PGF

Overall, these data strongly suggest that eEF1A is involved in triggering aggresome formation as a sensor of the cellular levels of DRiPs

January 6, 2023

Overall, these data strongly suggest that eEF1A is involved in triggering aggresome formation as a sensor of the cellular levels of DRiPs. Discussion In our previous work, we found that the formation of aggresomes by SynCGFP in response to proteasome inhibition does not depend on the cellular levels of this protein. a marginal effect on aggresome formation. Furthermore, SidI reduced the threshold of the aberrant ribosomal products for triggering aggresome formation. Therefore, eEF1A binds defective polypeptides released from ribosomes, which generates a signal that triggers aggresome formation. toxin SidI, which was recently described to specifically bind to eEF1A. The unique feature of SidI is that while inhibiting the eEF1A function in translation, it does not prevent eEF1A-mediated signaling to Hsf1 (Shen et al., 2009). Considering the apparent similarity in activation of Hsf1 and induction of aggresome formation, we tested whether effects of SidI on eEF1A-mediated translation and on putative eEF1A-mediated triggering of the aggresome formation could also be differentiated. In this series of experiments, we first compared the effects of SidI and emetine on translation. Because the efficiency of HeLa transfection was less than 100%, we could not use radioactive labeling to assess the extent of inhibition of translation, and had to monitor expression of a co-transfected polypeptide. Accordingly, we transfected HeLa cells with a plasmid encoding EGFP, and co-transfected with a plasmid encoding either SidI or the Metiamide vector. Various concentrations of emetine were added to the cells co-transfected with the empty vector before they accumulated any detectable amounts of EGFP. The levels of EGFP were assessed 20 hours after the end of the transfection. As seen in Fig. 4A, 2 M emetine almost completely inhibited translation of EGFP, whereas 100 nM emetine caused about a 40% inhibition. Co-expression of SidI had very strong inhibitory effect, reducing the yield of translation by 97% (Fig. 4A). Of note, SidI inhibited its own translation, and thus was expressed at almost undetectable levels, whereas a SidI mutant, which cannot interact with eEF1A (Shen et al., 2009), was expressed at high levels (not shown). Open in a separate window Fig. 4. SidI reduces the threshold of DRiPs necessary to trigger aggresome formation. (A) Effects of various emetine concentrations and SidI on protein synthesis. HeLa cells were transfected for 3 hours with a plasmid encoding EGFP and co-transfected with either a plasmid encoding SidI or an empty vector. 1.5 hours after the end of the transfection indicated amounts of emetine were added to some samples. The levels of synthesized EGFP were assessed by immunoblotting 20 hours after the end of the transfection. (B) Divergent effects of SidI and emetine on induction of Hsp70. HeLa cells were transfected with a plasmid encoding SidI or an empty vector and on the next day incubated with or without 10 M MG132 and the indicated concentrations of emetine for 7 hours. RNA was isolated from the cells and the levels of mRNA were assessed Metiamide with RT-PCR. The effect of SidI was adjusted for the efficiency of transfection. (C) Divergent effects of SidI and emetine on aggresome formation. HeLa cells stably expressing SynCGFP were transfected with a plasmid encoding SidI or an empty vector. On the next day cells were incubated for 4 hours with 10 M MG132 and to some samples the indicated amounts of emetine were added; the aggresome formation was analyzed with a fluorescence microscope. The effect of SidI was adjusted for the efficiency of transfection. (D) Canavanine relieves inhibition of the aggresome formation by low concentration of emetine. Cells stably expressing SynCGFP were incubated for 2 hours with 5 M MG132 and 50 nM emetine with or without 20 mM canavanine. The extent of the aggresome formation was analyzed with a fluorescence microscope. (E) The remaining protein synthesis is necessary for aggresome formation in the presence of SidI. HeLa cells stably Mouse monoclonal to CD4.CD4 is a co-receptor involved in immune response (co-receptor activity in binding to MHC class II molecules) and HIV infection (CD4 is primary receptor for HIV-1 surface glycoprotein gp120). CD4 regulates T-cell activation, T/B-cell adhesion, T-cell diferentiation, T-cell selection and signal transduction expressing SynCGFP were transfected with a plasmid encoding either SidI or an empty vector. On the next day, cells were incubated for 4 hours with 10 M MG132 and 5 M emetine was added to one sample. The extent of the aggresome formation was analyzed with a fluorescence microscope. Scale bars: 20 m. We then compared the inhibitory effects of emetine and SidI on the activation of Hsf1 in response to inhibition of the proteasome. The activation was monitored by the Hsf1-controlled induction of the mRNA, isolated after 7 hours of proteasome inhibition. High concentration (2 M) of emetine.4A). does not prevent eEF1A-mediated signaling to Hsf1 (Shen et al., 2009). Considering the apparent similarity in activation of Hsf1 and Metiamide induction Metiamide of aggresome formation, we tested whether effects of SidI on eEF1A-mediated translation and on putative eEF1A-mediated triggering of the aggresome formation could also be differentiated. In this series of experiments, we first compared the effects of SidI and emetine on translation. Because the efficiency of HeLa transfection was less than 100%, we could not use radioactive labeling to assess the extent of inhibition of translation, and had to monitor expression of a co-transfected polypeptide. Accordingly, we transfected HeLa cells with a plasmid encoding EGFP, and co-transfected with a plasmid encoding either SidI or the vector. Various concentrations of emetine were added to the cells co-transfected with the empty vector before they accumulated any detectable amounts of EGFP. The levels of EGFP were assessed 20 hours after the end of the transfection. As seen in Fig. 4A, 2 M emetine almost completely inhibited translation of EGFP, whereas 100 nM emetine caused about a 40% inhibition. Co-expression of SidI had very strong inhibitory effect, reducing the yield of translation by 97% (Fig. 4A). Of note, SidI inhibited its own translation, and thus was expressed at almost undetectable levels, whereas a SidI mutant, which cannot interact with eEF1A (Shen et al., 2009), was expressed at high levels (not shown). Open in a separate window Fig. 4. SidI reduces the threshold of DRiPs necessary to trigger aggresome formation. (A) Effects of various emetine concentrations and SidI on protein synthesis. HeLa cells were transfected for 3 hours with a plasmid encoding EGFP and co-transfected with Metiamide either a plasmid encoding SidI or an empty vector. 1.5 hours after the end of the transfection indicated amounts of emetine were added to some samples. The levels of synthesized EGFP were assessed by immunoblotting 20 hours after the end of the transfection. (B) Divergent effects of SidI and emetine on induction of Hsp70. HeLa cells were transfected with a plasmid encoding SidI or an empty vector and on the next day incubated with or without 10 M MG132 and the indicated concentrations of emetine for 7 hours. RNA was isolated from the cells and the levels of mRNA were assessed with RT-PCR. The effect of SidI was adjusted for the efficiency of transfection. (C) Divergent effects of SidI and emetine on aggresome formation. HeLa cells stably expressing SynCGFP were transfected with a plasmid encoding SidI or an empty vector. On the next day cells were incubated for 4 hours with 10 M MG132 and to some samples the indicated amounts of emetine were added; the aggresome formation was analyzed with a fluorescence microscope. The effect of SidI was adjusted for the efficiency of transfection. (D) Canavanine relieves inhibition of the aggresome formation by low concentration of emetine. Cells stably expressing SynCGFP were incubated for 2 hours with 5 M MG132 and 50 nM emetine with or without 20 mM canavanine. The extent of the aggresome formation was analyzed with a fluorescence microscope. (E) The remaining protein synthesis is necessary for aggresome formation in the presence of SidI. HeLa cells stably expressing SynCGFP were transfected with a plasmid encoding either SidI or an empty vector. On the next day, cells were incubated for 4 hours with 10 M MG132 and 5 M emetine was added to one sample. The extent of the aggresome formation was analyzed with a fluorescence microscope. Scale bars: 20 m. We then compared the inhibitory effects of emetine and SidI on the activation of Hsf1 in response to inhibition of the proteasome. The activation was monitored by the Hsf1-controlled induction of the mRNA, isolated after 7 hours of proteasome inhibition. High concentration (2 M) of emetine blocked Hsf1 activation almost completely, and 100 nM.