Mechanistic Basis of Residue Specificity in Targeted Electrophilic Modification

Abstract

Our group recently developed the T‐REX (Targeted Reactive Electrophiles and Oxidants) technology that enables selective delivery of reactive electrophilic signals to a protein of interest (POI) in cells. T‐REX exploits photo‐uncaging chemistry to liberate bioactive electrophiles such as 4‐hydroxynonenal (HNE) at a precise time from the photocaged HNE‐precursor that is covalently bound to the genetically encodable HaloTag (HT) fused to POI. The liberated HNE is captured by the reactive cysteine(s) (Cys) on POI fused to HT. With T‐REX, we have shown the activation of antioxidant response (AR) pathway by selectively HNEylating Keap1—a redox sensitive protein and a key player in mammalian AR, against a backdrop of otherwise unperturbed cell. My project seeks to answer residue specificity in Keap1 HNEylation enabled by T‐REX. Specifically, I aim to elucidate whether residue specificity is dependent on the position of the HT. T‐REX on HT at the N‐terminus of Keap1 in cells resulted in HNEylation of two specific Cys's. I now aim to test the extent to which residue specificity is altered when HT is at the C‐terminus. Toward this goal, I have recently cloned, expressed and purified recombinant Keap1‐HT. I have begun to test residue specificity in T‐REX targeting using Keap1‐HT in vitro and that expressed in human cells. I also aim to characterize residue specificity in vitro when Keap1 is in complex with two known binding partners—Nrf2 transcription factor, and CUL3 ubiquitin E3 ligase protein. Ultimately, these studies will shed light on the Cys residue specificity manifested by the unique T‐REX system and how this property changes upon association with Nrf2 and CUL3 as well as changes in Keap1 conformation in vitro and in cells.