Uncovering the Mechanism of Protein Sulfination in Regulating Atherosclerotic Plaque Calcification via Fluorescence Imaging

Atherosclerotic plaque calcification is an oxidative-stress-dependent process involved in the onset of plaque disruption and subsequent atherothrombotic events. Excessive reactive oxygen species (ROS) production determines the structural modification of protein cysteine sulfhydryl (Cys-SH), leading to the alteration of protein functions and redox signaling outputs. Although these redox-centered signaling pathways have been found to play important roles in plaque calcification, the extent of protein Cys-SH modifications and the regulatory regions of specific proteins remains unclear. This is due to the lack of tools that can visually distinguish and characterize the oxidation products of different Cys-SH proteins, especially protein sulfination (Cys-SO 2 H). Herein, we present a novel “turn-on” fluorescent probe (Z-1) for the in situ visualization of Cys-SO 2 H modifications and investigate its effects on the initiation of vascular smooth muscle cell (VSMC) calcification. In vitro and in vivo imaging with Z-1, cigarette smoking-induced VSMC calcification, display a significant increase in protein Cys-SO 2 H levels. Protein spectrum analysis reveals that Cys-SO 2 H modification occurred at the sulfhydryl active sites of the Kelch-like ECH-associated protein (Keap1). These oxidative modifications are desired to dysregulate the ROS/Keap1/nuclear factor-E2-related factor 2 (Nrf2) antioxidant signaling pathway and accelerate the calcification process. Furthermore, elevated levels of Cys-SO 2 H observed in serum samples from patients with acute myocardial infarction and cerebral infarction give clinical evidence of the relevance of protein Cys-SO 2 H modification in pathological calcification.