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Nature Methods Apr 2009We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and...
We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.
Topics: Gene Expression Profiling; Immunoblotting; Isotope Labeling; Protein Biosynthesis; Proteins; Puromycin; Staining and Labeling
PubMed: 19305406
DOI: 10.1038/nmeth.1314 -
Angewandte Chemie (International Ed. in... Jun 2023Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me N surrogates, exhibit translation...
Puromycin derivatives containing an emissive thieno[3,4-d]-pyrimidine core, modified with azetidine and 3,3-difluoroazetidine as Me N surrogates, exhibit translation inhibition and bactericidal activity similar to the natural antibiotic. The analogues are capable of cellular puromycylation of nascent peptides, generating emissive products without any follow-up chemistry. The 3,3-difluoroazetidine-containing analogue is shown to fluorescently label newly translated peptides and be visualized in both live and fixed HEK293T cells and rat hippocampal neurons.
Topics: Rats; Animals; Humans; Puromycin; HEK293 Cells; Peptides
PubMed: 36973168
DOI: 10.1002/anie.202216784 -
Journal of Medicinal Chemistry Mar 2023Overexpression of the selenoprotein thioredoxin reductase (TrxR) has been documented in malignant tissues and is of pathological significance for many types of tumors....
Overexpression of the selenoprotein thioredoxin reductase (TrxR) has been documented in malignant tissues and is of pathological significance for many types of tumors. The antibiotic puromycin (Puro) is a protein synthesis inhibitor causing premature polypeptide chain termination during translation. The well-defined action mechanism of Puro makes it a useful tool in biomedical studies. However, the nonselective cytotoxicity of Puro limits its therapeutic applications. We report herein the construction and evaluation of two Puro prodrugs, that is, S1-Puro with a five-membered cyclic disulfide trigger and S2-Puro with a linear disulfide trigger. S1-Puro is selectively activated by TrxR and shows the TrxR-dependent cytotoxicity to cancer cells, while S2-Puro is readily activated by thiols. Furthermore, S1-Puro displays higher stability in plasma than S2-Puro. We expect that this prodrug strategy may promote the further development of Puro as a therapeutic agent.
Topics: Thioredoxin-Disulfide Reductase; Prodrugs; Puromycin
PubMed: 36855911
DOI: 10.1021/acs.jmedchem.2c01509 -
Cell Reports Oct 2018Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to...
Ribosome profiling, or Ribo-seq, is based on large-scale sequencing of RNA fragments protected from nuclease digestion by ribosomes. Thanks to its unique ability to provide positional information about ribosomes flowing along transcripts, this method can be used to shed light on mechanistic aspects of translation. However, current Ribo-seq approaches lack the ability to distinguish between fragments protected by either ribosomes in active translation or inactive ribosomes. To overcome this possible limitation, we developed RiboLace, a method based on an original puromycin-containing molecule capable of isolating active ribosomes by means of an antibody-free and tag-free pull-down approach. RiboLace is fast, works reliably with low amounts of input material, and can be easily and rapidly applied both in vitro and in vivo, thereby generating a global snapshot of active ribosome footprints at single nucleotide resolution.
Topics: Animals; Cell Line; High-Throughput Nucleotide Sequencing; Humans; Mice; Microspheres; Puromycin; RNA, Messenger; Ribosomes
PubMed: 30355487
DOI: 10.1016/j.celrep.2018.09.084 -
ELife Aug 2020Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in...
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.
Topics: Animals; Caenorhabditis elegans; Cell Nucleus; Emetine; Peptides; Protein Biosynthesis; Protein Synthesis Inhibitors; Puromycin; RNA, Transfer; Rabbits; Ribosomes; Single-Cell Analysis
PubMed: 32844748
DOI: 10.7554/eLife.60303 -
Journal of the American Chemical Society Nov 2022Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products...
Translation is an elementary cellular process that involves a large number of factors interacting in a concerted fashion with the ribosome. Numerous natural products have emerged that interfere with the ribosomal function, such as puromycin, which mimics an aminoacyl tRNA and causes premature chain termination. Here, we introduce a photoswitchable version of puromycin that, in effect, puts translation under optical control. Our compound, termed , features a diazocine that allows for reversible and nearly quantitative isomerization and pharmacological modulation. Its synthesis involves a new photoswitchable amino acid building block. shows little activity in the dark and becomes substantially more active and cytotoxic, in a graded fashion, upon irradiation with various wavelengths of visible light. In vitro translation assays confirm that inhibits translation with a mechanism similar to that of puromycin itself. Once incorporated into nascent proteins, reacts with standard puromycin antibodies, which allows for tracking protein synthesis using western blots and immunohistochemistry. As a cell-permeable small molecule, can be used for nascent proteome profiling in a variety of cell types, including primary mouse neurons. We envision as a useful biochemical tool for the optical control of translation and for monitoring newly synthesized proteins in defined locations and at precise time points.
Topics: Animals; Mice; Puromycin; Light; Blotting, Western; RNA, Transfer, Amino Acyl; Amino Acids
PubMed: 36394560
DOI: 10.1021/jacs.2c07374 -
Progress in Brain Research 1980
Review
Topics: Animals; Dendrites; Electrophoresis, Polyacrylamide Gel; Haplorhini; Nerve Regeneration; Nerve Tissue Proteins; Puromycin; Rats; Spinal Cord; Synapses
PubMed: 6779347
DOI: 10.1016/S0079-6123(08)60055-2 -
Nature Oct 1966
Topics: Animals; Carbon Isotopes; Culture Techniques; DNA; Drug Stability; HeLa Cells; Humans; Intestine, Small; Leucine; Male; Mice; Protein Biosynthesis; Puromycin; Thymidine; Tritium
PubMed: 5972219
DOI: 10.1038/212196a0 -
European Journal of Medicinal Chemistry Oct 2017Substantial progress has been described in the study of puromycin and its analogs for antibiotic properties. However, the peptidase inhibitory activity of related...
Substantial progress has been described in the study of puromycin and its analogs for antibiotic properties. However, the peptidase inhibitory activity of related analogs has not been explored as extensively. Specifically, inhibiting aminopeptidases for achieving antitumor effect has been sparsely investigated. Herein, we address this challenge by reporting the synthesis of a series of analogs based on the structural template of puromycin. We also present exhaustive biochemical and in vitro analyses in support of our thesis. Analyzing the structure-activity relationship revealed a steric requirement for maximum potency. Effective inhibitors of Puromycin-Sensitive Aminopeptidase (PSA) are disclosed here. These potential therapeutic agents display superior in vitro antitumor potency against two leukemic cell lines, as compared to known inhibitors of aminopeptidases.
Topics: Aminopeptidases; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Enzyme Inhibitors; HL-60 Cells; Hematologic Neoplasms; Humans; Molecular Structure; Puromycin; Structure-Activity Relationship; Vero Cells
PubMed: 28803047
DOI: 10.1016/j.ejmech.2017.07.048 -
Contributions To Nephrology 1988
Review
Topics: Animals; Disease Models, Animal; Doxorubicin; Kidney Failure, Chronic; Puromycin; Puromycin Aminonucleoside; Rats
PubMed: 3278861
DOI: 10.1159/000414793