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

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 Cys(148) and Cys(206) of STING in cells. Molecular analyses led us to establish a model in which Cys(148) oxidation is constitutive, whereas Cys(206) oxidation is inducible by oxidative stress or by the natural ligand of STING, 2'3'-cGAMP. Our data suggest that the oxidation of Cys(206) 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.

Automated summary

The reversible oxidation of cysteine residues plays a crucial role in regulating protein function, especially in virus-host interactions. This study focused on the oxidation of STING protein and revealed the impact of different oxidation sites on its function. These findings could be valuable for the design of therapies targeting STING in various medical applications.