Name: monitoring on-target signaling responses in larval zebrafish - z-rex unmasks precise mechanisms of electrophilic drugs and metabolites
Uploaded: 2023-06-02T14:55:00
Duration: 5 min 28 s
Description: Watch this Scientific Journal Video about Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites at JoVE.com

Funding: Novartis FreeNovation, NCCR, and EPFL.

<ol>
	<li>Long, M. J. C., Ly, P., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+primer+on+harnessing+non-enzymatic+post-translational+modifications+for+drug+design.">A primer on harnessing non-enzymatic post-translational modifications for drug design.</a> RSC Medicinal Chemistry. 12 (11), 1797-1807 (2021).</li><li>Parvez, S., Long, M. J. C., Poganik, J. R., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+signaling+by+reactive+electrophiles+and+oxidants.">Redox signaling by reactive electrophiles and oxidants.</a> Chemical Reviews. 118 (18), 8798-8888 (2018).</li><li>Parvez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-REX+on-demand+redox+targeting+in+live+cells.">T-REX on-demand redox targeting in live cells.</a> Nature Protocols. 11 (12), 2328-2356 (2016).</li><li>Long, M. J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+electrophile+tagging+in+Caenorhabditis+elegans.">Precision electrophile tagging in Caenorhabditis elegans.</a> Biochemistry. 57 (2), 216-220 (2018).</li><li>Van Hall-Beauvais, A., Zhao, Y., Urul, D. A., Long, M. J. C., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-protein-specific+redox+targeting+in+live+mammalian+cells+and+C.+elegans.">Single-protein-specific redox targeting in live mammalian cells and C. elegans.</a> Current Protocols in Chemical Biology. 10 (3), 43 (2018).</li><li>Long, M. J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Akt3+is+a+privileged+first+responder+in+isozyme-specific+electrophile+response.">Akt3 is a privileged first responder in isozyme-specific electrophile response.</a> Nature Chemical Biology. 13 (3), 333-338 (2017).</li><li>Poganik, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wdr1+and+cofilin+are+necessary+mediators+of+immune-cell-specific+apoptosis+triggered+by+Tecfidera.">Wdr1 and cofilin are necessary mediators of immune-cell-specific apoptosis triggered by Tecfidera.</a> Nature Communications. 12 (1), 5736 (2021).</li><li>Lin, H. -. Y., Haegele, J. A., Disare, M. T., Lin, Q., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+generalizable+platform+for+interrogating+target-+and+signal-specific+consequences+of+electrophilic+modifications+in+redox-dependent+cell+signaling.">A generalizable platform for interrogating target- and signal-specific consequences of electrophilic modifications in redox-dependent cell signaling.</a> Journal of the American Chemical Society. 137 (19), 6232-6244 (2015).</li><li>Schmittgen, T. D., Livak, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+real-time+PCR+data+by+the+comparative+CT+method.">Analyzing real-time PCR data by the comparative CT method.</a> Nature Protocols. 3 (6), 1101-1108 (2008).</li><li>Wakabayashi, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keap1-null+mutation+leads+to+postnatal+lethality+due+to+constitutive+Nrf2+activation.">Keap1-null mutation leads to postnatal lethality due to constitutive Nrf2 activation.</a> Nature Genetics. 35 (3), 238-245 (2003).</li><li>Huang, K. -. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Z-REX:+Shepherding+reactive+electrophiles+to+specific+proteins+expressed+either+tissue-specifically+or+ubiquitously,+and+recording+the+resultant+functional+electrophile-induced+redox+responses+in+larval+fish.">Z-REX: Shepherding reactive electrophiles to specific proteins expressed either tissue-specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish.</a> Nature Protocols. , (2023).</li><li>Long, M. J. C., Assari, M., Aye, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hiding+in+plain+sight:+The+issue+of+hidden+variables.">Hiding in plain sight: The issue of hidden variables.</a> ACS Chemical Biology. 17 (6), 1285-1292 (2022).</li></ol>

Isoform-specific small-molecule kinase inhibitors, the discovery of which were enabled by REX technologies, have been filed for patent application

Z-REX described in this protocol demonstrates a robust strategy for electrophile-target pair investigation and signaling pathway deconvolution in live fish. The proximity-directed delivery enables dosage and spatial control of the electrophilic compound treatment. Unlike conventional bolus dosing methods, in which the supraphysiological concentrations of electrophile deployed often lead to off-target issues, the relatively minor amount of electrophile released to the system renders Z-REX largely noninvasive. We have used 0.1-6 &#181;M Ht-PreHNE(alkyne) in zebrafish embryos, and the results showed the treatment is not detrimental to embryo development11.
The Z-REX procedure is generally longer than T-REX, a technique for screening/studying electrophile-sensing proteins in cultured cells. Suppose the experiment&#39;s purpose is to screen for electrophile-target interactions; we suggest first performing extensive screening by T-REX in cultured cells and using Z-REX for in vivo validation and phenotypic/pathway analysis. Compared to cell culture, requirements for performing Z-REX are basic fish husbandry techniques in addition to biochemical experimental skills required by T-REX. A typical timeframe for Z-REX (from fish crossing to light-inducible electrophile delivery) is 2-3 days, which is no more than 1 day longer than the typical time for a T-REX experiment on transfected live cells. Live imaging for phenotypic analysis can be performed 2-10 h after light illumination; click coupling with biotin-azide for pull-down assay takes 3 days; qRT-PCR for assaying transcriptional response takes 3 days; IF staining takes 5 days. These steps are roughly similar to their cell culture equivalents, although the interpretation of data requires an understanding of fish physiology and reporter strains.
As a multiple variable procedure12, several control groups are necessary for Z-REX to exclude uncertainties in the results (Figure 1D). Common control groups are: (1) DMSO/vehicle treatment only; (2) probe treatment, but without light illumination; (3) light illumination, but without probe treatment; (4) P2A-split construct, in which Halo and the POI are expressed separately, so the proximity delivery is ablated; and (5) hypomorphic mutants, whose electrophile-sensing residue(s) is/are mutated, such as Akt3 (C119S)6 and Keap1 (C151S, C273W, C288E)5, which we have used in previous studies.
If downstream assays involve western blot analysis, deyolking must be performed before harvest. The yolk proteins reduce the fidelity of lysate-concentration assessments and may bind non-specifically to antibodies. When performing live fish imaging or whole-mount IF staining, we have also observed non-specific fluorescent signals in the yolk sac, likely resulting from autofluorescent proteins in the yolk sac, or non-specific binding of the antibodies themselves. If the autofluorescence signal interferes with the signal, we suggest excluding the yolk sac from quantification, or quantifying different regions separately. Dechorination is necessary for live fish imaging and whole-mount IF staining assay. The chorion can interfere with imaging, and later with quantification/cell counting. However, dechorination is only applicable to embryos older than 1 dpf; younger embryos at blastulation/gastrulation/segmentation stages are too fragile to be dechorionated.
The Z-REX protocol described here is based on mRNA-driven ectopic POI expression. The procedure is rapid compared to using/generating transgenic fish lines. mRNA-driven expression is ubiquitous and transient, and lasts for at least 2 days for mRNAs used in this protocol. However, the duration of expression is likely to vary in other cases. Thus, this approach provides a fast, and more global investigative window into the effects of a specific electrophile-labeling event, compatible with several high-throughput/high-content assays. Transgenic lines with stable Halo-POI expression in specific tissues are also compatible with Z-REX11. Such lines are best used when a more precise question needs to be asked, for instance, when a phenotype in a specific organ is predicted from cell-culture data, or when screening from mRNA-injection experiments predicts that a specific organ is sensitive to an electrophile-labeling event. A heart-specific antioxidant response induction through Z-REX was demonstrated using Tg(gstp1:GFP;DsRed-P2A-myl7:Halo-TeV-Keap1) fish in our previous publication11. It may also be possible to perform Z-REX on transgenic fish older than 2 dpf.

A myriad of cellular signaling events involve reactions between small reactive molecules (endogenously produced in the cell or xenobiotics/xenometabolites, such as drugs) and their protein target. In many instances, a substoichiometric level of such covalent binding events can trigger cellular responses, leading to changes in, for instance, development, metabolism, apoptosis, and/or immune response1. However, deconstructing the mode of action (MOA) by linking specific binding events to their phenotypic consequences has proven challenging. Traditional bolus dosing methods that involve the introduction of high concentrations of the reactive species often result in a multitude of proteins being modified, as well as excessive toxicity to the model organism2. Such conditions are far from ideal. A method was developed to resolve these issues in cell culture using precision localized electrophile delivery in a native cellular context, named T-REX (targetable reactive electrophiles and oxidants)3. In the intervening years, the focus has turned to experiments in whole organisms, which allow the opportunity to study proteins in specific cellular contexts in non-transformed cells. Thus, we have extended the technique to be compatible with several models, including Danio rerio embryo models. Herein, we present Z-REX (zebrafish targeting reactive electrophiles and oxidants) (Figure 1).
To understand Z-REX, this article first presents REX technologies and their underlying concepts. At their core, these techniques model endogenous physiological reactive electrophilic species (RES) signaling by mimicking how natural electrophiles are locally produced in vivo with spatiotemporal precision. The protein of interest (POI) is expressed as a fusion construct to Halo; the latter anchors the tissue-permeable and inert probe bearing the photocaged RES in a 1:1 stoichiometry. One such endogenous RES is 4-hydroxynonenal (HNE hereafter), which is photocaged in the probe Ht-PreHNE. In many instances, we use an alkyne-functionalized version of HNE [i.e., HNE(alkyne)], which has essentially identical biological properties to HNE, but can be labeled by click chemistry. The probe, which is also functionalized with a chloroalkane for reactivity with Halo, is referred to as Ht-PreHNE(alkyne). The complex of the Halo-POI fusion and the probe thus formed allows proximal delivery of RES to the fused POI upon irradiation with UV light. If the POI reacts rapidly with the liberated RES, the resulting covalent labeling of the POI with RES allows us to identify kinetically-privileged cysteines.
Z-REX takes the aforementioned advantages of REX technologies and applies them broadly to study specific signaling pathways in live fish. This protocol has been optimized for zebrafish (D. rerio), since they are genetically-tractable vertebrate organisms that are transparent during development, and thus ideal for opto-chemical/-genetic techniques like REX technologies. Nevertheless, a similar strategy is also likely to work well on other genetically-tractable fish species, since the broad applicability of the method is due to the pseudo-intramolecular mechanism of lipid-derived electrophile (LDE) delivery. Indeed, the procedure is highly biocompatible, since fish can be treated with the Z-REX photocaged-electrophile [e.g., Ht-PreHNE(alkyne)] for at least 48 h without any noticeable impacts on development. A similar protocol functions in C. elegans4,5.
The protocol first describes the use of mRNA injection to produce a transient expression of a non-native Halo-POI fusion construct in embryonic zebrafish models, 1-1.5 days post-fertilization (dpf). This results in the expression of the ectopic protein in the majority of cells within the fish (hereafter referred to as &#39;ubiquitous&#39;), rather than in specific tissues or locales; however, the data shows that cell-specific effects can be observed in certain cases. Following injection, the embryos are incubated with a low concentration [0.3-5 &#181;M Ht-PreHNE(alkyne)] of the probe for up to 30.5 h post fertilization (hpf). Then, at a user-prescribed time, delivery of the RES to the POI within fish is achieved by photouncaging for 2-5 min. Following photouncaging of the RES, a variety of downstream phenotypic assays can be performed over the next 2-10 h: 1) live imaging of reporter lines (Figure 2A); 2) target-labeling assessment by western blot analysis (Figure 3); 3) transcriptomic analysis (Figure 4); or 4) whole-mount immunofluorescence (Figure 5).
As an example of live imaging of reporter lines, Z-REX is demonstrated in tandem with live imaging of fish lines, Tg(lyz:TagRFP) and Tg(mpeg1:eGFP), to measure how RES modification of a specific electrophile-sensor POI (namely Keap1) decreases neutrophil and macrophage levels, respectively, with no observable effects on other cells in the fish. However, we have shown previously that the POI-labeling and the consequential pathway signaling from T-REX studies can be reproduced using Z-REX for several proteins: Akt36, Keap17, and Ube2v26. Overall, with Z-REX, scientists can study the consequence(s) of covalent modification of POIs by RES in the context of several complex redox pathways. This technique is primed to pinpoint targets and their functional residues for covalent drug design and novel drug mechanisms in a more contextually-relevant whole animal model.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol (BME)</td>
			<td>Sigma-Aldrich</td>
			<td>M6250</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5-mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-150-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>10-cm Petri dishes</td>
			<td>Celltreat</td>
			<td>229692</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mL microcentrifuge tube</td>
			<td>Axygen</td>
			<td>MCT-200-C-S</td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide and bis-acrylamide solution</td>
			<td>BioRad</td>
			<td>1610154</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Celltreat</td>
			<td>229506</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Fisher</td>
			<td>A/0600/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>GoldBio</td>
			<td>A201-100</td>
			<td></td>
		</tr>
		<tr>
			<td>All Blue Prestained Protein Standards</td>
			<td>BioRad</td>
			<td>1610373</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotin-dPEG11-azide</td>
			<td>Quanta Biodesign</td>
			<td>102856-142</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Dye</td>
			<td>BioRad</td>
			<td>5000205</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Capillary tubes</td>
			<td>VWR</td>
			<td>HARV30-00200</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Supelco, Inc</td>
			<td>1.02445.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail</td>
			<td>Roche</td>
			<td>11836170001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cu(TBTA)</td>
			<td>Lumiprobe</td>
			<td>21050</td>
			<td></td>
		</tr>
		<tr>
			<td>CuSO4</td>
			<td>Sigma</td>
			<td>209198</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Fisher</td>
			<td>D128-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150107</td>
			<td>1:800 (IF)</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit-Alexa Fluor 647</td>
			<td>Abcam</td>
			<td>ab150075</td>
			<td>1:1000 (IF)</td>
		</tr>
		<tr>
			<td>Donkey anti-rat-AlexaFluor 568</td>
			<td>Abcam</td>
			<td>ab175475</td>
			<td>1:1000 (IF)</td>
		</tr>
		<tr>
			<td>ECL substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>32106</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL-Plus substrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>32132</td>
			<td></td>
		</tr>
		<tr>
			<td>End-to-end rotator</td>
			<td>FinePCR</td>
			<td>Rotator AG</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>E/0650DF/15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium bromide</td>
			<td>Sigma</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescence stereomicroscope</td>
			<td>Leica</td>
			<td>M165 FC</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps (blunt)</td>
			<td>self made</td>
			<td></td>
			<td>self made by blunting sharp forceps (Fine Science Tools, Dumont #5, 11252-40)</td>
		</tr>
		<tr>
			<td>Forceps (sharp)</td>
			<td>Fine Science Tools</td>
			<td>Dumont #5, 11252-40</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel loading tip</td>
			<td>Fisher</td>
			<td>02-707-181</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel/blot imager</td>
			<td>Vilber</td>
			<td>Fusion FX imager</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass beads</td>
			<td>Sigma</td>
			<td>45-G1145</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass stage micrometer</td>
			<td>Meiji Techno.</td>
			<td>MA285</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat inactivated FBS</td>
			<td>Sigma</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>Heated ultrasonic bath</td>
			<td>VWR</td>
			<td>89375-470</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>High capacity streptavidin agarose</td>
			<td>Thermo Fisher Scientific</td>
			<td>20359</td>
			<td></td>
		</tr>
		<tr>
			<td>Ht-PreHNE alkyne probe</td>
			<td>self-made</td>
			<td>-</td>
			<td>Parvez, S. et al. T-REX on-demand redox targeting in live cells. Nat Protoc. 11 (12), 2328-2356, (2016).</td>
		</tr>
		<tr>
			<td>Imaging plate (10% HBSS buffer, for live embryos)</td>
			<td></td>
			<td></td>
			<td>Made with Petri dish, and 2% agarose in 10% HBSS buffer</td>
		</tr>
		<tr>
			<td>Imaging plate (PBS, for formaldehyde-fixed embryos)</td>
			<td></td>
			<td></td>
			<td>Made with Petri dish, and 2% agarose in PBS</td>
		</tr>
		<tr>
			<td>Injection plate</td>
			<td></td>
			<td></td>
			<td>Made with microinjection mold, Petri dish, and 2% agarose in 10% HBSS buffer</td>
		</tr>
		<tr>
			<td>LDS</td>
			<td>Apollo</td>
			<td>APOBI3331</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>VWR</td>
			<td>20864.32</td>
			<td></td>
		</tr>
		<tr>
			<td>Microinjection mold</td>
			<td>Adaptive Science Tools</td>
			<td>TU-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloader tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>Narishige</td>
			<td>MN-153</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope for micro-injection</td>
			<td>Olympus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope light source</td>
			<td>Olympus</td>
			<td>&#160;KL 1600 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M3516</td>
			<td></td>
		</tr>
		<tr>
			<td>mMessage mMachine SP6 Transcription Kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti- &#946;-actin-HRP</td>
			<td>Sigma</td>
			<td>A3854</td>
			<td>1:20000 (WB)</td>
		</tr>
		<tr>
			<td>Mouse anti-HaloTag</td>
			<td>Promega</td>
			<td>G921A</td>
			<td>1:500 (IF)</td>
		</tr>
		<tr>
			<td>Multi-mode reader</td>
			<td>BioTek Instruments</td>
			<td>Cytation 5</td>
			<td></td>
		</tr>
		<tr>
			<td>Nucleic acid agarose gel electrophoresis apparatus</td>
			<td>Biorad</td>
			<td>Mini-Sub Cell GT Systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo(dT)20</td>
			<td>Integrated DNA Technologies</td>
			<td>customized: (dT)20</td>
			<td></td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>Sigma</td>
			<td>O3756</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>pCS2+8 Halo-2XHA-P2A-TeV-Keap1-2xHA&#160;</td>
			<td>self-cloned</td>
			<td>-</td>
			<td>Available from Prof. Yimon AYE&#39;s group at EPFL</td>
		</tr>
		<tr>
			<td>pCS2+8 Halo-TeV-Keap1-2xHA</td>
			<td>self-cloned</td>
			<td>-</td>
			<td>Available from Prof. Yimon AYE&#39;s group at EPFL</td>
		</tr>
		<tr>
			<td>Pneumatic PicoPump</td>
			<td>WPI</td>
			<td>SYS-PV820</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein electrophoresis equipment and supplies</td>
			<td>Biorad</td>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-active Caspase-3</td>
			<td>BD Pharmingen&#160;</td>
			<td>559565</td>
			<td>1:800 (IF)</td>
		</tr>
		<tr>
			<td>Rat anti-HA-HRP</td>
			<td>Sigma</td>
			<td>H3663</td>
			<td>1:500 (IF and WB)</td>
		</tr>
		<tr>
			<td>Rat anti-RFP</td>
			<td>ChromoTek</td>
			<td>5F8</td>
			<td>1:800 (IF)</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>Eppendorf</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAseOUT recombinant ribonuclease inhibitor</td>
			<td>ThermoFisher Scientific</td>
			<td>10777019</td>
			<td></td>
		</tr>
		<tr>
			<td>RnaseZap RNA decontamination solution</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9780</td>
			<td></td>
		</tr>
		<tr>
			<td>Rocking Shaker</td>
			<td>DLAB</td>
			<td>SK-R1807-S</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Teknova</td>
			<td>S9974</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript III reverse transcriptase</td>
			<td>ThermoFisher Scientific</td>
			<td>18080085</td>
			<td></td>
		</tr>
		<tr>
			<td>t-Butanol</td>
			<td>Sigma</td>
			<td>471712</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Gold Biotechnology</td>
			<td>TCEP1</td>
			<td></td>
		</tr>
		<tr>
			<td>TeV protease (S219V)</td>
			<td>self-made</td>
			<td>-</td>
			<td>Parvez, S. et al. T-REX on-demand redox targeting in live cells. Nat Protoc. 11 (12), 2328-2356, (2016).</td>
		</tr>
		<tr>
			<td>Tg(lyz:TagRFP)</td>
			<td>Zebrafish International Resource Center (ZIRC)</td>
			<td>uwm11Tg (ZFIN)</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tg(mpeg1:eGFP)</td>
			<td>Zebrafish International Resource Center (ZIRC)</td>
			<td>gl22Tg (ZFIN)</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Thermal cycler</td>
			<td>Analytik Jana</td>
			<td>846-x-070-280</td>
			<td></td>
		</tr>
		<tr>
			<td>TMEDA</td>
			<td>Sigma</td>
			<td>T7024</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Fisher</td>
			<td>13-711-9D</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Apollo</td>
			<td>APOBI2888</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
			<td>Sigma</td>
			<td>T9003</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher</td>
			<td>BP337-500</td>
			<td></td>
		</tr>
		<tr>
			<td>UV lamp with 365-nm light</td>
			<td>Spectroline</td>
			<td>ENF 240C</td>
			<td></td>
		</tr>
		<tr>
			<td>UV meter&#160;</td>
			<td>Spectroline</td>
			<td>XS-365</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortexer</td>
			<td>Scientific Industries, Inc.</td>
			<td>Vortex-Genie 2</td>
			<td></td>
		</tr>
		<tr>
			<td>Western Blotting Transfer equipment and supplies</td>
			<td>Biorad</td>
			<td>Mini Trans-Blot or Criterion Blotter&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Zebrafish husbandry and breeding equipment</td>
			<td></td>
			<td></td>
			<td>in house</td>
		</tr>
		<tr>
			<td>Zirconia beads</td>
			<td>BioSpec</td>
			<td>11079107zx</td>
			<td></td>
		</tr>
	</tbody>
</table>

monitoring on-target signaling responses in larval zebrafish - z-rex unmasks precise mechanisms of electrophilic drugs and metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Live imaging of Z-REX-treated transgenic neutrophil/macrophage reporter fish, Tg(lyz:TagRFP) and Tg(mpeg1:EGFP). Induction of neutrophil/macrophage apoptosis through Keap1 HNEylation. (See also Figure 2). The effect of electrophile labeling of Keap1 on neutrophil and macrophage levels was assessed by injecting heterozygous transgenic embryos derived from Tg(lyz:TagRFP) or Tg(mpeg1:EGFP) with mRNA encoding Halo-Keap1, and then treating with Ht-PreHNE(alkyne). Following the procedures for step 6.1-downstream assay Option 1-HNE(alkyne) was liberated and Keap1 was labeled. Neutrophil and macrophage levels were assessed by live imaging of reporter lines, Tg(lyz:TagRFP) and Tg(mpeg1:eGFP), respectively. The level of both cell types decreased by 30%-40% after Z-REX treatment, in which HNE was delivered to Keap1. On the contrary, no loss of neutrophils or macrophages was seen in Z-REX technical control groups [without light and Ht-PreHNE(alkyne), light alone, or Ht-PreHNE(alkyne) alone] (Figure 1D and Figure 2A-D).
The induction of neutrophil/macrophage apoptosis indicated successful HNE delivery to Keap1 through Z-REX. Details for the pathway analysis and apoptosis mechanism have been published5. To account for off-target effects of HNE(alkyne), several controls were used. (1) Under the same experimental conditions, instead of Halo-TeV-Keap1 mRNA, embryos were injected with Halo-P2A-Keap1 mRNA. P2A linker allowed the Halo and Keap1 proteins to be expressed independently. In this scenario, HNE(alkyne) released from Halo could not label Keap1, as it was no longer proximal to Halo (Figure 1D); hence, the apoptosis signaling pathway was not triggered. No changes in macrophage or neutrophil levels were observed in this group (Figure 2A,B). (2) The same experimental conditions were performed using mRNA encoding Halo-TeV-Keap1(C151S,C273W,C288E), a mutant of Keap1 that does not respond to HNE(alkyne) (Figure 1D). No changes in macrophage or neutrophil levels were observed (Figure 2G,H).
Biotin azide-click coupling and biotin pull-down assay. Target-labeling assessment. (See also Figure 3). The target-labeling assessment was carried out using WT embryos, injected with mRNA encoding either Halo-TeV-Keap1-2xHA (Halo-POI fusion construct) or Halo-2xHA-P2A-Keap1-2xHA (P2A-split construct, in which Halo and Keap1 are not fused; Figure 1D). Labeled Keap1 protein was only pulled down in the group expressing fusion protein and treated with Z-REX (second lane in the top anti-HA blot), but not in other control groups (no mRNA injection, fusion construct without Z-REX, or P2A-split construct). The results indicate the HNE(alkyne) was successfully delivered to Keap1, and the modified Keap1 was subsequently conjugated with biotin through click reaction, and the biotin-labeled Keap1 was pulled down by streptavidin resin.
Transcriptional analysis. RNA-seq and qRT-PCR. (See also Figure 4). The transcriptional change after Z-REX treatment was assessed by RNA-seq and qRT-PCR. In RNA-seq, several immune-related genes were downregulated after Z-REX. In contrast, many antioxidant response(AR)-related genes were upregulated after Z-REX, which resulted from the induction of the Keap1-Nrf2-AR pathway upon HNEylation on&#160;Keap110 (Figure 4A). In qRT-PCR analysis, similar results were found when analyzing three immune-related genes (lyz, mpeg1.1, and coro1a) (Figure 4B). The up- and down-regulation of the respective genes showed the successful induction of pathways mediated by Keap1 HNEylation.
Whole-mount (co-)immunofluorescence staining assay and colocalization analysis. (See also Figure 5). The exogenous Halo-TeV-Keap1-2xHA and Halo-2xHA-P2A-Keap1-2xHA expression were assessed by whole-mount immunofluorescence (IF) staining (Figure 5A,B). The P2A-split-construct had two times the number of HA tags than the TeV-fusion-construct, which corresponds to a twofold higher anti-HA signal in the P2A-split-construct-mRNA-injected group than the other, indicating the expression level of the two constructs were similar (Figure 5C). The expression levels of the Halo-TeV-Keap1 (wt) and Halo-TeV-Keap1(C151S,C273W,C288E) were also found similar when probing with anti-Halo (Figure&#160;5D,E). Colocalization of neutrophils and active caspase 3 in Z-REX-treated Tg(lyz:TagRFP) was observed by co-immunostaining with anti-RFP and anti-active-Caspase 3 (Figure 5F). Active Caspase 3 is an indicator of apoptosis events.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64846/64846fig01.jpg" /> 
Figure 1: Z-REX workflow. (A,B) A 1-4 cell stage zebrafish embryo is injected with (morpholino and) mRNA encoding Halo-POI (e.g., Halo-Keap1). Injected embryos are then treated with a probe composed of a HaloTag ligand and a photocaged electrophile appended with an alkyne functional group, such as Ht-PreHNE(alkyne) in B. After removing the excess amount of probe, the embryo is exposed to light to release the electrophile of interest [e.g., HNE or its analog, HNE(alkyne)]. The downstream analysis is performed at a given/user-defined timepoint. (C) Design and mechanism of the Ht-PreLDE probe, which is applicable to different lipid-derived electrophiles (LDE). (D) Negative/technical control groups for Z-REX. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64846/64846fig02.jpg" /> 
Figure 2: Live imaging of transgenic neutrophil/macrophage reporter fish subjected to Z-REX. Z-REX-mediated Keap1 HNEylation induces neutrophil/macrophage apoptosis. (A) Representative images of Tg(lyz:TagRFP) fish expressing either Halo-TeV-Keap1 (fusion construct) or Halo-P2A-Keap1 (split construct), and subjected to negative control conditions [no treatment, light alone, or Ht-PreHNE(alkyne) alone or Z-REX]. Embryo age: 36 hpf. (B) Quantitation of neutrophil levels in A. (C) Representative images of Tg(mpeg1:eGFP) fish expressing Halo-TeV-Keap1 with or without Z-REX treatment. Embryo age: 34 hpf. (D) Quantitation of macrophage levels in C. (E,F) Time-course measurement of (E) neutrophil and (F) macrophage levels after Z-REX treatment. (G) Similar experiment as in A in fish expressing either Halo-TeV-Keap1 (WT) or Halo-TeV-Keap1 (C151S, C273W, C288E), a mutant that does not have HNE-sensing capability. (H) Quantitation of neutrophil levels in G. Scale bars: 500 &#181;m. All the graphs are presented with mean &#177; SEM. p values were calculated with one-way ANOVA (blue) and two-tailed Student&#39;s t-test (black). This figure has been modified from Poganik et al.7. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64846/64846fig03.jpg" /> 
Figure 3: Biotin pull-down assay. WT embryos expressing Halo-TeV-Keap1-2XHA or Halo-2XHA-P2A-Keap1-2XHA were treated with Z-REX or respective negative control conditions (no probe treatment in this case). After harvest, embryos were lysed and treated with TeV protease before the biotin pull-down assay. The results were analyzed by western blotting. This figure has been modified from Huang et al. Z-REX: shepherding reactive electrophiles to specific proteins expressed either tissue-specifically or ubiquitously, and recording the resultant functional electrophile-induced redox responses in larval fish. This figure has been modified from Huang et al.11. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64846/64846fig04.jpg" /> 
Figure 4: Transcriptional analysis. (A) RNA-seq results of Z-REX-treated versus non-treated embryos. Statistically significant differentially-expressed (SDE) genes are highlighted. Immunity-related SDE genes are colored red. Antioxidant response (AR)-related genes are colored green. Other SDE genes are colored blue. All p values were calculated with CuffDiff. (B-D) Three immunity-related SDE genes from A: (B) lyz, (C) mpeg1.1, and (D) coro1a were further analyzed with qRT-PCR, and only the Z-REX-treated embryos showed the suppression of these transcripts. All the graphs are presented with mean &#177; SEM. p values were calculated with one-way ANOVA (blue) and two-tailed Student&#39;s t-test (black). This figure has been modified from Poganik et al.7. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/64846/64846fig05.jpg" /> 
Figure 5: Whole-mount immunofluorescence staining assay. (A,B) Representative images of embryos expressing either (A) Halo-TeV-Keap1-2xHA or (B) Halo-2xHA-P2A-Keap1-2xHA immunostained with anti-HA and secondary antibody conjugated with AlexaFluor568. mRNA-injected fish were compared to age-matched non-injected fish. (C) Quantification of anti-HA signal in (A,B). (D) Representative images of embryos expressing Halo-TeV-Keap1 (WT) or Halo-TeV-Keap1 (C151S, C273W, C288E) immunostained with anti-Halo and secondary antibody conjugated with AlexaFluor647. mRNA-injected fish were compared to age-matched non-injected fish. (E) Quantification of anti-Halo signal in D. p values were calculated with two-tailed Student&#39;s t-test. (F) Tg(lyz:TagRFP) embryos subjected to Z-REX were co-immunostained with anti-RFP and anti-active Caspase 3, and respective fluorophore-conjugated secondary antibodies. The white box marks the magnified area. White arrows indicate colocalizations of neutrophils and active Caspase 3. Scale bars: 500 &#181;m. All the graphs are presented with mean &#177; SEM. This figure has been modified from Poganik et al.7. and Huang et al.11. <a href="https://www.jove.com/files/ftp_upload/64846/64846fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Table 1: List of buffers used in this study. <a href="https://www.jove.com/files/ftp_upload/64846/Supplementary Table 1.xlsx" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites at JoVE.com

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

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