,biology ,TOR Serine-Threonine Kinases ,Intracellular Signaling Peptides and Proteins ,Proteins ,AMPK ,Regulatory-Associated Protein of mTOR ,Cell Biology ,Phosphoproteins ,Cell biology ,Gene Expression Regulation ,Multiprotein Complexes ,biology.protein ,RNA Interference ,Carrier Proteins ,Protein Binding ,Signal Transduction ,Transcription Factors - Abstract
The mTORC1 protein kinase complex consists of mTOR, raptor, mLST8/GbetaL and PRAS40. Previously, we reported that mTOR plays an important role in regulating protein synthesis in response to alcohol (EtOH). However, the mechanisms by which EtOH regulates mTORC1 activity have not been established. Here, we investigated the effect of EtOH on the phosphorylation and interaction of components of mTORC1 in C2C12 myocytes. We also examined the specific role that PRAS40 plays in this process. Incubation of myocytes with EtOH (100 mM, 24 h) increased raptor and PRAS40 phosphorylation. Likewise, there were increased levels of the PRAS40 upstream regulators Akt and IRS-1. EtOH also caused changes in mTORC1 protein-protein interactions. EtOH enhanced the binding of raptor and PRAS40 with mTOR. These alterations occurred in concert with increased binding of 14-3-3 to raptor, while the PRAS40 and 14-3-3 interaction was not affected. The shRNA knockdown (KD) of PRAS40 decreased protein synthesis similarly to EtOH. PRAS40 KD increased raptor phosphorylation and its association with 14-3-3, whereas decreased GbetaL-mTOR binding. The effects of EtOH and PRAS40 KD were mediated by AMPK. Both factors increased in vitro AMPK activity towards the substrate raptor. In addition, KD enhanced the activity of AMPK towards TSC2. Collectively, our results indicate that EtOH stabilizes the association of raptor, PRAS40, and GbetaL with mTOR, while likewise increasing the interaction of raptor with 14-3-3. These data suggest a possible mechanism for the inhibitory effects of EtOH on mTOR kinase activity and protein synthesis in myocytes.
- Published
- 2010
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