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

Abstract

Brief summary of the main results.The function of proteins is regulated by post-translational modifications, among which reversible oxidation of Cys recently emerged as a key component. Comprehension of redox regulation of cellular responses requires identification of specific oxidation sites in vivo. Using a bioswitch method to specifically label Cys subjected to reversible oxidation coupled to mass spectrometry, we identified thousands of novel oxidation sites. Many are relevant to virus -host interaction pathways. Here, we focused on the oxid ation of STING, an adaptor critical for activating the innate immune type I interferon pathway engaged upon cytosolic DNA sensing. Molecular studies led us to establish a new model in which STING Cys 148 is oxidized at basal levels, while Cys 206 oxidation is induced by oxidative stress and ligand binding. We show that oxidation of Cys 206 has an inhibitory function to prevent STING hyperactivation. This study provides ground for novel research avenues aimed at designing therapeutics that target this pathway. AbstractProtein function is regulated by post-translational modifications, among which reversible oxidation of Cys (Cys ox-PTM) emerged as a key regulatory mechanism of cellular responses. The redox regulation of virus-host interactions is well documented, but in most cases, proteins subjected to Cys ox-PTM remain unknown. The identification of Cys ox-PTM sites in vivo is essential to underpin our understanding of the mechanisms of the redox regulation. In this study, we present a proteome-wide identification of reversible Cys ox-PTM sites in vivo during stimulation by oxidants using a maleimide-based bioswitch method coupled to mass spectrometry. We identified 2720 unique Cys ox-PTM sites encompassing 1473 proteins with distinct abundance, location and functions. Label-free quantification (LFQ)-based analysis revealed the enrichment of ox-PTM in numerous pathways, many relevant to virus-host interaction. Here, we focused on the oxidation of STING, the central adaptor of the innate immune type I interferon 2 pathway induced upon detection of cytosolic DNA. We provide the first in vivo demonstration of reversible oxidation of Cys 148 and Cys 206 of STING. Molecular analyses led us to establish a new model in which Cys 148 oxidation is constitutive, while Cys 206 oxidation is inducible by oxidative stress or by the natural ligand 2'3'-cGAMP. We show that oxidation of Cys 206 has an inhibitory function to prevent STING hyperactivation through the constraint of a conformational change associated with the formation of inactive polymers containing intermolecular disulfide bonds. This provides new ground for the design of therapies targeting STING relevant to autoinflammatory disorders, immunotherapies and vaccines.

Reactive oxygen species oxidize STING and suppress interferon production

“…Previous reports indicate that cysteine residues of STING may form disulfide-containing polymers ( Ergun et al, 2019 ; Jin et al, 2010 ; Jønsson et al, 2017 ; Li et al, 2015 ; Motani et al, 2015 ). A recent report identified redox regulation of C206 upon cGAMP binding to STING ( Cuervo et al, 2020 ). The authors also indicate the C148 may be oxidized under baseline conditions but did not characterize this oxidation.…”

Explorations in a galaxy of sialic acids: a review of sensing horizons, motivated by emerging biomedical and nutritional relevance

“…211 On the other hand, the participation of other co-receptors viz. CD147, 212 heparan sulfate, 213 etc. is also proposed for the initial recognition of the virus by the host cell.…”