Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum

Area of Science:

• Cell Biology
• Proteomics
• Biochemistry

Background:

• The endoplasmic reticulum (ER) is crucial for synthesizing and modifying proteins destined for secretion.
• Secretory pathway proteins rely on disulfide bonds, formed in the ER by oxidoreductases like protein disulfide isomerase (PDI), for stability in oxidative environments.
• Understanding cysteine reactivity and oxidation within the ER is key to deciphering protein folding and function.

Purpose of the Study:

• To establish chemoproteomic platforms for identifying oxidized and reduced cysteine residues in the ER.
• To quantify cysteine oxidation levels and identify functional cysteines involved in disulfide bonds or S-palmitoylation.
• To investigate the impact of ER stress and the unfolded protein response (UPR) on ER cysteine oxidation.

Main Methods:

• Utilized subcellular fractionation to enrich ER-localized proteins and cysteine residues.
• Employed reactive-cysteine profiling with an iodoacetamide-alkyne probe to assess cysteine reactivity.
• Applied a modified OxICAT protocol to quantify the percentage of oxidation for ER-localized cysteines.

Main Results:

• Identified approximately 900 reactive cysteines in the secretory pathway, including those involved in disulfide bonds and S-palmitoylation.
• Quantified oxidation percentages for around 700 ER-localized cysteines.
• Demonstrated that ER stress and UPR initiation alter ER-localized cysteine oxidation patterns.

Conclusions:

• Established a robust chemoproteomic platform for comprehensive analysis of ER cysteine reactivity and oxidation.
• Provided insights into the dynamic nature of cysteine oxidation in response to cellular stress.
• The platform facilitates further investigation into the functional roles of cysteines in the secretory pathway under various conditions.