Adaptation to stress in yeast: to translate or not?

For most eukaryotic organisms, including Saccharomyces cerevisiae, the rapid inhibition of protein synthesis forms part of a response to stress. In order to balance the changing conditions, precise stress-specific alterations to the cell's proteome are required. Therefore, in the background of a global down-regulation in protein synthesis, specific proteins are induced. Given the level of plasticity required to enable stress-specific alterations of this kind, it is surprising that the mechanisms of translational regulation are not more diverse. In the present review, we summarize the impact of stress on translation initiation, highlighting both the similarities and distinctions between various stress responses. Finally, we speculate as to how yeast cells generate stress-responsive programmes of protein production when regulation is focused on the same steps in the translation pathway.     

OPP Labeling Enables Total Protein Synthesis Quantification in CHO Production Cell Lines at the Single‐Cell Level

Accurate measurement of global and specific protein synthesis rates is becoming increasingly important, especially in the context of biotechnological applications such as process modeling or selection of production cell clones. While quantification of total protein translation across whole cell populations is easily achieved, methods that are capable of tracking population dynamics at the single‐cell level are still lacking. To address this need, we apply O‐propargyl‐puromycin (OPP) labeling to assess total protein synthesis in single recombinant Chinese hamster ovary (CHO) cells by flow cytometry. Thereby we demonstrate that global protein translation rates slightly increase with progression through the cell cycle during exponential growth. Stable CHO cell lines producing recombinant protein display similar levels of total protein synthesis as their parental CHO host cell line. Global protein translation does not correlate with intracellular product content of three model proteins, but the host cell line with high transient productivity has a higher OPP signal. This indicates that production cell lines with increased overall protein synthesis capacity can be identified by our method at the single‐cell level. In conclusion, OPP‐labeling allows rapid and reproducible assessment of global protein synthesis in single CHO cells, and can be multiplexed with DNA staining or any type of immunolabeling of specific proteins or markers for organelles.     

Effects of siRNA Targeting c‐Myc and VEGF on Human Colorectal Cancer Volo Cells

c‐Myc and vascular endothelial growth factor (VEGF) genes are frequently deregulated and overexpressed in this malignancy, and strategies designed to inhibit c‐Myc and VEGF expression in cancer cells may have considerable therapeutic value. In the present study, we design and use short interfering RNA (siRNA) to inhibit c‐Myc and VEGF expression in colorectal cancer Volo cells and validate their effects on cell proliferation, cell cycle, apoptosis, and cell metastasis. Upon transient transfection with plasmid‐encoding siRNA, it was found that expression of c‐Myc and VEGF was significantly downregulated in siRNA‐transfected cells and the downregulation of c‐Myc and VEGF inhibited cell growth and induced apoptosis and metastasis of Volo cells. c‐Myc and VEGF downregulation also increased cell population in the G0–G1 phase. In conclusion, the specific siRNA efficiently silenced the expression of c‐Myc and VEGF, further suppressed the cell proliferation, triggered cell apoptosis, and inhibited cell invasiveness of colorectal cancer Volo cells. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:499‐505, 2012;Viewthis article online at wileyonlinelibrary.com . DOI 10.1002/jbt.21455     

LncRNA‐MIF,a c‐Myc‐activated long non‐coding RNA,suppresses glycolysis by promoting Fbxw7‐mediated c‐Myc degradation

The c‐Myc proto‐oncogene is activated in more than half of all human cancers. However, the precise regulation of c‐Myc protein stability is unknown. Here, we show that the lncRNA‐MIF (c‐Myc inhibitory factor), a c‐Myc‐induced long non‐coding RNA, is a competing endogenous RNA for miR‐586 and attenuates the inhibitory effect of miR‐586 on Fbxw7, an E3 ligase for c‐Myc, leading to increased Fbxw7 expression and subsequent c‐Myc degradation. Our data reveal the existence of a feedback loop between c‐Myc and lncRNA‐MIF, through which c‐Myc protein stability is finely controlled. Additionally, we show that the lncRNA‐MIF inhibits aerobic glycolysis and tumorigenesis by suppressing c‐Myc and miR‐586.