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.

Precision Electrophile Tagging in Caenorhabditis 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 cells 1−5 and fish. 6 These results in C. elegans , one of the most powerful and genetically tractable systems for the study of stress and aging, 7,8 represent a key step in developing model systems for interrogating simultaneously the genetics and biochemistry of electrophile signaling.…”

“…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 demand, 23 giving first refusal to the Halo-fused POI.…”

A Generalizable Platform for Interrogating Target- and Signal-Specific Consequences of Electrophilic Modifications in Redox-Dependent Cell Signaling

“…3a,6 The alkyne functionalization at the chain terminus enables tracking of proteins covalently bound (via HaloTag, 16 Figure 1b) to the caged precursors, or quantitative assessment of the extent to which specific proteins are modified by the liberated electrophile (by using Click coupling 17 with Cy5-azide). 12 Photocaged precursors to alkyne-functionalized alkenals—ONE 2 , dHNE 3 , HHE 4 , HDE 5 , HDDE 6 , and 2-HD 7 —( 11 – 16 , Figure 2) (Scheme S2–S7) were selected. These alkenals not only regulate various physiologic responses 3a,6 but they also cover a range of chain lengths and electronically diverse Michael acceptors allowing us to investigate the tolerance of T-REX.…”

“…Our initial synthetic strategy was centered upon extending the previously reported synthesis of Ht-Pre-HNE 17 12 (Figure 3a). In this approach, Williamson ether synthesis 18 furnishes the caged electrophile from anthraquinone 18 and allylic bromide 19 —accessed from known aldehyde 20 and the corresponding Grignard reagent, or other simple precursors.…”

“…12 We subsequently extended this method to interrogate whether specific HNEylation of Keap1 in low stoichiometry could elicit an ARE response, or whether subsidiary factors were required. 13 These pilot studies unambiguously demonstrated that Keap1 is a key redox sensor along the Nrf2–ARE cascade—specifically, HNEylation of Keap1 is alone biologically sufficient to elicit an ARE response of magnitude similar to that observed under whole-cell HNE flooding.…”