Z-REX: shepherding reactive electrophiles to specific proteins expressed tissue specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.

Details

Serval ID
serval:BIB_FE6B92D5DC45
Type
Article: article from journal or magazin.
Publication sub-type
Review (review): journal as complete as possible of one specific subject, written based on exhaustive analyses from published work.
Collection
Publications
Institution
Title
Z-REX: shepherding reactive electrophiles to specific proteins expressed tissue specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.
Journal
Nature protocols
Author(s)
Huang K.T., Poganik J.R., Parvez S., Raja S., Miller B., Long MJC, Fetcho J.R., Aye Y.
ISSN
1750-2799 (Electronic)
ISSN-L
1750-2799
Publication state
Published
Issued date
05/2023
Peer-reviewed
Oui
Volume
18
Number
5
Pages
1379-1415
Language
english
Notes
Publication types: Journal Article ; Review ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
Publication Status: ppublish
Abstract
This Protocol Extension describes the adaptation of an existing Protocol detailing the use of targetable reactive electrophiles and oxidants, an on-demand redox targeting toolset in cultured cells. The adaptation described here is for use of reactive electrophiles and oxidants technologies in live zebrafish embryos (Z-REX). Zebrafish embryos expressing a Halo-tagged protein of interest (POI)-either ubiquitously or tissue specifically-are treated with a HaloTag-specific small-molecule probe housing a photocaged reactive electrophile (either natural electrophiles or synthetic electrophilic drug-like fragments). The reactive electrophile is then photouncaged at a user-defined time, enabling proximity-assisted electrophile-modification of the POI. Functional and phenotypic ramifications of POI-specific modification can then be monitored, by coupling to standard downstream assays, such as click chemistry-based POI-labeling and target-occupancy quantification; immunofluorescence or live imaging; RNA-sequencing and real-time quantitative polymerase chain reaction analyses of downstream-transcript modulations. Transient expression of requisite Halo-POI in zebrafish embryos is achieved by messenger RNA injection. Procedures associated with generation of transgenic zebrafish expressing a tissue-specific Halo-POI are also described. The Z-REX experiments can be completed in <1 week using standard techniques. To successfully execute Z-REX, researchers should have basic skills in fish husbandry, imaging and pathway analysis. Experience with protein or proteome manipulation is useful. This Protocol Extension is aimed at helping chemical biologists study precision redox events in a model organism and fish biologists perform redox chemical biology.
Keywords
Animals, Zebrafish/metabolism, Larva/metabolism, Proteins/metabolism, Oxidation-Reduction, Oxidants/metabolism
Pubmed
Web of science
Create date
11/04/2023 15:55
Last modification date
09/12/2023 7:02
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