Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum

The endoplasmic reticulum (ER) is the initial site of biogenesis of secretory pathway proteins, including proteins localized to the ER, Golgi, lysosomes, intracellular vesicles, plasma membrane, and extracellular compartments. Proteins within the secretory pathway contain a high abundance of disulfide bonds to protect against the oxidative extracellular environment. These disulfide bonds are typically formed within the ER by a variety of oxidoreductases, including members of the protein disulfide isomerase (PDI) family. Here, we establish chemoproteomic platforms to identify oxidized and reduced cysteine residues within the ER. Subcellular fractionation methods were utilized to enrich for the ER and significantly enhance the coverage of ER-localized cysteine residues. Reactive-cysteine profiling ranked ∼900 secretory pathway cysteines by reactivity with an iodoacetamide–alkyne probe, revealing functional cysteines annotated to participate in disulfide bonds, or S-palmitoylation sites within proteins. Through application of a variation of the OxICAT protocol for quantifying cysteine oxidation, the percentages of oxidation for each of ∼700 ER-localized cysteines were calculated. Lastly, perturbation of ER function, through chemical induction of ER stress, was used to investigate the effect of initiation of the unfolded protein response (UPR) on ER-localized cysteine oxidation. Together, these studies establish a platform for identifying reactive and functional cysteine residues on proteins within the secretory pathway as well as for interrogating the effects of diverse cellular stresses on ER-localized cysteine oxidation.内质网（endoplasmic reticulum, ER）是分泌途径蛋白生物发生的初始位点，此类蛋白涵盖定位于ER、高尔基体、溶酶体、细胞内囊泡、质膜及细胞外区域的蛋白质。分泌途径内的蛋白富含二硫键，以抵御氧化性的细胞外环境。这类二硫键通常在ER内由多种氧化还原酶催化形成，包括蛋白质二硫键异构酶（protein disulfide isomerase, PDI）家族成员。本研究建立了化学蛋白质组学平台，用于鉴定ER内的氧化型与还原型半胱氨酸残基；我们采用亚细胞分级分离方法对ER进行富集，显著提升了定位于内质网的半胱氨酸残基的检测覆盖度。我们通过基于碘乙酰胺-炔基探针反应性的反应性半胱氨酸表征分析，对约900个分泌途径半胱氨酸残基的反应性进行排序，揭示了被注释为参与二硫键形成或蛋白内S-棕榈酰化位点的功能性半胱氨酸残基。通过应用改良版的OxICAT半胱氨酸氧化定量方案，我们计算了约700个定位于ER的半胱氨酸残基各自的氧化占比。最后，我们通过化学诱导ER应激以扰动ER功能，以此探究未折叠蛋白反应（unfolded protein response, UPR）的激活对定位于内质网的半胱氨酸氧化的影响。综上，本研究建立了一套可用于鉴定分泌途径蛋白上反应性与功能性半胱氨酸残基的研究平台，同时也可用于探究多种细胞应激对ER定位半胱氨酸氧化的调控效应。