Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Reest, Jiska van der; Lilla, Sérgio; Zheng, Liang; Zanivan, Sara; Gottlieb, Eyal

doi:10.1038/s41467-018-04003-3

Cited by 214 publications

(217 citation statements)

References 60 publications

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“…The understanding of cell signaling regulation by redox processes has largely benefitted from the recent development of labeling techniques aimed at identifying pro tein subjected to reversible Cys ox-PTMs (7,15). Although these techniques have allowed identification of numerous human proteins undergoing Cys ox-PTMs in vivo, the report of specific oxidation sites by MS is very often limited to proteins highly abundant and/or highly susceptible to oxidation (13,14,67). In most cases, identification of the oxidized residue by MS relied on the production of recombinant proteins and in vitro oxidation (19)(20)(21).…”

Section: Discussionmentioning

confidence: 99%

“…Accurate identification of sites of Cys ox-PTM in vivo in mammalian cells in a proteomewide fashion by Mass spectrometry (MS) has proved to be challenging due to lack of sensitivity (14,44). Here, we used a proven bioswitch method with a Maleimide-(Polyethylene Glycol)2-Biotin (Mal-PEG 2 -Bio) probe to specifically label Cys subjected to reversible ox -PTM independently of the oxidation type ( Fig.…”

Section: Proteome-wide Identification Of Cys Ox-ptm Sites In Basal Anmentioning

confidence: 99%

“…Reversible Cys ox-PTMs consist of a variety of modifications, the most widely studied being S-sulfenylation (Cys-SOH), S-glutathionylation (Cys-SSG) and disulfide (S-S) (7). Iodoacetamide-or maleimide (Mal)-based bioswitch methods, allowing labeling of oxidized Cys independently of the type of reversible oxidation, coupled to immunoblot or mass spectrometry (MS) have dramatically evolved and made it possible to identify numerous proteins subjected to redox regulation in vivo (10)(11)(12)(13)(14). However, identification of the corresponding Cys residues subjected to ox -PTM by MS has proven to be challenging and consequently, except in the case of very abundant or highly reactive redox proteins, most often relies on the analysis of the recombinant protein oxidized in vitro (7, 12, 13, 15, 16, 17 , 18-21).…”

Section: Introductionmentioning

confidence: 99%

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

Cuervo

Fortin

Caron

et al. 2020

Preprint

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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.

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Section: Discussionmentioning

confidence: 99%

Section: Proteome-wide Identification Of Cys Ox-ptm Sites In Basal Anmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Cuervo

Fortin

Caron

et al. 2020

Preprint

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“…TMT-labelled peptides were analysed as previously described (van der Reest et al, 2018) with minor modifications. First peptides were fractionated using high pH reverse phase chromatography on a C18 column (150 × 2.1 mm i.d.…”

Section: Lc/ms/ms Protocolmentioning

confidence: 99%

The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

Cao

Lilla

Cao

et al. 2019

Preprint

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Folding of proteins entering the mammalian secretory pathway requires the insertion of the correct disulfide bonds. Disulfide formation involves both an oxidative pathway for their insertion and a reductive pathway to remove incorrectly formed disulfides. Reduction of these disulfides is critical for correct folding and degradation of misfolded proteins. Previously, we showed that the reductive pathway is driven by NADPH generated in the cytosol. Here, by reconstituting the pathway using purified proteins and ER microsomal membranes, we demonstrate that the thioredoxin reductase system provides the minimal cytosolic components required for reducing proteins within the ER lumen. In particular, saturation of the pathway and its protease sensitivity demonstrates the requirement for a membrane protein to shuttle electrons from the cytosol to the ER lumen. These results provide compelling evidence for the critical role of the cytosol in regulating ER redox homeostasis to ensure correct protein folding and to facilitate the degradation of misfolded ER proteins.

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“…Even with the success of these approaches, there are significant limitations for specific applications imposed by the requirement for co-factors, an exogenous biotin-phenol probe, and high levels of H2O2 to initiate labeling, which could bias efficient labeling in specific cellular and chemical environments. Additionally, the requirement for peroxide limits application of this approach with proteins or pathways that involve redox regulation, which likely includes a large percentage of the proteome [14][15][16] . Therefore, new methods that can label proximal proteins in live cells with high spatial and temporal control, ideally without significant perturbation to the cellular environment, would provide a significant advantage to mapping PPIs.…”

Section: Introductionmentioning

confidence: 99%

Photoproximity Profiling of Protein-Protein Interactions in Cells

McCutcheon

Lee

Carlos

et al. 2019

Preprint

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We report a novel photoproximity protein interaction (PhotoPPI) profiling method to map protein-protein interactions in vitro and in live cells. This approach utilizes a bioorthogonal, multifunctional chemical probe that can be targeted to a genetically encoded protein of interest (POI) through a modular SNAP-Tag/benzylguanine covalent interaction. A first generation photoproximity probe, PP1, responds to 365 nm light to simultaneously cleave a central nitroveratryl linker and a peripheral diazirine group, resulting in diffusion of a highly reactive carbene nucleophile away from the POI. We demonstrate facile probe loading, and subsequent interaction-and light-dependent proximal labeling of a model protein-protein interaction (PPI) in vitro. Integration of the PhotoPPI workflow with quantitative LC-MS/MS enabled unbiased interaction mapping for the redox regulated sensor protein, KEAP1, for the first time in live cells. We validated known and novel interactions between KEAP1 and the proteins PGAM5 and HK2, among others, under basal cellular conditions. By contrast, comparison of Pho-toPPI profiles in cells experiencing metabolic or redox stress confirmed that KEAP1 sheds many basal interactions and becomes associated with known lysosomal trafficking and proteolytic proteins like SQSTM1, CTSD and LGMN. Together, these data establish PhotoPPI as a method capable of tracking the dynamic sub-cellular and protein interaction "social network" of a redox-sensitive protein in cells with high temporal resolution. Figure 4. PhotoPPI detects differential KEAP1 localization and protein interactors in response to metabolic and redox stress. A-B)Volcano plots graph of streptavidin-enriched protein SILAC ratios and P-values for both SNAP-KEAP1 (red) and KEAP1-SNAP (blue) expressing cells treated with CBR-470-1 (A) or tertbutylhydroperoxide (tBuOOH, B), followed by treatment of heavy cells with PP1 probe, irradiation of both heavy and light and proteomic processing as above in Fig. 3. Only the marker proteins KEAP1, MGMT (SNAP-Tag) and PGAM5 are labeled. C) Graphical depiction of specific target proteins that were significantly enriched in KEAP1 PhotoPPI profiles. Shown are mean enrichment ratios (y-axis) for each protein across all of the runs in each biological condition (x-axis). Proteins that were enriched as KEAP1 interactors among all conditions are labeled in black; proteins that were enriched in the basal profile but lost in stressed conditions are labeled red; proteins that were selectively enriched only in stressed conditions are labeled in green. When a protein was not detected among any runs in a specific condition, a SILAC ratio of 1 (i.e. no enrichment) was assigned and the point labeled as not detected (N.D.). D) Schematic depicting the altered sub-cellular localization and specific protein interactors detected under basal (left) and metabolic/redox stressed conditions (right).

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Proteome-wide analysis of cysteine oxidation reveals metabolic sensitivity to redox stress

Cited by 214 publications

References 60 publications

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

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

The Mammalian Cytosolic Thioredoxin Reductase Pathway Acts via a Membrane Protein to Reduce ER-localised Proteins

Photoproximity Profiling of Protein-Protein Interactions in Cells

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