| Autor: |
Evans HT; Center for Neural Science, New York University, New York, New York 10003, United States., Ko T; Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States., Oliveira MM; Center for Neural Science, New York University, New York, New York 10003, United States., Yu A; Center for Neural Science, New York University, New York, New York 10003, United States., Kalavai SV; Center for Neural Science, New York University, New York, New York 10003, United States., Golhan EN; Center for Neural Science, New York University, New York, New York 10003, United States., Polavarapu A; Center for Neural Science, New York University, New York, New York 10003, United States., Balamoti E; Center for Neural Science, New York University, New York, New York 10003, United States., Wu V; Center for Neural Science, New York University, New York, New York 10003, United States., Klann E; Center for Neural Science, New York University, New York, New York 10003, United States., Trauner D; Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. |
| Abstrakt: |
Elucidating the mechanisms by which protein synthesis contributes to complex biological processes has remained a challenging endeavor. This is particularly true in the field of neuroscience, where multiple, tightly regulated periods of new protein synthesis in different cell-types are thought to facilitate intricate neurological functions, such as memory formation. Current methods for labeling the de novo proteome have lacked the spatial and temporal resolution to accurately discriminate these overlapping and often competing windows of mRNA translation. To address this technological limitation, here we describe a novel, light-inducible specific method for labeling newly synthesized proteins within a targeted cell-type.By developing Opto-ANL, a photocaged version of the nonendogenous amino acid azidonorleucine (ANL), we can selectively label newly synthesized proteins in specific cell-types through the targeted expression of a mutant methionyl-tRNA synthetase (L274G-MetRS). We demonstrate that Opto-ANL can be rapidly uncaged by UV light treatment in both cell culture and mouse brain slices, with Opto-ANL labeled proteins being able to be visualized via fluorescent noncanonical amino acid tagging. We also reveal that pretreatment with Opto-ANL not only allows for the period of de novo proteomic labeling to be tightly controlled, but also improves labeling efficiency compared to regular ANL. To demonstrate the potential applications of this novel technique, we use Opto-ANL to detect insulin-induced increases in protein synthesis and to label the excitatory neuronal de novo proteome in mouse brain slices. We believe that this application of photopharmacology will allow researchers to generate novel insights into how the translational landscape is altered across cell-types during complex neurological phenomena such as memory formation. |
