The science of puromycin: From studies of ribosome function to applications in biotechnology

Aviner, Ranen

doi:10.1016/j.csbj.2020.04.014

Cited by 139 publications

(142 citation statements)

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“…After entering the A site, it causes a premature, factor free termination of polypeptide synthesis [7,11]. The activity of puromycin is well studied; its fluorescent and biotin derivatives are widely used for the visualization and quantitative analysis of newly synthesized proteins by many modern techniques such as PUNCH P, SUnSET, Puro PLA, RiboLace, and RPM, as well as for mRNA display [126]. Treating the cells with puromycin in a com bination with cycloheximide leads to the accumulation of ribosomes exclusively on start codons, which facilitates their identification by ribosome profiling [127].…”

Section: Inhibitors Of Eukaryotic Ribosomementioning

confidence: 99%

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

Dmitriev

Vladimirov

Lashkevich

2020

Biochemistry Moscow

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Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru/) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.

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Section: Inhibitors Of Eukaryotic Ribosomementioning

confidence: 99%

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

Dmitriev

Vladimirov

Lashkevich

2020

Biochemistry Moscow

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“…Puromycin is a potent translational inhibitor that binds to ribosomes from all domains of life and has been used as a chemical probe and selectable marker for decades ( Aviner, 2020 ; Yarmolinsky and Haba, 1959 ). Puromycin is unique among translational inhibitors in that it is itself a substrate of the ribosomal peptidyl-transferase reaction ( Nathans, 1964 ).…”

Section: Introductionmentioning

confidence: 99%

Puromycin reactivity does not accurately localize translation at the subcellular level

Enam

Zinshteyn

Goldman

et al. 2020

eLife

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Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Using in vitro and in vivo experiments in various eukaryotic systems, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in vivo.

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“…Puromycin treatment causes premature chain termination during translation and the accumulation of defective ribosomal products ( 29 , 30 ). Defective ribosomal products are frequently sequestered into punctate, ubiquitin-positive structures in the cytosol referred to as ALIS ( 16 , 31 ).…”

Section: Resultsmentioning

confidence: 99%

AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner

Klickstein

Mukkavalli

Raman

2020

Journal of Biological Chemistry

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Protein quality control is maintained by a number of integrated cellular pathways that monitor the folding and functionality of the cellular proteome. Defects in these pathways lead to the accumulation of misfolded or faulty proteins that may become insoluble and aggregate over time. Protein aggregates significantly contribute to the development of a number of human diseases such as Amyotrophic lateral sclerosis, Huntington’s and Alzheimer’s Disease. In vitro, imaging-based, cellular studies have defined key biomolecular components that recognize and clear aggregates; however, no unifying method is available to quantify cellular aggregates limiting our ability to reproducibly and accurately quantify these structures. Here we describe an ImageJ macro called AggreCount to identify and measure protein aggregates in cells. AggreCount is designed to be intuitive, easy to use and customizable for different types of aggregates observed in cells. Minimal experience in coding is required to utilize the script. Based on a user defined image, AggreCount will report a number of metrics: (i) total number of cellular aggregates, (ii) percent of cells with aggregates, (iii) aggregates per cell, (iv) area of aggregates and (v) localization of aggregates (cytosol, perinuclear or nuclear). A data table of aggregate information on a per cell basis as well as a summary table is provided for further data analysis. We demonstrate the versatility of AggreCount by analyzing a number of different cellular aggregates including aggresomes, stress granules and inclusion bodies caused by Huntingtin polyQ expansion.

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The science of puromycin: From studies of ribosome function to applications in biotechnology

Cited by 139 publications

References 100 publications

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

A Quick Guide to Small-Molecule Inhibitors of Eukaryotic Protein Synthesis

Puromycin reactivity does not accurately localize translation at the subcellular level

AggreCount: an unbiased image analysis tool for identifying and quantifying cellular aggregates in a spatially defined manner

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