Developing Tools to Investigate Protein Sulfenylation in Living Cells.

Abstract

Oxidation of cysteine to sulfenic acid has emerged as a biologically relevant post-translational modification with particular importance in redox-mediated signal transduction; however, the identity of modified proteins remains largely unknown. In the present study we report the development of DAz-1, a cell-permeable chemical probe capable of detecting sulfenic acid- modified proteins directly in living cells. We then describe DAz-2, an analog of DAz-1 that exhibits significantly improved potency in vitro and in cells. Application of this new probe for global analysis of the sulfenome in a tumor cell line identifies most known sulfenic acid-modified proteins – 14 in total, plus more than 175 new candidates, with further testing confirming oxidation in several candidates. The newly identified proteins have roles in signal transduction, DNA repair, metabolism, protein synthesis, redox homeostasis, nuclear transport, vesicle trafficking, and ER quality control. Next we employ DAz-2 to discover two thioredoxin-related proteins that protect single cysteines from irreversible oxidation to sulfinic and sulfonic acid in Escherichia coli, which have functional homologues in eukaryotic cells. Finally we further refine these sulfenic acid probes to develop redox based probes of protein tyrosine phosphatases xxvii (PTPs) which show greatly increased sensitivity towards PTP sulfenic acid modification over previous probes. The combination of selective chemical enrichment and live-cell compatibility makes these sulfenic acid probes powerful new tools with the potential to reveal new regulatory mechanisms in signaling pathways, and identify new therapeutic targets.