Evaluation of mTOR-regulated mRNA translation
mTOR, the mammalian target of rapamycin, regulates protein synthesis (mRNA translation) by affecting the phosphorylation or activity of several translation factors. Here, we describe methods for studying the impact of mTOR signalling on protein synthesis, using inhibitors of mTOR such as rapamycin (which impairs some of its functions) or mTOR kinase inhibitors (which probably block all functions).To assess effects of mTOR inhibition on general protein synthesis in cells, the incorporation of radiolabelled amino acids into protein is measured. This does not yield information on the effects of mTOR on the synthesis of specific proteins. To do this, two methods are described. In one, stable-isotope labelled amino acids are used, and their incorporation into new proteins is determined using mass spectrometric methods. The proportions of labelled vs. unlabeled versions of each peptide from a given protein provide quantitative information about the rate of that protein's synthesis under different conditions. Actively translated mRNAs are associated with ribosomes in polyribosomes (polysomes); thus, examining which mRNAs are found in polysomes under different conditions provides information on the translation of specific mRNAs under different conditions. A method for the separation of polysomes from non-polysomal mRNAs is described.
Available from: Barak Rotblat
- "Under such conditions, determining real-time rates of protein synthesis is expected to be a better predictor of protein abundance than measurements of respective mRNA levels. Stable isotope labelling by amino acids in cell culture (SILAC) is a highly robust tool for quantitatively comparing different proteomes, and pulsed SILAC approaches have been developed to directly quantify protein synthesis on a proteome-wide scale  . However, such methods are not optimal for measuring changes in protein synthesis over very short timescales due to technical and biological variability , or because the signal from newly synthesized proteins are generally too low to be within the quantitative dynamic range of most mass analysers. "
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ABSTRACT: Messenger RNA-binding translational regulatory proteins determine in large part the spectrum of transcripts that are translated under specific cellular contexts. Y-box binding protein-1 (YB-1) is a conserved eukaryotic translational regulator that is implicated in cancer progression. To identify specific proteins that are translationally regulated by YB-1, we established a pulse-labelling approach combining Click chemistry and stable isotope labelling by amino acids in cell culture (SILAC). The proteome of TC32 human Ewing sarcoma cells, which robustly express YB-1, was compared with or without YB-1 siRNA knockdown. Cells labelled with light or heavy isotopologs of Arg and Lys were then cotranslationally pulsed with the methionine derivative, azidohomoalanine (AHA). Cells were lysed and newly synthesized proteins were selectively derivatized via a Click (3+2 cycloaddition) reaction to add an alkyne biotin tag. They were then affinity purified and subjected to liquid chromatography-tandem mass spectrometry. This combined Click-SILAC approach enabled us to catalog and quantify newly synthesized proteins regulated by YB-1 after only 45min of labelling. Bioinformatic analysis revealed that YB-1 regulated proteins are involved in diverse biological pathways. We anticipate that this Click-SILAC strategy will be useful for studying short-term protein synthesis in different cell culture systems and under diverse biological contexts.
Journal of proteomics 09/2012; 77. DOI:10.1016/j.jprot.2012.08.019 · 3.89 Impact Factor
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ABSTRACT: Few targeted therapies have been developed for ovarian granulosa cell tumor (GCT), even though it represents 5% of all malignant ovarian tumors in women. As misregulation of PI3K/AKT signaling has been implicated in GCT development, we hypothesized that the AKT signaling effector mammalian target of rapamycin (mTOR) may play a role in the pathogenesis of GCT and could represent a therapeutic target. Analyses of human GCT samples showed an increase in protein levels of mTOR and its downstream effectors RPS6KB1, RPS6, eIF4B and PPARG relative to normal granulosa cells, suggestive of an increase in mTOR pathway activity and increased translational activity and/or protein stability. We next sought to evaluate mTOR as a GCT therapeutic target using the Pten (tm1Hwu/tmiHwu);Ctnnb1 (tm1Mmt/+);Amhr2 (tm3(cre)Bhr/+) (PCA) mouse model, in which mTOR, RPS6KB1, eIF4B and PPARG are upregulated in tumor cells in a manner similar to human GCT. Treatment of PCA mice with the mTOR-specific inhibitor everolimus reduced tumor growth rate (1.5-fold; P < 0.05) and also reduced total tumor burden (4.7-fold; P < 0.05) and increased survival rate (78 versus 44% in the vehicle group) in a PCA surgical model of GCT peritoneal carcinomatosis. Everolimus decreased tumor cell proliferation and tumor cell volume relative to controls (P < 0.05), whereas apoptosis was unaffected. Phosphorylation of RPS6KB1 and RPS6 were decreased (P < 0.05) by everolimus, but RPS6KB1, RPS6, eIF4B and PPARG expressions were not affected. These results suggest that mTOR is a valid and clinically useful pharmacological target for the treatment of GCT, although its inhibition does not reverse all consequences of aberrant PI3K/AKT signaling in the PCA model.
Carcinogenesis 08/2012; 33(11). DOI:10.1093/carcin/bgs263 · 5.33 Impact Factor
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ABSTRACT: Cachexia is a multifactorial syndrome of atrophy of skeletal muscle and adipose tissue, resulting in progressive loss of body weight associated with low quality of life and poor prognosis in cancer. Studies on experimental animal models and observations on patients have shown that the soluble factors secreted by tumor cells and tissues of the patient can participate in regulation of the wasting process. Cachexia is often accompanied by anorexia, which is caused by predominance of signals inhibiting appetite in the hypothalamus, such as release of proopiomelanocortin and anorexigenic action of proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α). Cachexia is also accompanied by extensive metabolic changes consisting of increase of resting energy expenditure and disturbance of carbohydrate, protein and lipid metabolism. Increased expression of protein uncoupling phosphorylation leads to increased thermogenesis in skeletal muscle. Tumor tissue hypoxia caused by its growth beyond blood vessels activates the transcription factor HIF-1, which results in increase in glycolysis, and leads to lactic acid accumulation and activation of the energy inefficient Cori cycle. Loss of fat tissue is caused by increase of lipolysis induced by lipid-mobilizing factor (LMF) and proinflammatory cytokines. Skeletal muscle wasting in cachexia is caused by a reduction of protein synthesis at the stage of initiation and elongation of translation and the simultaneous increase of protein degradation via ubiquitin-dependent and lysosomal pathways. The main mediators of skeletal muscle wasting in cancer are proteolysis-inducing factor (PIF), proinflammatory cytokines, and angiotensin II acting through increased levels of reactive oxygen species (ROS) and nuclear factor NF-κB activation, as well as glucocorticoid activated FOXO transcription factors and myostatin. Understanding of the complexity of the interaction of factors produced by the tumor and the patient›s body may form the basis for the development of effective treatments for cachexia in cancer and other pathological conditions.
Postępy Higieny i Medycyny Doświadczalnej (Advances in Hygiene and Experimental Medicine) 01/2013; 67:1397-409. DOI:10.5604/17322693.1085135 · 0.57 Impact Factor
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