Precision Electrophile Tagging in Caenorhabditis elegans

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Precision electrophile tagging in C. elegans Marcus J. C. Long, Daniel A Urul, Shivansh Chawla, HongYu Lin, Yi Zhao, Joseph A Haegele, Yiran Wang, and Yimon Aye Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b00642 • Publication Date (Web): 31 Aug 2017 Downloaded from http://pubs.acs.org on September 5, 2017

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Biochemistry

Precision electrophile tagging in C. elegans Marcus J. C. Long,‡,¶ Daniel A. Urul,‡ ,¶ Shivansh Chawla,‡ ,¶ Hong-Yu Lin,‡,† Yi Zhao,‡,† Joseph A. Haegele,‡ Yiran Wang,‡ and Yimon Aye*,‡,§ ‡ §

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853, United States Department of Biochemistry, Weill Cornell Medicine, New York, New York 10065, United States

Supporting Information ¶

Co-lead authors, †Equal contributors

ABSTRACT: Electrophile adduction to privileged sensor

proteins and the resulting phenotypically-dominant responses are increasingly appreciated to be essential for metazoan health. Functional similarities between the biological electrophiles and electrophilic pharmacophores commonly found in covalent drugs further fortify the translational relevance of these small-molecule sentinels. Genetically-encodable or small-molecule-based fluorescent reporters along with redox-proteomics have revolutionized the observation and profiling of cellular redox states and electrophile-sensor proteins, respectively. However, precision mapping between specific redoxmodified targets and specific responses has only recently begun to be addressed and systems tractable to both genetic manipulation and on-target redox signaling in vivo remain largely limited. Here we engineer transgenic C. elegans expressing functional HaloTagged fusion proteins and use this system to develop a generalizable light-controlled approach to tag a prototypical electrophile-sensor protein with native electrophiles in vivo. The method circumvents issues associated with low uptake/distribution and toxicity/promiscuity. Given the validated success of C. elegans in aging studies, this optimized platform offers a new lens with which to scrutinize how on-target electrophile signaling influences redox-dependent lifespan regulation.

The purpose of this communication is to delineate a protocol to ultimately study the consequences of ontarget electrophile signaling in C. elegans. We describe several C. elegans lines amenable to this procedure, and show that single-protein targeting with diffusible reactive signaling electrophiles can be achieved in live worms with no observable negative effects. We have shown similar results in cultured cells1-5 and fish6. These results in C. elegans—one of the most powerful and genetically tractable systems for studying of stress and aging7,8—represents a key step in developing model systems to interrogate simultaneously the genetics and biochemistry of electrophile signaling. Electrophilic pharmaceuticals9,10 as well as endogenous electrophilic signals11,12 are key factors in disease treatment and signaling regulation/fitness promotion, respectively. The burgeoning interest in both facets of electrophiles has established reactive electrophilic species (RES)—particularly sp2-carbon-based electrophiles of common prevalence in modern covalent drugs and native electrophilic signals10—as important areas of study. With the growing appreciation to identify ligandable cyteines13, the emergence of successful covalent drugs10,14, and the role of precision endogenous RES signaling15, the most recent years have witnessed no letup in the push to ID new sensors/signaling pathways. Numerous powerful technologies to interrogate targets of electrophilic drugs/biomolecules are now available16. Figure 1. C. elegans T-REX platform. Indicated transgenic Halo worms following transgene induction were treated with bioinert photocaged precursors (1–3) to specific bioactive lipid-derived electrophiles (LDEs) (HNE 4, dHNE 5, and HDE 6)3. At a user-defined time, photouncaging liberates the respective alkyne-functionalized LDEs, 4–6) and proximity enhancement21 enables labeling of a quintessential electrophile sensor protein Keap1 (See also Fig. S1A). Halo functionality, probe specificity, and LDE-differential modification efficiency on target are assessed by 3 independent readouts.

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Biochemistry

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Unfortunately, many of these protocols are restricted to cultured cells and/or offer limited spatiotemporal resolution. Many also require extensive downstream processing. Few methods can easily report on phenotypic outputs directly attributable to one modification. Most methods rely upon bolus dosing of RES to a specific specimen, with little regard for metabolic processing, tissue/organelle-segregation, or off-target effects. These issues present significant impediments to progress in the field. Functional impacts of canonical enzyme-mediated post-translational modifications (PTMs)—such as phosphorylation and ubiquitination, have benefited from the combination of small-molecule-based tools17,18 and classical genetic/biochemical approaches such as targetedknockdown, mutagenesis, and phenotypic screening19,20. Indeed, the synergy between biochemistry/chemical biology and genetics/cell biology is arguably magnified in redox signaling15,16,21,22. This is because many RES are: toxic at elevated concentrations; promiscuous; engage with specific proteins without mediator-enzymes; and can be converted to numerous products11. Thus, genetic approaches alone are unable to address precision mapping of non-enzymatic RES-signal input on a single target to specific signaling output15. Indeed, balancing electrophile dose/time/target/locale to mirror physiologic signaling in vivo has proven difficult. We recently developed a chemistry-driven tool, called T-REX (Targetable Reactive Electrophiles and Oxidants), to begin to rigorously investigate electrophilic signaling (Figure S1)1-6. We achieved target-specific RES-labeling by tethering a photocaged-RES to the commercial-protein-tag HaloTag fused to a protein of interest (POI). Light-exposure released unfettered RES on demand23, giving first refusal to the Halo-fused POI. Thus, if the POI is a sensor protein, RES-labeling occurs: if the POI is not a sensor, this minute amount of RES (stoichiometric to Halo-POI) unleashed is metabolized or averaged over the whole proteome. This method constitutes a stringent assay for RES-sensing, and because little RES is released, it does not perturb the cell significantly. Built-in controls account for off-target effects and/or artefactual modifications/responses4,6. We recently adapted this platform to transient expression in larval zebrafish6—a popular vertebrate research model, and uncovered conserved signaling nodes that interchange RES-based non-enzyme-mediated-PTM and conventional enzyme-regulated-PTMs at a single-POIprecision scale10,15. This model provided new insights into redox signaling carried out by specific Akt-kinase isoforms in developing fish, but is currently incompatible with adult fish, restricting studies on aging. It would thus be adventitious if T-REX were shown to work in a model with shorter lifespan, and that retains transparency throughout its life. C. elegans is one system that fits these requirements.

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Figure 2. (A) Photocaged precursors selectively bind to functional HaloTag expressed in live worms. Top right: schematic of blocking experiment (Readout A, Fig. 1). See SI for the detailed procedure. Left panel: representative data analyzed by in-gel fluorescence (top) and western blot (lower) using anti-actin and anti-Halo antibodies respectively confirm protein loading and inducible halo::tev::keap1 expression. (halo::tev::keap1 full construct MW~105 kDa). See also Figure S2 for additional replicates. Bottom right: Quantitation. Ratios of the fluorescence signal to anti-Halo western blot signal were normalized to DMSO within each independent gel before averaging across multiple replicates. Errors designate s.d., n = 8 independent biological replicates. (B) Fluorescence imaging of the heat shockinduced live worms further confirms transgene expression. Fluorescence of tom70-MLS-localized mcherry::halo is visible throughout the worm (bottom row). While halo::tev::keap1 does not itself feature a fluorescent marker, it is co-expressed with a constitutive dominant marker (mec7p::mrfp), which displays fluorescence localized to touch-receptor neurons (top row, white arrows). Scale bars, 50 µm.

Establishing T-REX in C. elegans requires three criteria to be met: (1) transgenic lines expressing Halo fused to a specific RES-sensitive POI; (2) Halo must be active in live worms and be able to be labeled by the photoactivatable precursors; (3) delivery of RES to the POI must be possible in live worms. Using standard protocols, we generated several transgenic animals [pHSP16::tom70::mcherry::halo; pHSP16::halo::tev::keap1, where “tev” is a recognition site for the tobacco etch virus (TEV) protease; and pHSP16::gfp::halo] that upregulate Halo-protein upon heat shock (HS) (Figure 1). We used the dominant genetic marker pmec7::mrfp that gives constitutive expression in six touch-receptor neurons to mark transgenic worms24. The HS-promoter (HSP16p)25 was chosen because it is inducible, and gives high expression of many transgenes. Importantly, the HS-promoter can give high


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Biochemistry

expression without need for codon optimization or C. elegans introns. We chose human Keap1 protein (keap1 hereafter) because it is an established RES-sensor26 and validated to be kinetically-privileged to react with various RES signals delivered under T-REX-driven electrophile-limited conditions1-5, and there are few bona fide RES-sensors in C. elegans to serve as robust proof of concept. Expression was validated by both western blot (Figure 2A and S2), and in the case of tom70::mcherry::halo, fluorescence (Figure 2B). Halo blot showed that expression of tom70::mcherry::halo was approximately 5-fold higher than halo::tev::keap1 (Figure S2B). Next, we evaluated if the Halo protein could selectively bind our photocaged-probes in vivo (Readout A, Figure 1). We have reported a fleet of probes that can deliver various endogenous bioactive lipid-derived electrophiles (LDEs) to Keap1 upon light exposure in mammalian cells3,4. As proofs of principle in extending the platform to C. elegans, we chose precursors to 4hydroxynonenal (HNE) 4, nonenal (or dehydroxyhydroxynonenal) (dHNE) 5 and 4-hydroxydodecenal (HDE) 6 (Ht-PreHNE 1, Ht-PredHNE 2, Ht-PreHDE 3, respectively) (Figure 1 and S1A), all of which give similar levels of HaloTag conjugation in mammalian cells, and similar levels of Keap1-targeted LDEylation following light exposure3. These photocaged precursors were produced via Williamson ether synthesis, using anthraquinone and a corresponding Grignard-generated allylic bromide3. Using an optimized protocol, worms were treated with 30 µM probe for 6 h in liquid media with OP50 bacteria (food), then washed 3 times. After this point, worms were lysed by freeze-thaw/vortexing with beads. Halo protein activity in lysates was assayed by treatment of normalized lysates with TMR-Halo 7 (or FAM-Halo 8), ligands that react irreversibly with Halo (Figure S1B, 2A, and S2). FAM-Halo was used for quantifying labeling of tom70::mcherry::halo due to bleed-through of residual mCherry interfering with TMR in the gel (Figure S2B). The reaction of Halo with TMRor FAM-Halo-ligand gives a 1:1 complex that can be resolved on SDS-PAGE, giving a fluorescent band that migrates similarly to the unlabeled Halo-fusion protein. Bolus HNE 4 (30 µM, 1 h) treatment did not affect Halo activity in vivo (Figure 2A and S2C). However, pretreatment with Ht-PreHNE/Ht-PredHNE/Ht-PreHDE reduced TMR (or FAM) signal relative to untreated controls (Figure 2A and S2). These ligands all contained an alkyne appendage, to facilitate direct detection by Click reaction27, although conjugation of halo::tev::keap1 by Ht-PreHNE (with no alkyne-functionalization) (1-na) was similar regardless of the presence of alkyne functionalization (Figure 2A and S2C). Surprisingly, there were subtle differences between Halo-conjugation of these probes, but occupancy was >50% for each (Figure 2A, inset). Worms were not negatively affected by this protocol. Using Click chemistry, we validated this result

Figure 3. The T-REX concept of proximity-targeted on-demand release of HNE in situ under electrophile-limited conditions selectively HNEylate Keap1 protein expressed in live C. elegans. (A) Schematic of optimized biotin-Click pulldown conditions compatible with C. elegans lysate is shown (Readout C, Fig. 1). “B” indicates biotin. See SI methods for details. (B) Inset shows quantitation of the extent of HNEylated-Keap1 under indicated conditions [n ≥ 5 independent biological replicates (Fig. S5). Errors indicate s.e.m.]. Bulk HNE exposure and T-REX give different extent of HNEylation on Keap1, suggesting that uptake/metabolism is a more significant variable in living model organisms than in living cells where HNEylation efficiencies are largely found to be comparable between the two conditions3. (C) A representative data set is shown for input and elution samples. Actin serves as loading control. Induction designates heat shock for transgene expression. Also see Figure S5.

of Halo-occupancy by photocaged probes in vivo (Readout B, Figure 1). Using a protocol similar to that above, lysates were exposed to Click conditions with FAM-azide (Figure S3). A similar extent of Haloconjugation was observed. Our final aim in this communication was to evaluate whether T-REX-targeted LDE delivery to Keap1 could occur upon light exposure in live C. elegans. We first evaluated in vivo photouncaging efficiency of the HtPreHNE covalently bound to Halo, by measuring the time-dependent loss of alkyne label from gfp::halo (Figure 1) pre-conjugated to Ht-PreHNE 1, enabled by Click coupling in lysates post light exposure to live worms (Figure S4). The half-life of HNE release in vivo under these conditions was on par with that previously assessed in cultured cells (t1/2 < 1–2 min)3. Since expression of halo:: tev::keap1 transgene was relatively low, we opted to determine the targeted HNEylation efficiency to Keap1 using biotin/streptavidin enrichment (Readout C, Figure 1) that gives a higher signal:noise output compared to Click assay with azido-dyes in worm lysates. In this biotinpulldown protocol, C. elegans expressing halo::tev::keap1 are treated with the desired photocaged LDE-precursor for 6 h, and after washout, the LDE is


Biochemistry

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delivered to Keap1 by photouncaging. C. elegans are then lysed, halo::tev::keap1 is then separated into the two domains (halo and keap1 using TEV protease), lysates are precleared with streptavidin agarose to remove biotinylated proteins, then biotin is conjugated to alkynated proteins using biotin-azide Click coupling. After precipitation, and resolubilization, biotinylated proteins are enriched using streptavidin agarose beads. Using this assay (Figure 3 and S5), keap1 was significantly enriched relative to controls in which (i) Ht-PreHNE (with no alkyne-functionalization) (1-na) was used in place of alkyne-functionalized Ht-PreHNE 1 under otherwise identical conditions, and (ii) no compound treatment was performed. We were also keen to evaluate how our protocol compared to bolus dosing. We thus evaluated the amount of keap1 pulled down by Ht-PreHNE 1 and HNE 4 treatment. Strikingly, treatment with HNE 4 (30 µM), even over 1 h, did not HNEylate keap1 as significantly as that achieved under T-REX involving only 5-min incubation post light-driven HNE-release within the proximity of halo::tev::keap1 in vivo [Figure 3 and S2A (inset)]. These data demonstrate that T-REX likely by-passes absorption, distribution, metabolism effects of reactive electrophiles, and directs the HNE delivery to a specific protein, demonstrating that potential LDE-sensors could be identified using this method. In conclusion, we have established a protocol that will enable us to ultimately interrogate on-target electrophile signaling in a key model organism in which in vivo chemical biology is under-represented. Our protocol is robust: photocaged LDE probes (1–3) give highoccupancy conjugation of the Halo-protein functionally expressed in vivo, and targeted HNEylation can be accomplished at a user-defined time. The protocol can also be extended to POIs that lack accessible antibodies, by epitope tagging, for instance. Based on our recent work in cultured cells1-5 and zebrafish6, we can use this procedure to either validate sensors in vivo, or measure downstream signaling ramifications of signaling at a specific protein, specifically with an eye on interrogating the precision redox events of conserved importance in metazoan lifespan.

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ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author

*[email protected] Author Contributions

Y.A. designed and oversaw the project. M.J.C.L. generated transgenic worm strains. M.J.C.L., D.A.U., S.C., and J.A.H. maintained worm culture. M.J.C.L., D.A.U. and S.C. performed all experiments except time-dependent photo-liberation studies, which were performed by J.A.H. H.L., Y.Z., and Y.W. performed chemical syntheses. Y.A. wrote the paper with assistance from M.J.C.L., S.C., and D.A.U. ¶,† These authors contributed equally as shared first and second authors, respectively. Funding Sources No competing financial interests have been declared. Research instrumentation, supplies, and personnel in this work are partly or fully supported by NSF CAREER (CHE1351400), Sloan Fellowship (FG-2016-6379), and Beckman Young Investigator, ONR Young Investigator (N00014-17-1-2529), NIH New Innovator (1DP2GM114850) (to Y.A.); Cornell University Graduate School Fellowship and NSF Graduate Research Fellowship (DGE-1650441, to D.A.U); ACS Division of Organic Chemistry Summer Undergraduate Research Fellowship and Barry Goldwater Scholarship (to S.C.); NIH CBI Fellowship (to J.A.H.) (NIGMS T32-GM008500, PI: H. Lin); NSF MRI (CHE-1531632, PI: Y.A.) for Cornell University core-facility NMR instrumentation; NIH 1S10RR025502 (PI: R. M. Williams) for Cornell Imaging Center.

ACKNOWLEDGMENT Professor Jun (Kelly) Liu, Mr. Steve Sammons, and the Liu laboratory for the worm genetic techniques transfer, early guidance, and the use of microinjection facility; and Sanjna L. Surya and Alexandra Van Hall-Beauvais (Aye Lab) for TEV-protease preparation.

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Biochemistry

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1. Fang, X., Fu, Y., Long, M. J., Haegele, J. A., Ge, E. J., Parvez, S., and Aye, Y. (2013) J. Am. Chem. Soc. 135, 14496-14499. 2. Parvez, S., Fu, Y., Li, J., Long, M. J., Lin, H. Y., Lee, D. K., Hu, G. S., and Aye, Y. (2015) J. Am. Chem. Soc. 137, 10-13. 3. Lin, H. Y., Haegele, J. A., Disare, M. T., Lin, Q., and Aye, Y. (2015) J. Am. Chem. Soc. 137, 6232-6244. 4. Parvez, S., Long, M. J., Lin, H. Y., Zhao, Y., Haegele, J. A., Pham, V. N., Lee, D. K., and Aye, Y. (2016) Nat. Protoc. 11, 23282356. 5. Long, M. J. C., Lin, H.-Y., Parvez, S., Zhao, Y., Poganik, J. R., Huang, P., and Aye, Y. (2017) Cell Chem. Biol. 24, 1-14. 6. Long, M. J., Parvez, S., Zhao, Y., Surya, S. S., Wang, Y., Zhang, S., and Aye, Y. (2017) Nat. Chem. Biol. 13, 333-338. 7. Antoshechkin, I., and Sternberg, P. W. (2007) Nat. Rev. Genet. 8, 518-532. 8. Volovik, Y., Marques, F. C., and Cohen, E. (2014) Methods 68, 458-464. 9. Prosperini, L., and Pontecorvo, S. (2016) Ther. Clin. Risk Manag. 12, 339-350. 10. Long, M. J. C., and Aye, Y. (2017) Cell Chem. Biol. 10.1016/j.chembiol.2017.05.023. 11. Schopfer, F. J., Cipollina, C., and Freeman, B. A. (2011) Chem. Rev. 111, 5997-6021. 12. Jacobs, A. T., and Marnett, L. J. (2010) Acc. Chem. Res. 43, 673-683. 13. Backus, K. M., Correia, B. E., Lum, K. M., Forli, S., Horning, B. D., Gonzalez-Paez, G. E., Chatterjee, S., Lanning, B. R., Teijaro, J. R., Olson, A. J., Wolan, D. W., and Cravatt, B. F. (2016) Nature 534, 570-574.

14. Blewett, M. M., Xie, J., Zaro, B. W., Backus, K. M., Altman, A., Teijaro, J. R., and Cravatt, B. F. (2016) Science signaling 9, rs10. 15. Long, M. J., and Aye, Y. (2016) Chem. Res. Toxicol. 29, 1575– 1582. 16. Long, M. J., Poganik, J. R., Ghosh, S., and Aye, Y. (2017) ACS Chem. Biol. 12, 586-600. 17. Lai, A. C., and Crews, C. M. (2017) Nat. Rev. Drug Discov. 16, 101-114. 18. Tarrant, M. K., and Cole, P. A. (2009) Annu. Rev. Biochem 78, 797-825. 19. Bishop, A., Buzko, O., Heyeck-Dumas, S., Jung, I., Kraybill, B., Liu, Y., Shah, K., Ulrich, S., Witucki, L., Yang, F., Zhang, C., and Shokat, K. M. (2000) Annu. Rev. Biophys. Biomol. Struct. 29, 577606. 20. Aghajan, M., Jonai, N., Flick, K., Fu, F., Luo, M., Cai, X., Ouni, I., Pierce, N., Tang, X., Lomenick, B., Damoiseaux, R., Hao, R., Del Moral, P. M., Verma, R., Li, Y., Li, C., Houk, K. N., Jung, M. E., Zheng, N., Huang, L., Deshaies, R. J., Kaiser, P., and Huang, J. (2010) Nat. Biotechnol. 28, 738-742. 21. Dickinson, B. C., and Chang, C. J. (2011) Nat. Chem. Biol. 7, 504-511. 22. Brewer, T. F., Garcia, F. J., Onak, C. S., Carroll, K. S., and Chang, C. J. (2015) Annu. Rev. Biochem. 84, 765-790. 23. Long, M. J., Poganik, J. R., and Aye, Y. (2016) J. Am. Chem. Soc. 138, 3610-3622. 24. Chalfie, M. (1994) Curr. Biol. 4, 443-443. 25. Stringham, E. G., Dixon, D. K., Jones, D., and Candido, E. P. (1992) Mol. Biol. Cell 3, 221-233. 26. Hayes, J. D., and Dinkova-Kostova, A. T. (2014) Trends Biochem. Sci. 39, 199-218. 27. Kolb, H. C., Finn, M. G., and Sharpless, K. B. (2001) Angew. Chem. Int. Ed. 40, 2004-2021.


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Halo transgenic worms Precision redox targeting in vivo

Precision redox signaling events in lifespan control

Halo

Halo POI Redox-responsive protein of interest

POI

Native reactive redox signal

This study


Organelle/ tissue-specific stress responses Locale-specific privileged first responders

Future capabilities

Figure 1

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T-REX on-demand electrophile targeting in live C. elegans Produce transgenic worms

O

Introduce probe O

halo::tev::keap1

O N H

Cl

Keap1

R1 R3

O R2

R4

X, TEV-protease site

tom70::mcherry::halo TOM70

gfp::halo

mCherry

Halo

GFP

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O

SH

Light

X Keap1 1, Ht-PreHNE(alkyne) Halo 1-na, Ht-PreHNE(na) Worm lysis 2, Ht-PredHNE(alkyne) 3, Ht-PreHDE(alkyne)

Readouts: A) Competition with flourescent Halo-ligand à  Halo in vivo activity à  Binding specificity

Proof-ofconcept LDEs: 4, HNE(alkyne)

Cl

Halo

X

O

Cl

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(n,1; R, OH) O n R

5, dHNE(alkyne) B)

C)

Click coupling with fluorescent azide à  T-REX probe specificity

TEV-treatment & biotin-Click pulldown à  Validation of worm T-REX


(n,1; R, H) 6, HDE(alkyne) (n,4; R, OH)

Figure 2 A)

+ − − − + −

+ − − − − +

+ − − + − −

In live C. elegans

− + − + − − −

+ + − − − −

+ − − − − −

+ − − − − −

Halo

Keap1

TMR-Halo ligand (7)

< 0.0001 < 0.0001

37

150 100

0.75 0.50 0.25 0.00

50

mRFP Channel

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tom70::mcherry::halo

halo::tev::keap1

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DMSO 1-na 1 2 3 4

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Relative extent of blocking of the Halo binding-site

100

50

ns

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Halo

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Halo-targetable photocaged LDE (1, 2, 3, or 1-na) Cl

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Cl

1 2 3 4 Induction 5 6 TMR-Halo (7) 7 HNE(alkyne) (4) 8 Ht-PreHNE(na) (1-na) 9 Ht-PreHNE(alkyne) (1) 10 11 Ht-PredHNE(alkyne) (2) 12 Ht-PreHDE(alkyne) (3) 13 14 15 TMR channel 16 17 18 halo::tev::keap1 à 19 20 21 22 23 24 25 26 27 actin 28 29 30 31 32 33 Halo 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Keap1

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A)

In live worms

Target Keap1

Light Halo Keap1

Halo Keap1

Keap1

-- Lysis, -- TEV cleavage,

Halo Keap1

Halo

-- Preclearance of biotinylated

Keap1

ns

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< 0.0001

0 .5 0 0 .2 5

DMSO

0 .7 5

H t - P r e H N E ( a lk y n e ) (1 )

H t - P r e H N E ( n a ) (1 - n a )

DMSO

H N E ( a lk y n e ) (4 )

actin

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C)

Biotin azide (Click)

Keap1

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HNE(alkyne) (4)

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B

HNE(alkyne) (4)

Streptavidin enrichment

Halo

H N E y la te d K e a p 1

Halo

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Elution and analysis

R e la tiv e a m o u n t o f

Keap1

proteins

Halo Keap1

Halo Keap1

Halo Keap1

DMSO

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Biochemistry

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Figure S1 1 2 3 4 In cultured mammalian and E. coli cells, zebrafish (recent work) and worms (this work) 5 6 7 Photo8 O 9 uncaging Covalent O 10 O 11 binding to O N 12 H O R1 O Halo 13 3 Cl R 14 + R2 15 4 R 16 Ht-PreLDE (1−3) 17 Halo POI Halo POI 18 Halo POI 19 LDE, lipid-derived reactive 20 signaling electrophiles 21 22 POI, any redox-responsive 23 protein of interest 24 Proximity Post photo-uncaging 25 targeting 26 27 OH A representative LDE: 28 O 29 OH O 30 O N O H 31 OH OH 32 O O 33 Halo POI HNE(alkyne) 34 35 36 Remaining “empty cage” Liberated HNE(alkyne) (4) 37 38 covalently bound to HaloTag 39 40 active site Asp residue 41 42 43 44 45 Me Me Me O O O Me 46 N O N Me O O 47Me O 48 O H 49 O2C N O H 50 O Cl N O 51 O O Cl 52 O 53 TMR Halo ligand 7 diAc-FAM Halo ligand 8 54 55 HO O OH POI 56 57 O 58 59 O SH 60 365 nm @

A)

B)

O

HN N3

Cy5 azide (suflo version)

FAM azide


500 μW−5 mW/cm2 t1/2 < 1−2 min

Page 11 of 14

Biochemistry

Figure S2

A)

+

Induction TMR-Halo (7)

+

+

+

+


−

+

−

−

Ht-PredHNE(alkyne) (2)

+

−

−

−

MW/ kDa 150

TMR channel

halo

B)

−

−

+

−

diAcFAM-Halo (8)

+

+

+

+

+

+

+

+


−

+

−

−

−

+

−

−

Ht-PreHDE(alkyne) (3)

+

−

−

−

+

−

−

−

MW/ kDa 100 75

ß

100 75 50 37

+

Induction

FAM channel

ß ß

50 37

ß

100 75 50 37

ß

100 75

halo

ß

50 37

actin actin halo::tev::keap1

tom70:: mcherry:: halo

halo::tev:: keap1

C)

Induction TMR-Halo (7) HNE(alkyne) (4) Ht-PreHNE(na) (1-na) Ht-PreHNE(alkyne) (1)

TMR channel

+ − + −

+ + − −

L

+ − − −

In live C. elegans

+ − − −

Halo-targetable photocaged LDE (1, 2, 3, or 1-na) Halo

X

Keap1

Halo

ß

TMR-Halo ligand (7) or diAcFAM-Halo ligand (8)


Cl

halo

75 50 37 100

+ − − +

−

Cl

MW/ 150 kDa 100

+

Cl

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

X

Keap1

Biochemistry

Figure S3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 14

+

+

+

−

+

+

+

Ht-PreHNE(alkyne) (1) −

+

−

−

−

+

−

Ht-PredHNE(alkyne) (2) +

−

−

−

+

−

−

Induction

In live worms Covalent binding to Halo

150 100 75 ⌘

ß

+

Halo POI

Halo POI

FAM channel

50

ß

37 25

POI, protein of interest Worm lysis

halo Click coupling with FAM-azide In-gel fluorescence and western blot analyses

MW/ kDa

150 100 75

⌘

50

ß

37 25

actin halo::tev:: keap1


tom70:: mcherry:: halo

ß

Page 13 of 14

Biochemistry

Figure S4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

HtPreHNE " (alkyne) "snagele(1) #.c"ebound vil" ni" ol a H:: P F G" m o r f" e n y kl a $ E N H e r P" g n i g a c n u $ o t o h P . 3 "?smrow"evil"nito "enHalo y kl a $ E N H e r P" m o r f" e n y kl a $ E N H" e s a e l e r" e w" n a C . a Whole-worm "

HNE(alkyne) (4) "snagele#.c"evilGFP "ni"olaH::PFHalo G" m o r f" e n y kl a $ E N H e r P" g n i g a c n u $ o t o h P . 3 flooding " ? s m r o w" e vil" ni" e n y kl a $ E N H e r P" m o r f" e n y kl a $ E N H" e s a e l e r" e w" n a C . a

"

"

MW/ kDa 100 75 ß

gfp::halo

50 35

Cy5 Channel 5

0.5

0.05

0

HNE-alkyne (mM)

0

1

3

5

15

Time of illumination (min)

WB (Anti-GFP)

" " " "

"

" " "

"7 " "7 "


Elution

keap1

keap1

halo::tev::keap1

halo::tev::keap1

Input

actin

actin −

+

HNE(alkyne) (4)

DMSO

III

Elution


−


Induction

keap1

halo::tev::keap1

Input actin −


DMSO

II




HNE(alkyne) (4)

I DMSO

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 14

HNE(alkyne) (4)

Biochemistry

Figure S5

+


+

Precision Electrophile Tagging in Caenorhabditis elegans

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