Inhibition of Protein Synthesis in Intact HeLa Cells

Tscherne, Joan S.; Pestka, Sidney

doi:10.1128/aac.8.4.479

Cited by 59 publications

(33 citation statements)

References 35 publications

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“…Pactamycin and harringtonine, the other drugs that were tested, are both protein synthesis inhibitors that lead to depletion of polysomes in eukaryotic cells (Kappen et al 1973;Tscherne and Pestka 1975;Fresno et al 1977). As with puromycin, we found that both reduced the BiFC signal in extracts (Fig.…”

Section: The Bifc Interaction Between Rps18 and Rpl11 Occurs In The 8mentioning

confidence: 63%

“…To assess more directly the dependence of the BiFC signal on translation, we first compared the effects of emetine and puromycin. These translation inhibitor drugs have different mechanisms of action (Tscherne and Pestka 1975): Emetine blocks ribosomal polypeptide elongation, freezes 80S, and stabilizes polysomes (Grollman 1968), whereas puromycin is an aminoacyl-tRNA-like molecule that causes premature translation termination by serving as acceptor for the peptidyl-tRNA and leads to rapid breakdown of polysomes (Nathans and Lipmann 1961). As exemplified in Supplemental Figure S3D, puromycin causes dissociation of the two subunits at least in vitro (Blobel and Sabatini 1971).…”

Section: The Bifc Interaction Between Rps18 and Rpl11 Occurs In The 8mentioning

confidence: 99%

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Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

Al-Jubran

Wen

Abdullahi

et al. 2013

RNA

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In eukaryotes the 40S and 60S ribosomal subunits are assembled in the nucleolus, but there appear to be mechanisms preventing mRNA binding, 80S formation, and initiation of translation in the nucleus. To visualize association between ribosomal subunits, we tagged pairs of Drosophila ribosomal proteins (RPs) located in different subunits with mutually complementing halves of fluorescent proteins. Pairs of tagged RPs expected to interact, or be adjacent in the 80S structure, showed strong fluorescence, while pairs that were not in close proximity did not. Moreover, the complementation signal is found in ribosomal fractions and it was enhanced by translation elongation inhibitors and reduced by initiation inhibitors. Our technique achieved 80S visualization both in cultured cells and in fly tissues in vivo. Notably, while the main 80S signal was in the cytoplasm, clear signals were also seen in the nucleolus and at other nuclear sites. Furthermore, we detected rapid puromycin incorporation in the nucleolus and at transcription sites, providing an independent indication of functional 80S in the nucleolus and 80S association with nascent transcripts.

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Section: The Bifc Interaction Between Rps18 and Rpl11 Occurs In The 8mentioning

confidence: 63%

Section: The Bifc Interaction Between Rps18 and Rpl11 Occurs In The 8mentioning

confidence: 99%

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

Al-Jubran

Wen

Abdullahi

et al. 2013

RNA

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“…1C, FIG 2 CBF␤ does not affect translational activity on Vif mRNA. Polysome analysis was done using a modification of a previously described protocol (63). Briefly, HeLa-KD cells (9 ϫ 10 6 in a 75-cm 2 flask) were transfected with 12 g of pcDNA-hVif together with either 3 g of pcDNA-CBF␤ or 3 g of empty vector DNA.…”

Section: Resultsmentioning

confidence: 99%

CBFβ Enhances De Novo Protein Biosynthesis of Its Binding Partners HIV-1 Vif and RUNX1 and Potentiates the Vif-Induced Degradation of APOBEC3G

Miyagi

Kao

Yedavalli

et al. 2014

J Virol

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Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBF␤ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBF␤ remains unclear. Several studies noted reduced Vif expression in CBF␤ knockdown cells while others saw no significant impact of CBF␤ on Vif stability. Here, we confirmed that CBF␤ increases Vif steady-state levels. CBF␤ affected expression of neither viral Gag nor Vpu protein, indicating that CBF␤ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBF␤ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBF␤ to interact with Vif. Importantly, at comparable Vif levels, CBF␤ further enhanced A3G degradation, suggesting that CBF␤ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBF␤ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBF␤ had no detectable effect on RUNX1 metabolic stability. We propose that CBF␤ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. IMPORTANCEIn this study, we show that CBF␤ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBF␤. Kinetic studies revealed that CBF␤ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBF␤ were nonresponsive to CBF␤. Our data suggest that CBF␤ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.

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“…3B). The Golgi morphology of compound-treated cells also differed from the characteristic Golgi architecture of cells treated with anisomycin, a peptidyl transferase inhibitor shown to induce apoptosis in HeLa cells (57). Other small-molecule compounds known to affect Golgi morphology include nocodazole (10), a microtubule-disrupting agent, and BFA, a known trafficking inhibitor that causes complete dispersal of the Golgi apparatus (24,42).…”

Section: Vol 75 2007 Small-molecule Inhibitors Of Toxin Transport 4555mentioning

confidence: 99%

Identification and Characterization of Small Molecules That Inhibit Intracellular Toxin Transport

2007

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Shiga toxin (Stx), cholera toxin (Ctx), and the plant toxin ricin are among several toxins that reach their intracellular destinations via a complex route. Following endocytosis, these toxins travel in a retrograde direction through the endosomal system to the trans-Golgi network, Golgi apparatus, and endoplasmic reticulum (ER). There the toxins are transported across the ER membrane to the cytosol, where they carry out their toxic effects. Transport via the ER from the cell surface to the cytosol is apparently unique to pathogenic toxins, raising the possibility that various stages in the transport pathway can be therapeutically targeted. We have applied a luciferase-based high-throughput screen to a chemical library of small-molecule compounds in order to identify inhibitors of Stx. We report two novel compounds that protect against Stx and ricin inhibition of protein synthesis, and we demonstrate that these compounds reversibly inhibit bacterial transport at various stages in the endocytic pathway. One compound (compound 75) inhibited transport at an early stage of Stx and Ctx transport and also provided protection against diphtheria toxin, which enters the cytosol from early endosomes. In contrast, compound 134 inhibited transport from recycling endosomes through the Golgi apparatus and protected only against toxins that access the ER. Small-molecule compounds such as these will provide insight into the mechanism of toxin transport and lead to the identification of compounds with therapeutic potential against toxins routed through the ER.

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Inhibition of Protein Synthesis in Intact HeLa Cells

Cited by 59 publications

References 35 publications

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

Visualization of the joining of ribosomal subunits reveals the presence of 80S ribosomes in the nucleus

CBFβ Enhances De Novo Protein Biosynthesis of Its Binding Partners HIV-1 Vif and RUNX1 and Potentiates the Vif-Induced Degradation of APOBEC3G

Identification and Characterization of Small Molecules That Inhibit Intracellular Toxin Transport

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