An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells

Vinogradova, Ekaterina V.; Zhang, Xiaoyu; Remillard, David; Lazar, Daniel C.; Suciu, Radu M.; Wang, Yujia; Bianco, Giulia; Yamashita, Yu; Crowley, Vincent M.; Schafroth, Michael A.; Yokoyama, Minoru; Konrad, David; Lum, Kenneth M.; Simón, Gabriel; Kemper, Esther K.; Lazear, Michael R.; Yin, Sifei; Blewett, Megan M.; Dix, Melissa M.; Nguyen, Nhan; Shokhirev, Maxim N.; Chin, Emily; Lairson, Luke L.; Melillo, Bruno; Schreiber, Stuart L.; Forli, Stefano; Teijaro, John R.; Cravatt, Benjamin F.

doi:10.1016/j.cell.2020.07.001

Cited by 209 publications

(332 citation statements)

References 101 publications

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“…After statistical filtering, we identified a total of 1097 cysteines in 691 proteins that were sensitive to IA-alkyne labelling ( Table S1 ). Similar to previous studies 4,23,24 , individual cysteines displayed a range of inherent reactivity towards the probe ( Fig. 1b ).…”

Section: Main Textsupporting

confidence: 87%

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Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Benns

Storch

Falco

et al. 2021

Preprint

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Nucleophilic amino acids are important in covalent drug development yet underutilized as antimicrobial targets. Over recent years, several chemoproteomic technologies have been developed to mine chemically-accessible residues via their intrinsic reactivity toward electrophilic probes. However, these approaches cannot discern which reactive sites contribute to protein function and should therefore be prioritized for drug discovery. To address this, we have developed a CRISPR-based Oligo Recombineering (CORe) platform to systematically prioritize reactive amino acids according to their contribution to protein function. Our approach directly couples protein sequence and function with biological fitness. Here, we profile the reactivity of >1,000 cysteines on ~700 proteins in the eukaryotic pathogen Toxoplasma gondii and prioritize functional sites using CORe. We competitively compared the fitness effect of 370 codon switches at 74 cysteines and identify functional sites in a diverse range of proteins. In our proof of concept, CORe performed >800 times faster than a standard genetic workflow. Reactive cysteines decorating the ribosome were found to be critical for parasite growth, with subsequent target-based screening validating the apicomplexan translation machinery as a target for covalent ligand development. CORe is system-agnostic, and supports expedient identification, functional prioritization, and rational targeting of reactive sites in a wide range of organisms and diseases.

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Section: Main Textsupporting

confidence: 87%

“…1c; Table S2, S1a ), which were not correlated with general enrichment of abundant proteins ( Fig. S1b ) and absent from similar datasets obtained from other eukaryotic cell systems 4,23,24 .…”

Section: Main Textmentioning

confidence: 79%

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Benns

Storch

Falco

et al. 2021

Preprint

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“…Vinogradova et al 175 used chemical proteomic activity-based protein profiling to map 3,000 cysteines in T cells that were reactive with a library of electrophilic small molecules. Mass-spectroscopic analysis showed that acrylamides BPK-21 and BPK-25 formed adducts with Cys91 of STING, as well as with cysteines of other immune-related proteins.…”

Section: Therapeutic Targeting Of Cgas–stingmentioning

confidence: 99%

The cGAS–STING pathway as a therapeutic target in inflammatory diseases

et al. 2021

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The cGAS–STING signalling pathway has emerged as a key mediator of inflammation in the settings of infection, cellular stress and tissue damage. Underlying this broad involvement of the cGAS–STING pathway is its capacity to sense and regulate the cellular response towards microbial and host-derived DNAs, which serve as ubiquitous danger-associated molecules. Insights into the structural and molecular biology of the cGAS–STING pathway have enabled the development of selective small-molecule inhibitors with the potential to target the cGAS–STING axis in a number of inflammatory diseases in humans. Here, we outline the principal elements of the cGAS–STING signalling cascade and discuss the general mechanisms underlying the association of cGAS–STING activity with various autoinflammatory, autoimmune and degenerative diseases. Finally, we outline the chemical nature of recently developed cGAS and STING antagonists and summarize their potential clinical applications.

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“…This protocol illuminates dynamic immune state-dependent alterations in cysteine reactivity, revealing chemoselective and stereoselective small-molecule interactions with cysteines in structurally and functionally diverse proteins that lack chemical probes. For complete details on the use and execution of this protocol, please refer to Vinogradova et al. (2020) .…”

mentioning

confidence: 99%

“…For complete details on the use and execution of this protocol, please refer to Vinogradova et al. (2020) .…”

mentioning

confidence: 99%

Multiplexed proteomic profiling of cysteine reactivity and ligandability in human T cells

Vinogradova

Cravatt

2021

STAR Protocols

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Summary Differential amino acid reactivity with chemical probes can provide valuable information on the functionality and ligandability of proteins in native biological systems. Here, we present a quantitative, multiplexed chemical proteomic protocol for in-depth reactivity and ligandability profiling of cysteines in proteins in quiescent and stimulated T cells. This protocol illuminates dynamic immune state-dependent alterations in cysteine reactivity, revealing chemoselective and stereoselective small-molecule interactions with cysteines in structurally and functionally diverse proteins that lack chemical probes. For complete details on the use and execution of this protocol, please refer to Vinogradova et al. (2020) .

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An Activity-Guided Map of Electrophile-Cysteine Interactions in Primary Human T Cells

Cited by 209 publications

References 101 publications

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

Prioritization of antimicrobial targets by CRISPR-based oligo recombineering

The cGAS–STING pathway as a therapeutic target in inflammatory diseases

Multiplexed proteomic profiling of cysteine reactivity and ligandability in human T cells

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