1558055-04-1Relevant articles and documents
A New Photocaged Puromycin for an Efficient Labeling of Newly Translated Proteins in Living Neurons
Elamri, Isam,Heumüller, Maximilian,Herzig, Lisa-M.,Stirnal, Elke,Wachtveitl, Josef,Schuman, Erin M.,Schwalbe, Harald
, p. 2458 - 2464 (2018)
Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7-N,N-(diethylaminocumarin-4-yl)-methoxycarbonyl-puromycin (DEACM-puromycin) was synthesized and compared with the previously developed 6-nitroveratryloxycarbonyl puromycin (NVOC-puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two-photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light-controlled puromycylation to visualize new translated proteins in neurons.
Design of photocaged puromycin for nascent polypeptide release and spatiotemporal monitoring of translation
Buhr, Florian,Kohl-Landgraf, J?rg,Tomdieck, Susanne,Hanus, Cyril,Chatterjee, Deep,Hegelein, Andreas,Schuman, Erin M.,Wachtveitl, Josef,Schwalbe, Harald
supporting information, p. 3717 - 3721 (2015/03/18)
The antibiotic puromycin, which inhibits protein translation, is used in a broad range of biochemical applications. The synthesis, characterization, and biological applications of NVOC-puromycin, a photocaged derivative that is activated by UV illumination, are presented. The caged compound had no effect either on prokaryotic or eukaryotic translation or on the viability of HEK 293 cells. Furthermore, no significant release of ribosome-bound polypeptide chains was detected invitro. Upon illumination, cytotoxic activity, invitro translation inhibition, and polypeptide release triggered by the uncaging of NVOC-puromycin were equivalent to those of the commercial compound. The quantum yield of photolysis was determined to be 1.1±0.2 % and the NVOC-puromycin was applied to the detection of newly translated proteins with remarkable spatiotemporal resolution by using two-photon laser excitation, puromycin immunohistochemistry, and imaging in rat hippocampal neurons. On like a light: The antibiotic puromycin (green) is a translation inhibitor that triggers the release of the nascent polypeptide chain (red) from the ribosome (yellow) and it is used in a number of applications. A photocaged puromycin derivative, NVOC-puromycin, was synthesized and characterized. Both functional recovery upon UV illumination and biological inactivity invitro and invivo were demonstrated.
