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Cell Reports Methods Apr 2022The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to...
The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in , they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact , we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.
Topics: Animals; Caenorhabditis elegans; Protein Biosynthesis; Puromycin; Microscopy, Fluorescence
PubMed: 35497499
DOI: 10.1016/j.crmeth.2022.100203 -
Nature Protocols Feb 2019Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and...
Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging. The synthesis of O-propargyl-puromycin (OP-Puro), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OP-Puro enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Incorporated OP-Puro can be detected through a click-chemistry reaction that links it to a fluorescently tagged azide molecule. In this protocol, we describe how to administer OP-Puro to mice, obtain cells of interest (here, we use bone marrow cells) just 1 h later, and quantify the amount of protein synthesized per hour by flow cytometry on the basis of OP-Puro incorporation. We have used this approach to show that hematopoietic stem cells (HSCs) exhibit an unusually low rate of protein synthesis relative to other hematopoietic cells, and it can be easily adapted to quantify cell-type-specific rates of protein synthesis across diverse mammalian tissues in vivo. Measurement of protein synthesis within bone marrow cells in a cohort of six mice can be achieved in 8-10 h.
Topics: Animals; Azides; Bone Marrow Cells; Click Chemistry; Flow Cytometry; Fluorescent Dyes; Hematopoietic Stem Cells; Injections, Intraperitoneal; Mice; Mice, Inbred C57BL; Organ Specificity; Protein Biosynthesis; Puromycin; Rhodamines; Ribosomes; Single-Cell Analysis; Staining and Labeling; Sulfonic Acids
PubMed: 30610239
DOI: 10.1038/s41596-018-0100-z -
Nature Chemical Biology Sep 2016Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis....
Improved methods for studying intracellular reactive Fe(II) are of significant interest for studies of iron metabolism and disease-relevant changes in iron homeostasis. Here we describe a highly selective reactivity-based probe in which a Fenton-type reaction with intracellular labile Fe(II) leads to unmasking of the aminonucleoside puromycin. Puromycin leaves a permanent and dose-dependent mark on treated cells that can be detected with high sensitivity and precision using a high-content, plate-based immunofluorescence assay. Using this new probe and screening approach, we detected alteration of cellular labile Fe(II) in response extracellular iron conditioning, overexpression of iron storage and/or export proteins, and post-translational regulation of iron export. We also used this new tool to demonstrate that labile Fe(II) pools are larger in cancer cells than in nontumorigenic cells.
Topics: Ferrous Compounds; Fluorescent Antibody Technique; Fluorescent Dyes; Humans; Molecular Structure; Puromycin; Spiro Compounds
PubMed: 27376690
DOI: 10.1038/nchembio.2116 -
Journal of Natural Products Nov 2018The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B-E (1-4)] metabolites from a Himalayan isolate ( Streptomyces sp....
The isolation and structure elucidation of four new naturally occurring amino-nucleoside [puromycins B-E (1-4)] metabolites from a Himalayan isolate ( Streptomyces sp. PU-14-G, isolated from the Bara Gali region of northern Pakistan) is reported. Consistent with prior reports, comparative antimicrobial assays revealed the need for the free 2″-amine for anti-Gram-positive bacteria and antimycobacterial activity. Similarly, comparative cancer cell line cytotoxicity assays highlighted the importance of the puromycin-free 2″-amine and the impact of 3'-nucleoside substitution. These studies extend the repertoire of known naturally occurring puromycins and their corresponding SAR. Notably, 1 represents the first reported naturally occurring bacterial puromycin-related metabolite with a 3'- N-amino acid substitution that differs from the 3'- N-tyrosinyl of classical puromycin-type natural products. This discovery suggests the biosynthesis of 1 in Streptomyces sp. PU-14G may invoke a uniquely permissive amino-nucleoside synthetase and/or multiple synthetases and sets the stage for further studies to elucidate, and potentially exploit, new biocatalysts for puromycin chemoenzymatic diversification.
Topics: Gram-Positive Bacteria; Microbial Sensitivity Tests; Molecular Structure; Mycobacterium; Nucleosides; Pakistan; Puromycin; Streptomyces
PubMed: 30418763
DOI: 10.1021/acs.jnatprod.8b00720 -
STAR Protocols Sep 2022Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess...
Translational regulation is a fundamental step in gene expression with critical roles in biological processes within a cell. Here, we describe a protocol to assess translation activity in mammalian cells by incorporation of O-propargyl-puromycin (OP-Puro). OP-Puro is a puromycin analog that is incorporated into newly synthesized proteins and is detected by click chemistry reaction. We use OP-Puro labeling to assess translation activity between different cell types or cells under different growth conditions by confocal microscopy and flow cytometry. For complete details on the use and execution of this protocol, please refer to Hsu et al. (2021) and Hsu et al. (2022).
Topics: Animals; Cell Line; Click Chemistry; Mammals; Proteomics; Puromycin
PubMed: 36072758
DOI: 10.1016/j.xpro.2022.101654 -
Cells Feb 2022Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health...
Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ transplantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic options are urgently needed. Within the kidney, the glomeruli are involved in blood filtration and waste excretion and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be employed to induce acute glomerular damage and to model glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations revealed the disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell-death-related processes. We conclude that the treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated by an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine-stem-cell-derived kidney organoids can be used to model kidney-associated diseases and drug discovery.
Topics: Acute Kidney Injury; Humans; Induced Pluripotent Stem Cells; Kidney; Organoids; Puromycin Aminonucleoside
PubMed: 35203286
DOI: 10.3390/cells11040635 -
Proceedings of the National Academy of... Jan 2012Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation,...
Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation, or death. Methods that allow imaging and identification of nascent proteins are critical for dissecting regulation of translation, both spatially and temporally, particularly in whole organisms. We introduce a simple and robust chemical method to image and affinity-purify nascent proteins in cells and in animals, based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-puro). OP-puro forms covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized or captured by copper(I)-catalyzed azide-alkyne cycloaddition. Unlike methionine analogs, OP-puro does not require methionine-free conditions and, uniquely, can be used to label and assay nascent proteins in whole organisms. This strategy should have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.
Topics: Alkynes; Azides; Copper; Diagnostic Imaging; Magnetic Resonance Spectroscopy; Microscopy, Fluorescence; Molecular Structure; Protein Biosynthesis; Puromycin
PubMed: 22160674
DOI: 10.1073/pnas.1111561108 -
Zhongguo Ying Yong Sheng Li Xue Za Zhi... Mar 2022To construct the lentivirus overexpression vector with two label genes fused with CopGFP and Puro and to detect the emission of green fluorescence as well as resistance...
To construct the lentivirus overexpression vector with two label genes fused with CopGFP and Puro and to detect the emission of green fluorescence as well as resistance to puromycin in liver cancer cells infected with lentivirus packaged with the above vector. Firstly, two fragments containing copGFP and Puro coding sequences were amplified from pCDH-CMV-MCS-copGFP and pLKO.1 respectively; secondly, the two amplified regions were fused with each other by recombinant PCR; thirdly, the fusion DNA fragment was cut and inserted into pCDH-CMV-MCS-copGFP vector, which was linearized with the same restriction endonuclease as used to digest fusion DNA fragment: BamH Ⅰ and Sal Ⅰ. The fusion region in the constructed vector was confirmed by DNA sequencing. The checked vector was co-transfected with package assistant plasmids, namely PLP1, PLP2 and VSVG into in 293T cells and the culture supernatant was subjected to centrifuge and infect liver cancer MHCC97H cells, which were then used to detect their resistance to puromycin (infected cells were treated with 1 mg/ml puromycin for 7 days after infection) and to observe green fluorescence emission in microscope. To determine its efficiency in expressing foreign target protein, the Sp1 coding region was inserted into the MCS sites of the vector, and Sp1 mRNA and protein expression levels were compared with the vehicle vector by RT-qPCR and Western blot. The lentivirus overexpression vector with two label genes fused with CopGFP and Puro was successfully constructed, and the liver cancer cells infected with lentivirus packaged with the vector expressing two labeling genes fused with CopGFP and Puroshowed both emission of green fluorescence and resistance to puromycin simultaneously, while cells containing with the vector inserted with Sp1 coding region improved Sp1 mRNA level with 3.3 fold and protein level with 2.2 fold higher in comparison with cells containing the vehicle vector (<0.01). The fused label genes consisting of copGFP and Puro are correctly cloned into the lentivirus vector and confer cells with the ability to emission of green fluorescence and resistance to puromycin, besides, the vector may promote the expression of the target gene with long coding sequence.
Topics: Cytomegalovirus Infections; Genetic Vectors; Humans; Lentivirus; Liver Neoplasms; Puromycin; RNA, Messenger; Transfection
PubMed: 36031566
DOI: 10.12047/j.cjap.6215.2022.028 -
The Journal of Antibiotics Dec 1985Puromycin 2"-N-acetyltransferase was isolated from cell extracts of puromycin-producing Streptomyces alboniger KCC S-0309 by ammonium sulfate fractionation, heat...
Puromycin 2"-N-acetyltransferase was isolated from cell extracts of puromycin-producing Streptomyces alboniger KCC S-0309 by ammonium sulfate fractionation, heat treatment to eliminate contaminant proteins and chromatography on DEAE-Toyopearl 650S. After PAGE (polyacrylamide gel electrophoresis) of the final fraction, a single protein band corresponding to puromycin 2"-N-acetyltransferase was detected. The molecular weight of the enzyme determined by SDS-PAGE and Sephadex G-150 chromatography was about 21,000 and 85,000, respectively, suggesting that the enzyme consisted of four subunits. The isoelectric point and the optimum pH for reaction were 6.2 and 7.7, respectively. The Km values for puromycin and acetyl coenzyme A were 40 microM and 67 microM, respectively. The enzyme was thermostable up to 70 degrees C for 12 minutes. It was shown, by using an in vitro protein synthesizing system from a puromycin-susceptible organism S. flavotricini subsp. pseudochromogenes V-13-1, that the isolated puromycin 2"-N-acetyltransferase could protect polyphenylalanine synthesis from inhibition by puromycin.
Topics: Acetyltransferases; Bacterial Proteins; Hot Temperature; Hydrogen-Ion Concentration; Isoelectric Point; Kinetics; Molecular Weight; Puromycin; Streptomyces; Substrate Specificity
PubMed: 4093336
DOI: 10.7164/antibiotics.38.1761 -
Pharmacology Research & Perspectives Oct 2017Protein synthesis inhibitors are commonly used for measuring protein degradation rates, but may cause cytotoxicity via direct or indirect mechanisms. This study aimed to...
Protein synthesis inhibitors are commonly used for measuring protein degradation rates, but may cause cytotoxicity via direct or indirect mechanisms. This study aimed to identify concentrations providing optimal inhibition in the absence of overt cytotoxicity. Actinomycin D, cycloheximide, emetine, and puromycin were assessed individually, and in two-, three-, and four-drug combinations for protein synthesis inhibition (IC ) and cytotoxicity (CC ) over 72 h. Experiments were conducted in HepG2 cells and primary rat hepatocytes (PRH). IC for actinomycin D, cycloheximide, emetine, and puromycin were 39 ± 7.4, 6600 ± 2500, 2200 ± 1400, and 1600 ± 1200 nmol/L; with corresponding CC values of 6.2 ± 7.3, 570 ± 510, 81 ± 9, and 1300 ± 64 nmol/L, respectively, in HepG2 cells. The IC were 1.7 ± 1.8, 290 ± 90, 620 ± 920, and 2000 ± 2000 nmol/L, with corresponding CC values of 0.98 ± 1.8, 680 ± 1300, 180 ± 700, and 1600 ± 1000 (SD) nmol/L, respectively, in PRH. CC were also lower than the IC for all drug combinations in HepG2 cells. These data indicate that using pharmacological interference is inappropriate for measuring protein degradation over a protracted period, because inhibitory effects cannot be extricated from cytotoxicity.
Topics: Animals; Cells, Cultured; Cycloheximide; Dactinomycin; Emetine; Hep G2 Cells; Hepatocytes; Humans; Inhibitory Concentration 50; Male; Protein Synthesis Inhibitors; Proteolysis; Puromycin; Rats
PubMed: 28971619
DOI: 10.1002/prp2.359