Mitochondria-targeted Probes for Imaging Protein Sulfenylation

Abstract

Mitochondrial reactive oxygen species (ROS) are essential regulators of cellular signaling, metabolism and epigenetics underlying the pathophysiology of numerous diseases. Despite the critical function of redox regulation in mitochondria, currently there are limited methods available to monitor protein oxidation in this key subcellular organelle. Here, we describe compounds for imaging sulfenylated proteins in mitochondria: DCP-NEt2-Coumarin (DCP-NEt2C) and rhodamine-based DCP-Rho1. Side-by-side comparison studies are presented on the reactivity of DCP-NEt2C and DCP-Rho1 with a model protein sulfenic acid (AhpC-SOH) and mitochondrial localization to identify optimized experimental conditions for labeling and visualization of protein sulfenylation that would be independent of mitochondria membrane potential and would not impact mitochondrial function. These probes are applied to image mitochondrial protein sulfenylation under conditions of serum starvation and in a cell culture model of lung cancer exposed to ionizing radiation and silver nanoparticles, agents serving dual functions as environmental stressors and cancer therapeutics.

Sulforaphane promotes chlamydial infection by suppressing mitochondrial protein oxidation and activation of complement C3

“…2). The anti-and pro-oxidant activities of SFN and MitoPQ, respectively, were monitored in Figure 2 with the mitochondria-targeted protein sulfenylation probe, DCP-NEt 2 C. 37 This probe was previously shown to not interfere with mitochondrial respiration, a key property to avoid potential artifacts. Efforts to monitor selectively H 2 O 2 levels in mitochondria with Orp1-roGFP 48 or HyPer 49 genetically encoded probes were not successful due to the interference of SFN reaction with the reactive thiols in these reporter proteins.…”

[18F]DCP, First Generation PET Radiotracer for Diagnosis of Radiation Resistant Head and Neck Cancer

“…Overall, differences in redox metabolism are reflected in the DNA damage and protein oxidation profiles, with the radiation resistant rSCC-61 cells displaying lower level of single and double-strand DNA breaks and lower protein sulfenylation, a finding validated using ex vivo analysis of human HNSCC tumor specimens (10). 14 Analysis of protein sulfenylation in these studies was enabled by development of (2,4dioxocyclohexyl)propyl (DCP) family of sulfenic acid-selective chemical probes (47), which now include a variety of derivatives and tags such as: fluorophores (DCP-FL1, DCP-FL2) (16), biotin (DCP-Bio1, DCP-Bio2, DCP-Bio3 (16); BP1, containing the reactive 1,3-dicarbonyl DCP component (17)), click-ready tags (DAz/DYn series of azido-/alkyne-analogs of DCP (19,(48)(49)(50); linear alkyne betaketoesters (18)), and more recently mitochondrial-targeting components (DCP-NEt2C and DCP-Rho1) (14,16).…”

“…The goal of the work presented here was to build on the established selectivity of DCP-based reagents towards sulfenylated proteins (14,(16)(17)(18)47), and on the clinical evidence of increased protein sulfenylation in radiation sensitive HNSCC tumors (10) to develop a first-generation PET radiotracer for imaging protein sulfenylation in vivo.…”

“…Recombinant C165A mutant of Salmonella typhimurium AhpC was purified and oxidized as described in previous work (2,(14)(15)(16)(17)(18). The C165A AhpC-SOH (50 μM, also containing -SN and -SO2/3H species) was incubated with 5 mM dimedone or 5 mM [ 19…”

“…To facilitate the investigation of these redox processes, we reported on synthesis and applications of a series of chemical probes for selective labeling of sulfenylated proteins under both in vitro and in vivo conditions over the past years (14)(15)(16)(17)(18). These compounds include BP1, a biotin-tagged derivative of (2,4-dioxocyclohexyl)propyl (DCP) (17), which was crucial to our discovery of suppressed protein sulfenylation in clinical specimens of radiation resistant HNSCC (10,11).…”