Z-REX uncovers a bifurcation in function of Keap1 paralogs

Studying electrophile signaling is marred by difficulties in parsing changes in pathway flux attributable to on-target, vis-à-vis off-target, modifications. By combining bolus dosing, knockdown, and Z-REX—a tool investigating on-target/on-pathway electrophile signaling, we document that electrophile labeling of one zebrafish-Keap1-paralog (zKeap1b) stimulates Nrf2- driven antioxidant response (AR) signaling (like the human-ortholog). Conversely, zKeap1a is a dominant-negative regulator of electrophile-promoted Nrf2-signaling, and itself is nonpermissive for electrophile-induced Nrf2-upregulation. This behavior is recapitulated in human cells: (1) zKeap1b-expressing cells are permissive for augmented AR-signaling through reduced zKeap1b–Nrf2 binding following whole-cell electrophile treatment; (2) zKeap1a-expressing cells are non-permissive for AR-upregulation, as zKeap1a–Nrf2 binding capacity remains unaltered upon whole-cell electrophile exposure; (3) 1:1 ZKeap1a:zKeap1b-co-expressing cells show no Nrf2-release from the Keap1-complex following whole-cell electrophile administration, rendering these cells unable to upregulate AR. We identified a zKeap1a-specific point-mutation (C273I) responsible for zKeap1a’s behavior during electrophilic stress. Human-Keap1(C273I), of known diminished Nrf2-regulatory capacity, dominantly muted electrophile-induced Nrf2-signaling. These studies highlight divergent and interdependent electrophile signaling behaviors, despite conserved electrophile sensing.

For all that apply, please note where in the article the information is provided. Please note that we also collect information about data availability and ethics in the submission form.

Materials:
Newly created materials Indicate where provided: section/figure legend N/A The manuscript includes a dedicated "materials availability statement" providing transparent disclosure about availability of newly created materials including details on how materials can be accessed and describing any restrictions on access.

Randomisation
As indicated in Statistical Analysis & Reporting section (Main Manuscript, page 26), for zebrafish experiments, all injected embryos for a given injection condition were pooled and mixed prior to being divided equally into experimental groups without bias. Cellbased experiments were set up with cells derived from a common stock and divided equally without bias. Treatment groups for all experiments were assigned randomly.

Blinding
Blinding was not used in this study, consistent with widespread practice in the field for studies of this nature. Nevertheless, we ensured rigor by having multiple co-authors reproduce the discovered phenotypes independently.

Inclusion/exclusion criteria
No data were excluded.
Sample definition and in-laboratory replication Indicate where provided: section/figure legend N/A State number of times the experiment was replicated in the laboratory.
Indicated in corresponding figure legends. Briefly, all experiments were conducted with sufficient biological replicates to ensure rigor. Generally, the pathways / mode-of-mechanisms we identified were confirmed with orthogonal approaches (e.g., genetic knockdown/knockout and pharmacological modulators of key proteins), and pathway functionality was confirmed/replicated in multiple model systems (zebrafish and cultured mammalian cells). Additionally, identified phenotypes were confirmed in multiple zebrafish strains. We list the number of independent biological vs. technical replicates for each experiment in the corresponding figure legends Define whether data describe technical or biological replicates. Data availability Indicate where provided: section/submission form N/A For newly created and reused datasets, the manuscript includes a data availability statement that provides details for access (or notes restrictions on access).

Page 38, Main Manuscript
When newly created datasets are publicly available, provide accession number in repository OR DOI and licensing details where available.

Non-applicable
If reused data is publicly available provide accession number in repository OR DOI, OR URL, OR citation.

Non-applicable
Code availability Indicate where provided: section/figure legend N/A For any computer code/software/mathematical algorithms essential for replicating the main findings of the study, whether newly generated or re-used, the manuscript includes a data availability statement that provides details for access or notes restrictions.

Non-applicable
Where newly generated code is publicly available, provide accession number in repository, OR DOI OR URL and licensing details where available. State any restrictions on code availability or accessibility.

Non-applicable
If reused code is publicly available provide accession number in repository OR DOI OR URL, OR citation.

Non-applicable
Reporting: The MDAR framework recommends adoption of discipline-specific guidelines, established and endorsed through community initiatives.
Adherence to community standards Indicate where provided: section/figure legend N/A State if relevant guidelines (e.g., ICMJE, MIBBI, ARRIVE, STRANGE) have been followed, and whether a checklist (e.g., CONSORT, PRISMA, ARRIVE) is provided with the manuscript.
As described above and also briefly stated in the 'Statistical analysis & reporting section' in Main Manuscript (Page 26), our reporting protocol of experimental procedures, data analyses and interpretations are consistent with the 'ARRIVE essential 10' guidelines (as outlined in, for instance, Percie du Sert et al., 2020 PLOS Biology) * We provide the following guidance regarding transparent reporting and statistics; we also refer authors to Ten common statistical mistakes to watch out for when writing or reviewing a manuscript.

Sample-size estimation
• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use

Replicates
• You should report how often each experiment was performed • You should include a definition of biological versus technical replication • The data obtained should be provided and sufficient information should be provided to indicate the number of independent biological and/or technical replicates • If you encountered any outliers, you should describe how these were handled • Criteria for exclusion/inclusion of data should be clearly stated • High-throughput sequence data should be uploaded before submission, with a private link for reviewers provided (these are available from both GEO and ArrayExpress) Figure legends contain information pertaining to the following: • the exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement; • --about whether measurements were taken from distinct samples or whether the same sample was measured repeatedly; • --null hypothesis testing (two-tailed Students' t-test) with exact P values noted whenever suitable. • No statistical methods were used to pre-determine sample size. Size of datasets was chosen according to literature and based on our own experience, integrating similar methods of analysis. Number of technical replicates and biological replicates are reported in figure legends.

Statistical reporting
• Statistical analysis methods should be described and justified • Raw data should be presented in figures whenever informative to do so (typically when N per group is less than 10) • For each experiment, you should identify the statistical tests used, exact values of N, definitions of center, methods of multiple test correction, and dispersion and precision measures (e.g., mean, median, SD, SEM, confidence intervals; and, for the major substantive results, a measure of effect size (e.g., Pearson's r, Cohen's d) • Report exact p-values wherever possible alongside the summary statistics and 95% confidence intervals. These should be reported for all key questions and not only when the p-value is less than 0.05.

Group allocation
• Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis • Outliers were maintained in all data sets with error bars designating SEM and P-values from application of two-tailed Students' t-test included.
• Wherever applicable, figure legends contain information pertaining to SEM with associated P values, sample size (e.g., number of fish embryos analyzed, number of independent biological replicates). Representative raw images are included with accompanying quantitation where relevant.
• Summary information related to sample allocation/handling is detailed in the supplementary text.
• Briefly, no masking was used. Prior to beginning each experiment, cells/embryos were allocated into groups randomly, for each sample group. When an experiment was commenced, groups of cells/embryos were allocated into treatment groups without pattern or bias. This ensured that each treatment group in an experiment was identical to account for any variation across cells/fish breeding. Cell counting was performed at each step of the experiment whenever relevant, to rigorously standardize conditions both within each experiment and across different experiments. All procedures related to zebrafish studies conform to the respective NIH and European guidelines regarding animal experimentation and were approved by the corresponding Institutional Animal Care and Use committees at EPFL (Switzerland) and Cornell University (USA) (the authors' former institution where the initial work on this manuscript was performed). Casper strain zebrafish, wild-type zebrafish, and previously-validated reporter strains were used for the experiments involving embryos and for the latter transgenic reporter strains, at consistent zygosity. As zebrafish are believed to exhibit polygenic sex determination, at the age at which experiments were undertaken, the sex of the fish was unable to be determined, but likely account for 50:50 male: female.