Precision Electrophile Tagging in Caenorhabditis elegans

Abstract

Adduction of an electrophile to privileged sensor proteins and the resulting phenotypically dominant responses are increasingly appreciated as being 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 signals. Genetically encodable or small-molecule-based fluorescent reporters and redox proteomics have revolutionized the observation and profiling of cellular redox states and electrophile-sensor proteins, respectively. However, precision mapping between specific redox-modified 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 Caenorhabditis elegans expressing functional HaloTagged fusion proteins and use this system to develop a generalizable light-controlled approach to tagging 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 life span regulation.

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). At a user-defined time, photouncaging liberates the respective alkyne-functionalized LDEs (4–6) and proximity enhancement enables labeling of a quintessential electrophile–sensor protein Keap1 (see also Figure S1A). Halo functionality, probe specificity, and LDE differential modification efficiency on target are assessed by three independent readouts.

Figure 2

(A) Photocaged precursors selectively bind to functional HaloTag expressed in live worms. The schematic at the top right shows the blocking experiment (readout A, Figure 1). See the Supporting Information for the detailed procedure. The left panel shows representative data analyzed by in-gel fluorescence (top) and Western blot (bottom) using anti-actin and anti-Halo antibodies, respectively, that confirm protein loading and inducible halo::tev::keap1 expression (halo::tev::keap1 full construct molecular weight of ∼105 kDa). See also Figure S2 for additional replicates. In the bottom right panel, the ratios of the fluorescence signal to the anti-Halo Western blot signal were normalized to dimethyl sulfoxide within each independent gel before averaging across multiple replicates. Errors designate the standard deviation (n = 8 independent biological replicates). (B) Fluorescence images of the heat shock-induced live worms further confirm 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 are 50 μm.

Figure 3

T-REX concept of proximity-targeted on-demand release of HNE in situ under electrophile-limited conditions that selectively HNEylate Keap1 protein expressed in live C. elegans. (A) Schematic of optimized biotin-Click pull-down conditions compatible with the C. elegans lysate (readout C, Figure 1). “B” indicates biotin. See the methods in the Supporting Information for details. (B) The inset shows quantitation of the extent of HNEylated Keap1 under the indicated conditions [n ≥ 5 independent biological replicates (Figure S5); errors indicate the standard error of the mean]. Bulk HNE exposure and T-REX give different extents 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 conditions. (C) Representative data set for input and elution samples. Actin serves as a loading control. Induction designates heat shock for transgene expression. Also see Figure S5.