Efficient generation of recombinant hydrazides for the production of scarce proteins and development of chemical probes for detecting protein sulfenylation
Date
2020
Authors
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Publisher
University of Delaware
Abstract
The first half of the research work documented here follows an attempt to improve existing protein engineering methods to produce difficult or complicated protein targets. Here, we are using protein hydrazides, which are powerful thioester surrogates for chemical ligation. Hydrazides can also serve as an effective tool in introducing site-selective modifications. Currently, peptide hydrazides are generated mainly via solid phase peptide synthesis (SPPS), which comes with size or solubility limitations. Here we have introduced a recombinant approach to generate C-terminal hydrazides by protein trans splicing mediated by split inteins from Pyrococcus horikoshii (PhoRadA). The versatility of this recombinant hydrazide has been shown by its ability to undergo an aldehyde coupling reaction to produce a hydrazone. These hydrazides can further be utilized to generate thioesters for expressed protein ligation. ☐ In the second half, I describe a novel tool for studying oxidative stress in vivo. Cellular oxidative stress is a result of elevated levels of reactive oxygen species (ROS). In the cell, ROS are converted to less harmful forms such as hydrogen peroxide (H2O2), which subsequently lead to the formation of cysteine sulfenic acids (protein sulfenylation). Additionally, an increase in ROS is also implicated in a number of diseases including cancer, cardiovascular diseases and neurodegenerative diseases. As a result, there is great interest in studying protein sulfenylation stemming from its role in redox regulation and aberrant redox modifications in human disease. A number of compounds targeting sulfenic acids have been identified over the years including 5,5- dimethylcyclohexane-1,3-dione or dimedone and more recently 9-hydroxymethylbicyclo [6.1.0] nonyne (BCN). Unfortunately, current reagents suffer from sluggish reaction rates, lack of specificity towards sulfenic acids and poor cell permeability. Here we describe the use of a transcyclooctene (SAM-TCO) as a sulfenic acid reactive molecule, which renders a stable and irreversible thioether adduct with reactive sulfenic acids. We have illustrated its ability to enter live cells through western analysis. More importantly, we have used the rapid TCO-tetrazine reaction to bio-orthogonally quench excess probe inside cells. This will prevent detection of any artefactual sulfenylation that may occur due to unnatural stress introduced during sample processing. SAM-TCO will be used to site specifically modify sulfenic acids in cells and through chemoproteomics, to identify protein targets of cysteine sulfenylation. The identification of cellular sulfenylation sites will not only expand our knowledge of redox biology but also assists in identifying therapeutic targets for diseases implicated with oxidative stress.
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Keywords
Recombinant hydrazide, Scare protein, Chemical probe, Protein sulfenylation