Dataset Information

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

ABSTRACT: 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.

SUBMITTER: Bechtel TJ

PROVIDER: S-EPMC7650206 | biostudies-literature | 2020 Feb

REPOSITORIES: biostudies-literature

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Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

Bechtel Tyler J TJ Li Chun C Kisty Eleni A EA Maurais Aaron J AJ Weerapana Eranthie E

ACS chemical biology 20200115 2

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 disulfid ...[more]

PMID: 31899610

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Project description:Pharmacological activation of the activating transcription factor 6 (ATF6) arm of the Unfolded Protein Response (UPR) has proven useful for ameliorating proteostasis deficiencies in a variety of etiologically diverse diseases. Previous high-throughput screening efforts identified the small molecule AA147 as a potent and selective ATF6 activating compound that operates through a mechanism involving metabolic activation of its 2-amino- p -cresol substructure affording a quinone methide, which then covalently modifies a subset of ER protein disulfide isomerases (PDIs). Intriguingly, another compound identified in this screen, AA132, also contains a 2-amino- p -cresol moiety; however, this compound showed less transcriptional selectivity, instead globally activating all three arms of the UPR. Here, we show that AA132 activates global UPR signaling through a mechanism analogous to that of AA147, involving metabolic activation and covalent PDI modification. Chemoproteomic-enabled analyses show that AA132 covalently modifies PDIs to a greater extent than AA147. Paradoxically, activated AA132 reacts slower with PDIs, indicating it is less reactive than activated AA147. This suggests that the higher labeling of PDIs observed with activated AA132 can be attributed to its lower reactivity, which allows this activated compound to persist longer in the cellular environment prior to quenching by endogenous nucleophiles. Collectively, these results suggest that AA132 globally activates the UPR through increased engagement of ER PDIs. Consistent with this, reducing the cellular concentration of AA132 decreases PDI modifications and allows for selective ATF6 activation. Our results highlight the relationship between metabolically activatable-electrophile stability, ER proteome reactivity, and the transcriptional response observed with the enaminone chemotype of ER proteostasis regulators, enabling continued development of next-generation ATF6 activating compounds.

Dataset Information

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

Publications

Profiling Cysteine Reactivity and Oxidation in the Endoplasmic Reticulum.

Similar Datasets

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