Each Cellular Compartment Has a Characteristic Protein Reactive Cysteine Ratio Determining Its Sensitivity to Oxidation

Abstract

Signaling and detoxification of Reactive Oxygen Species (ROS) are important patho-physiologcal processes. Despite this, we lack comprehensive information on individual cells and cellular structures and functions affected by ROS, which is essential to build quantitative models of the effects of ROS. The thiol groups from cysteines (Cys) in proteins play a major role in redox defense, signaling, and protein function. In this study, we show that the proteins in each subcellular compartment contain a characteristic Cys amount. Using a fluorescent assay for -SH in thiolate form and amino groups in proteins, we show that the thiolate content correlates with ROS sensitivity and signaling properties of each compartment. The highest absolute thiolate concentration was found in the nucleolus, followed by the nucleoplasm and cytoplasm whereas protein thiolate groups per protein showed an inverse pattern. In the nucleoplasm, protein reactive thiols concentrated in SC35 speckles, SMN, and the IBODY that accumulated oxidized RNA. Our findings have important functional consequences, and explain differential sensitivity to ROS.

Temporal coordination of the transcription factor response to H2O2 stress

“…We next measured how protein and lipid oxidation scales with the concentrations of H 2 O 2 tested. For protein oxidation, we measured maleimide-488 incorporation in proteins and normalized to total protein content 39 . Maleimide-488 incorporation decreased at all doses tested, indicating a decrease in reactive thiol groups and high levels of protein oxidation (Supplementary Fig.…”

“…For protein oxidation experiments (Supplementary Fig. 1J ), reactive thiol groups and total proteins were stained using as described previously 39 . MCF7 cells were treated with different concentrations of H 2 O 2 for 3 h and then fixed, permeabilized, blocked, and incubated with 100 nM of Alexa Fluor 488 C5-maleimide (Invitrogen, A10254) in PBS for 10 min at room temperature.…”