-
Antimicrobial Agents and Chemotherapy Aug 2018Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and...
Schistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC) for schistosomula was 19 μg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development of -laid eggs (LC, 68 ng/ml) but was less effective against liver eggs (LC, 387 μg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LCs after 48 h of 4.9 and 17.3 μg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.
Topics: Animals; Animals, Genetically Modified; Female; Fluoresceins; Genomics; Male; Puromycin; Schistosoma mansoni; Schistosomiasis
PubMed: 29760143
DOI: 10.1128/AAC.02568-17 -
RNA (New York, N.Y.) Jul 2002Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the...
Peptidyl transferase inhibitors have generally been studied using simple systems and remain largely unexamined In in vitro translation extracts. Here, we investigate the potency, product distribution, and mechanism of various puromycin-oligonucleotide conjugates (1 to 44 nt with 3'-puromycin) In a reticulocyte lysate cell-free translation system. Surprisingly, the potency decreases as the chain length of the oligonucleotide is increased in this series, and only very short puromycin conjugates function efficiently (IC50 < 50 microM). This observation stands in contrast with work on isolated large ribosomal subunits, which Indicates that many of the puromycin-oligonucleotide conjugates we studied should have higher affinity for the peptidyl transferase center than puromycin itself. Two tRNA(Al)-derived minihelices containing puromycin provide an exception to the size trend, and are the only constructs longer than 4 nt with any appreciable potency (IC50 = 40-56 microM). However, the puromycin minihelices inhibit translation by sequestering one or more soluble translation factors, and do not appear to participate in detectable peptide bond formation with the nascent chain. In contrast, puromycin and other short derivatives act in a factor-independent fashion at the peptidyl transferase center and readily become conjugated to the nascent protein chain. However, even for the short derivatives, much of the translation inhibition occurs without peptide bond formation between puromycin and the nascent chain, a revision of the classical model for puromycin function. This peptide bond-independent mode is likely a combination of multiple effects including inhibition of initiation and failure to properly recycle translation complexes that have reacted with puromycin.
Topics: Animals; Base Sequence; Binding Sites; Carboxypeptidases; Cathepsin A; Cell-Free System; Globins; In Vitro Techniques; Models, Biological; Nucleic Acid Conformation; Oligoribonucleotides; Protein Biosynthesis; Puromycin; RNA, Messenger; Rabbits; Reticulocytes; Ribosomes
PubMed: 12166644
DOI: 10.1017/s1355838202022069 -
STAR Protocols Jun 2022Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation...
Gene functions can be assessed in mouse embryonic stem (ES) cells and in mutant mice derived from mutant ES cells. Here, we describe an approach for efficient isolation of the ES clones carrying deletion mutations at the target genes by CRISPR-Cas9. Two sgRNAs against a target gene are co-expressed with puromycin-resistant gene in ES cells through co-transfection followed by transient puromycin selection. Deletion mutations are identified by PCR from individual ES clones that are picked from puromycin-selected ES cells.
Topics: Animals; CRISPR-Cas Systems; Embryonic Stem Cells; Mice; Mouse Embryonic Stem Cells; Puromycin; Transfection
PubMed: 35693210
DOI: 10.1016/j.xpro.2022.101436 -
Chembiochem : a European Journal of... Sep 2022Peptidoyl RNAs are the products of ribosome-free, single-nucleotide translation. They contain a peptide in the backbone of the oligoribonucleotide and are interesting...
Peptidoyl RNAs are the products of ribosome-free, single-nucleotide translation. They contain a peptide in the backbone of the oligoribonucleotide and are interesting from a synthetic and a bioorganic point of view. A synthesis of a stabilized version of peptidoyl RNA, with an amide bond between the C-terminus of a peptide and a 3'-amino-2',3'-dideoxynucleoside in the RNA chain was developed. The preferred synthetic route used an N-Teoc-protected aminonucleoside support and involved a solution-phase coupling of the amino-terminal oligonucleotide to a dipeptido dinucleotide. Exploratory UV-melting and NMR analysis of the hairpin 5'-UUGGCGAAAGCdC-LeuLeu-AA-3' indicated that the peptide-linked RNA segments do not fold in a cooperative fashion. The synthetic access to doubly RNA-linked peptides on a scale sufficient for structural biology opens the door to the exploration of their structural and biochemical properties.
Topics: Amides; Dideoxynucleosides; Oligonucleotides; Oligoribonucleotides; Peptides; Puromycin Aminonucleoside; RNA
PubMed: 35867587
DOI: 10.1002/cbic.202200352 -
Beijing Da Xue Xue Bao. Yi Xue Ban =... Dec 2015To further demonstrate the interaction of a new 14-3-3 interaction protein hematopoietic progenitor kinase 1[HPK1]-interacting protein (HIP-55) and 14-3-3 proteins and...
OBJECTIVE
To further demonstrate the interaction of a new 14-3-3 interaction protein hematopoietic progenitor kinase 1[HPK1]-interacting protein (HIP-55) and 14-3-3 proteins and its potential biological function in HEK293 cells.
METHODS
PDEST-N-Venus-HIP-55WT (wild type),PDEST-N-Venus-HIP-55AA (mutants, S269A/T291A, abolishing the binding of HIP-55 to 14-3-3),PDEST-GST-HIP-55WT and PDEST-C-Venus-14-3-3τ plasmids were constructed by gateway system. Their expressions were demonstrated by Western blotting method. Then we used Bimolecular Fluorescence Complementation (BiFC) and co-immunoprecipitation (co-IP) methods to demonstrate the interaction of HIP-55 and 14-3-3 in HEK293 cells. Moreover, the 14-3-3 antagonist peptide, R18 and HIP-55 protein mutant plasmid HIP-55AA were used to detect the protein synthesis of HIP-55 at different time points induced by puromycin, an inhibitor of protein production.
RESULTS
The HEK293 cells expressed HIP-55 protein respectively, after being transected with PDEST-N-Venus-HIP-55WT,PDEST-N-Venus-HIP-55AA,PDEST-GST-HIP-55WT plasmids and expressed 14-3-3 protein after being transected with PDEST-C-Venus-14-3-3τ plasmids. We could detect venus fluorescence of venus-HIP-55 protein via confocal microscopy in HEK 293 cells transfected with N-Venus-HIP-55 and C-14-3-3τ plasmids by BiFC, but not in HEK 293 cells transfected with N-Venus-HIP-55 AA (mutants S269A/T291A) and C-14-3-3τ plasmids. The results of BiFC suggested that 14-3-3 interacted with HIP-55 through HIP-55 S269/T291 sites. At the same time, the data of co-IP showed that there were endogenous interactions between 14-3-3 and HIP-55. Furthermore, puromycin had no influence in HIP-55 protein synthesis at hours 0, 4, or 8 in HEK 293 cells expressing GST-HIP-55WT and 14-3-3 plasmids, while puromycin blocked HIP-55 protein synthesis in HEK 293 cells transfected with N-Venus-HIP-55AA (mutants S269A/T291A) and C-14-3-3τ plasmids. The results indicated that the 14-3-3/HIP-55 complex could contributed to the stability of HIP-55.
CONCLUSION
HIP-55 forms a complex with 14-3-3 and 14-3-3/HIP-55 interaction increases the stability of HIP-55.
Topics: 14-3-3 Proteins; Blotting, Western; HEK293 Cells; Humans; Microfilament Proteins; Microscopy, Confocal; Microscopy, Fluorescence; Plasmids; Protein Biosynthesis; Protein Stability; Puromycin; Transfection; src Homology Domains
PubMed: 26679646
DOI: No ID Found -
Journal of Lipid Research May 1980Adult rats of both sexes were prepared with indwelling drainage catheters in the left thoracic lymphatic duct, and with duodenal infusion catheters. Control and...
Adult rats of both sexes were prepared with indwelling drainage catheters in the left thoracic lymphatic duct, and with duodenal infusion catheters. Control and puromycin-treated animals were administered an aqueous test emulsion containing [7alpha-(3)H]cholesterol and [1-(14)C]oleic acid, followed two hours later, by a tracer dose of [1-(14)C]-leucine. Successive 2-hr lymph samples were subjected to ultracentrifugal separations of the major lipoprotein classes. These were specifically extracted for lipids, and for DNA- and lipid-free protein. In both sexes, oleic acid absorption was largely associated with the d < 1.006 g/ml chylomicron fraction throughout the 6-hr experimental period. Small but consistent levels of labeled fatty acid appeared in the 1.006 < d < 1.019 g/ml VLDL fraction. However, with both sexes 25-35% of the absorbed cholesterol appearing in lymph was recovered in the VLDL fraction. Furthermore, there were statistically greater levels of cholesterol in this lymph fraction in females than in males. Cumulative protein levels and leucine incorporation into chylomicron proteins was comparable in both sexes. However, VLDL protein in the female was significantly greater than in the male and this difference was mimicked by the greater incorporation of leucine into VLDL proteins in the female. In males, there were no significant effects of puromycin on cholesterol or oleic acid absorption, despite a marked inhibition in chylomicron protein levels and leucine incorporation into this fraction. There was also no effect of the inhibitor on VLDL protein levels or on leucine incorporation into VLDL peptides. Cholesterol but not oleic acid absorption in females was significantly depressed by administration of puromycin, and this was largely attributed to a decrease in VLDL transport of the sterol. Also, unlike males, leucine incorporation into VLDL peptides was inhibited by 75% by puromycin administration. These results emphasize the importance of non-chylomicron transport of cholesterol during absorption and suggest a hormonal influence on intestinal VLDL synthesis in female rats.-Vahouny, G. V., E. M. Blendermann, L. L. Gallo, and C. R. Treadwell. Differential transport of cholesterol and oleic acid in lymph lipoproteins: sex differences in puromycin sensitivity.
Topics: Animals; Biological Transport; Cholesterol; Chylomicrons; Female; Leucine; Lipoproteins; Lipoproteins, VLDL; Lymph; Male; Oleic Acids; Puromycin; Rats; Sex Factors
PubMed: 7381333
DOI: No ID Found -
Nucleic Acids Research Jun 2004Polyribosome sedimentation velocity centrifugation can be used to identify differential regulation of the translation of mRNAs. However, ultracentrifugation presents...
Polyribosome sedimentation velocity centrifugation can be used to identify differential regulation of the translation of mRNAs. However, ultracentrifugation presents practical limitations on the number of sedimentation velocity gradients that can be run simultaneously. A method for sedimentation velocity analysis of polyribosomes is presented that is based on low-speed centrifugation of sucrose gradients prepared in deep 96-well plates, the advantage of which is that hundreds of polyribosome fractionations can be performed simultaneously in a tabletop centrifuge.
Topics: Cell Fractionation; Cell Line, Tumor; Centrifugation, Density Gradient; Gene Expression Regulation; Humans; Polyribosomes; Protein Biosynthesis; Puromycin; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Ultracentrifugation
PubMed: 15173352
DOI: 10.1093/nar/gnh077 -
Proceedings of the National Academy of... Mar 2018Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in...
Regulation of gene expression at the level of protein synthesis is a crucial element in driving how the genetic landscape is expressed. However, we are still limited in technologies that can quantitatively capture the immediate proteomic changes that allow cells to respond to specific stimuli. Here, we present a method to capture and identify nascent proteomes in situ across different cell types without disturbing normal growth conditions, using O-propargyl-puromycin (OPP). Cell-permeable OPP rapidly labels nascent elongating polypeptides, which are subsequently conjugated to biotin-azide, using click chemistry, and captured with streptavidin beads, followed by digestion and analysis, using liquid chromatography-tandem mass spectrometry. Our technique of OPP-mediated identification (OPP-ID) allows detection of widespread proteomic changes within a short 2-hour pulse of OPP. We illustrate our technique by recapitulating alterations of proteomic networks induced by a potent mammalian target of rapamycin inhibitor, MLN128. In addition, by employing OPP-ID, we identify more than 2,100 proteins and uncover distinct protein networks underlying early erythroid progenitor and differentiation states not amenable to alternative approaches such as amino acid analog labeling. We present OPP-ID as a method to quantitatively identify nascent proteomes across an array of biological contexts while preserving the subtleties directing signaling in the native cellular environment.
Topics: Cell Differentiation; Chromatography, Liquid; Drug Discovery; Humans; K562 Cells; Protein Biosynthesis; Proteome; Proteomics; Puromycin; Signal Transduction; TOR Serine-Threonine Kinases; Tandem Mass Spectrometry
PubMed: 29467287
DOI: 10.1073/pnas.1707514115 -
PloS One 2022Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is...
Transgenic proteins can be routinely expressed in various mammalian cell types via different transgenic systems, but the efficiency of transgene expression is constrained by the complex interplay among factors such as the temporal consistency of expression and compatibility with specific cell types, including ocular cells. Here, we report a more efficient way to express an enhanced green fluorescent protein (EGFP) in human corneal fibroblasts, corneal epithelial cells, and conjunctival epithelial cells through a lentiviral expression system. The relative transducing unit criterion for EGFP-expressing pseudovirions was first determined in HEK-293T cells. Homogeneous populations of EGFP-positive and EGFP-negative cells could be isolated by cell sorting. The half-maximal inhibitory concentration (IC50) value for puromycin was calculated according to viability curves for each cell type. The results revealed that cell types differed with respect to EGFP expression efficiency after transduction with the same amount of EGFP-encoding pseudovirions. Using a cell sorter, the homogeneity of EGFP-positive cells reached >95%. In the initial sorting stage, however, the efficiency of EGFP expression in the sorted cells was noticeably reduced after two rounds of sequential culture, but repeated sorting for up to four rounds yielded homogeneous EGFP-positive human corneal fibroblasts that could be maintained in continuous culture in vitro. The sorted EGFP-positive cells retained their proper morphology and cell type-specific protein expression patterns. Puromycin resistance was found to depend on cell type, indicating that the IC50 for puromycin must be determined for each cell type to ensure the isolation of homogeneous EGFP-positive cells. Taken together, repeated cell sorting is an efficient means of obtaining homogeneous populations of ocular cells expressing a transgenic protein during continuous culture without the potential confounding effects of antibiotics.
Topics: Animals; Animals, Genetically Modified; Cell Separation; Flow Cytometry; Green Fluorescent Proteins; Humans; Mammals; Puromycin; Transgenes
PubMed: 35333876
DOI: 10.1371/journal.pone.0265183 -
Analytical Chemistry Jul 2020Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid...
Protein synthesis is quickly and tightly regulated in cells to adapt to the ever-changing extracellular and intracellular environment. Accurate quantitation of rapid protein synthesis changes can provide insights into protein functions and cellular activities, but it is very challenging to achieve because of the lack of effective analysis methods. Here, we developed an effective mass spectrometry-based method named quantitative O-propargyl-puromycin tagging (QOT) by integrating O-propargyl-puromycin (OPP) labeling, bioorthogonal chemistry, and multiplexed proteomics for global and quantitative analysis of rapid protein synthesis. The current method enables us to accurately quantitate rapid changes of newly synthesized proteins because, unlike amino acids and their analogs, OPP can be utilized by the ribosome immediately without being activated and conjugated to tRNA, and thus cell starvation or pretreatment is not required. This method was applied to quantitate rapid changes of protein synthesis in THP-1 macrophages treated with lipopolysaccharide (LPS). For 15-min labeling, >3000 proteins were quantitated, and the synthesis of 238 proteins was significantly altered, including transcription factors and cytokines. The results demonstrated that protein synthesis was modulated to facilitate protein secretion in macrophages in response to LPS. Considering the importance of protein synthesis, this method can be extensively applied to investigate rapid changes of protein synthesis in the biological and biomedical research fields.
Topics: Cell Differentiation; Cells, Cultured; Humans; Lipopolysaccharides; Macrophages; Proteins; Proteomics; Puromycin; THP-1 Cells
PubMed: 32531160
DOI: 10.1021/acs.analchem.0c01823