Pinpointing cysteine oxidation sites by high-resolution proteomics reveals a mechanism of redox-dependent inhibition of human STING

Abstract

Protein function is regulated by posttranslational modifications (PTMs), among which reversible oxidation of cysteine residues has emerged as a key regulatory mechanism of cellular responses. Given the redox regulation of virus-host interactions, the identification of oxidized cysteine sites in cells is essential to understand the underlying mechanisms involved. Here, we present a proteome-wide identification of reversibly oxidized cysteine sites in oxidant-treated cells using a maleimide-based bioswitch method coupled to mass spectrometry analysis. We identified 2720 unique oxidized cysteine sites within 1473 proteins with distinct abundances, locations, and functions. Oxidized cysteine sites were found in numerous signaling pathways, many relevant to virus-host interactions. We focused on the oxidation of STING, the central adaptor of the innate immune type I interferon pathway, which is stimulated in response to the detection of cytosolic DNA by cGAS. We demonstrated the reversible oxidation of Cys148 and Cys206 of STING in cells. Molecular analyses led us to establish a model in which Cys148 oxidation is constitutive, whereas Cys206 oxidation is inducible by oxidative stress or by the natural ligand of STING, 2′3′-cGAMP. Our data suggest that the oxidation of Cys206 prevented hyperactivation of STING by causing a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This finding should aid the design of therapies targeting STING that are relevant to autoinflammatory disorders, immunotherapies, and vaccines.

Sensitizing immune unresponsive colorectal cancers to immune checkpoint inhibitors through MAVS overexpression

“…These results support the reduced ability of these genes to polymerize, dimerize or phosphorylate after overexpression to elicit downstream IFN signaling, as has been previously reported with wild-type STING expression in the absence of ligand. 53 Collectively, these studies suggested that MAVS expression in CRCs was capable of eliciting innate immune signaling, but had a more marginal effect in stimulating apoptosis and directly suppressing cellular proliferation.…”

Protein posttranslational modifications in health and diseases: Functions, regulatory mechanisms, and therapeutic implications

“…Oxidative stress induces the intermolecular disulfide bond formation of TFEB/TFE3 in mammals, which enhances the activity of the TF 1370 . STING is a key regulator in the innate immune type I IFN pathway, and its C206 oxidation to form intermolecular disulfide bonds leads to a conformational change in the protein that prevents excessive activation of STING 1371 . In addition, cellular structure is also dependent on cysteine disulfides, as microtubule assembly is partly mediated by disulfide bonds 1345 …”

Thiol redox proteomics: Characterization of thiol‐based post‐translational modifications

“…Another recent study by Cuervo et al. reported the Cys sites for redox‐dependent inhibition of human STING [118], which is known for its role as a central adaptor of the interferon (IFN) pathway in innate immune defense when the system detects DNA from pathogen. In this study, a maleimide‐based BST approach coupled with MS was applied to identify ∼2700 Cys sites, and reversible oxidation of Cys148 and Cys206 in STING was observed.…”