Temporally controlled targeting of 4-hydroxynonenal to specific proteins in living cells

Abstract

In-depth chemical understanding of complex biological processes hinges upon the ability to systematically perturb individual systems. However, current approaches to study impacts of biologically relevant reactive small molecules involve bathing of the entire cell or isolated organelle with excess amounts, leading to off-target effects. The resultant lack of biochemical specificity has plagued our understanding of how biological electrophiles mediate signal transduction or regulate responses that confer defense mechanisms to cellular electrophilic stress. Here we introduce a target-specific electrophile delivery platform that will ultimately pave the way to interrogate effects of reactive electrophiles on specific target proteins in cells. The new methodology is demonstrated by photoinducible targeted delivery of 4-hydroxynonenal (HNE) to the proteins Keap1 and PTEN. Covalent conjugation of the HNE-precursor to HaloTag fused to the target proteins enables directed HNE delivery upon photoactivation. The strategy provides proof of concept of selective delivery of reactive electrophiles to individual electrophile-responsive proteins in mammalian cells. It opens a new avenue enabling more precise determination of the pathophysiological consequences of HNE-induced chemical modifications on specific target proteins in cells.

Figure 1

(a) Current approach to study impacts of reactive electrophiles on protein of interest (POI) involves treatment of entire cell/proteome with a reactive entity, unavoidably leading to multiple off-target effects. Red, HNE. (b) A new approach to interrogate with biochemical specificity effects of electrophiles on individual proteins. The strategy focuses on a time-resolved targetspecific delivery of electrophiles to a single POI against the entire cell/proteome. The synthetic delivery platform ‘dormant electrophile’ shown has 3 modules: gray, high affinity/specificity ligand to POI; blue, flexible linker; pink, inert warhead – a caged HNE.

Figure 2

Photoinducible HNE-targeting on HaloTag fusion proteins made possible by HaloTag PreHNE, HtPH (1, R = CH₂CH₃) and HaloTag PreHNE-alkyne, HtPHA (2, R = C₂H). Also see Figure S1. Color code refers to the schematic representation of dormant electrophile in Figure 1b.

Figure 3

(a) Designed ligand is cell-permeable. COS-1 cells transiently overexpressing cytosolic HaloKeap1 were treated with either (1) 5 µM HaloTag^® TMR alone, or (2) 50 µM HtPH (1, Figure 2) for 1 h prior to 5 µM HaloTag^® TMR. Scale bars, 20 µM. (b) Designed ligand is non-cytotoxic. Representative data from flow cytometry dye-exclusion assays on viability of HtPHA (2, Figure 2) treated (3 h) COS-1 cells. Error bars are SD (N=3).

Figure 4

(a) Directed HNE targeting strategy demonstrated by selective Keap1 targeting in living cells. COS-1 cells transiently expressing DsRed and Halo–Keap1 (Figure S2) were treated with 25 µM HtPHA (2) for 2.5 h. Subsequent to exposure to a 4 W 365 nm lamp (20 min), cells were lysed, lysate was treated with Tev protease, followed by Cy5-Click assay reagents. This set up enables quantitation of % targeting efficiency: Cy5 signal intensity on Halo band prior to light exposure serves as the input, and that on the Keap1 band post photo-uncaging and Tev-cleavage serves as the output. (b) A representative resultant SDS-PAGE with Coomassie staining. (c) In-gel Cy5-fluorescence analysis of the same SDS-PAGE. Lane 1, MW ladder; Lane 2, Cells not exposed to light; Lane 3, Cells exposed to light; Lane 4, No Tev cleavage in lysate. *, Halo–Keap1 (104 kDa); +, Keap1 (70 kDa); x, Halo (33 kDa). From independent replicates, average Cy5 signal intensity transfer from Halo band before light exposure, to Keap1 band after light exposure, is quantitated to be 55 ± 13%.