Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe

Jung, Ye‐Jin; Noda, Naotaka; Takaya, Junichiro; Abo, Masahiro; Toh, Kohei; Tajiri, Ken; Cui, Changyi; Zhou, Lu; Sato, Shin; Uesugi, Motonari

doi:10.1021/acschembio.1c00824

Cited by 11 publications

(16 citation statements)

References 52 publications

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“…GAPDH is an enzyme that canonically uses a pK-perturbed active-site cysteine to bind and reduce nicotinamide adenine dinucleotide (NAD) in glycolysis but consistent with its high abundance also engages in extensive moonlighting activities . Due to its high abundance and pK-perturbed active-site cysteines, GAPDH is a frequent conjugation target of electrophilic molecules. , GAPDH is found in Parkinson’s disease (PD)-associated aggregates, and its aggregation is promoted by electrophilic conjugation . Given its abundance and fold change in the DNAJB8 co-IP recovery, it is likely that GAPDH is the feature observed by silver stain at 37 kDa (Figure S4).…”

Section: Resultsmentioning

confidence: 99%

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

et al. 2023

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Herbicides in the widely used chloroacetanilide class harbor a potent electrophilic moiety, which can damage proteins through nucleophilic substitution. In general, damaged proteins are subject to misfolding. Accumulation of misfolded proteins compromises cellular integrity by disrupting cellular proteostasis networks, which can further destabilize the cellular proteome. While direct conjugation targets can be discovered through affinity-based protein profiling, there are few approaches to probe how cellular exposure to toxicants impacts the stability of the proteome. We apply a quantitative proteomics methodology to identify chloroacetanilide-destabilized proteins in HEK293T cells based on their binding to the H31Q mutant of the human Hsp40 chaperone DNAJB8. We find that a brief cellular exposure to the chloroacetanilides acetochlor, alachlor, and propachlor induces misfolding of dozens of cellular proteins. These herbicides feature distinct but overlapping profiles of protein destabilization, highly concentrated in proteins with reactive cysteine residues. Consistent with the recent literature from the pharmacology field, reactivity is driven by neither inherent nucleophilic nor electrophilic reactivity but is idiosyncratic. We discover that propachlor induces a general increase in protein aggregation and selectively targets GAPDH and PARK7, leading to a decrease in their cellular activities. Hsp40 affinity profiling identifies a majority of propachlor targets identified by competitive activity-based protein profiling (ABPP), but ABPP can only identify about 10% of protein targets identified by Hsp40 affinity profiling. GAPDH is primarily modified by the direct conjugation of propachlor at a catalytic cysteine residue, leading to global destabilization of the protein. The Hsp40 affinity strategy is an effective technique to profile cellular proteins that are destabilized by cellular toxin exposure. Raw proteomics data is available through the PRIDE Archive at PXD030635.

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Section: Resultsmentioning

confidence: 99%

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

et al. 2023

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“…The protein hits ALDH2, GSTO1, and GAPDH were further validated by pull-down and WB using the corresponding antibodies (Figure B). Notably, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a druggable target related to various diseases, , corresponds to the specific labeling band at low probe concentrations in different cancer cell lines (Figure E, right gel). Immunofluorescence experiments showed that the probe is co-localized well with GAPDH at the low probe concentration of 1 μM (Figure I).…”

Section: Resultsmentioning

confidence: 99%

Oxidant-Induced Bioconjugation for Protein Labeling in Live Cells

Liu

Zhang

et al. 2022

ACS Chem. Biol.

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Chemical proteomics is a powerful technology that can be used in the studies of the functions of uncharacterized proteins in the human proteome. It relies on a suitable bioconjugation strategy for protein labeling. This could be either a UV-responsive photo-crosslinker or an electrophilic warhead embedded in chemical probes that can form covalent bonds with target proteins. Here, we report a new protein-labeling strategy in which a nitrile oxide, a highly reactive intermediate that reacts with proteins, can be efficiently generated by the treatment of oximes with a water-soluble and a minimally toxic oxidant, phenyliodine bis (trifluoroacetate) (PIFA). The resulting intermediate can rapidly bioconjugate with amino acid residues of target proteins, thus enabling target identification of oxime-containing bioactive molecules. Excellent chemoselectivity of cysteine residues by the nitrile oxide was observed, and over 4000 reactive and/or accessible cysteines, including KRAS G12C, have been successfully characterized by quantitative chemical proteomics. Some of these residues could not be detected by conventional cysteine reagents, thus demonstrating the complementary utility of this method.

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“…GAPDH is an enzyme that canonically uses a susceptible active-site cysteine to bind and reduce nicotinamide adenine dinucleotide (NAD) in glycolysis 72 , but consistent with its high abundance also engages in extensive moonlighting activities 73 . Due to its high abundance and pK-perturbed active site cysteines, GAPDH is a frequent conjugation target of electrophilic molecules 74,75 .…”

Section: Small Structural Differences Have Been Shown To Have Large E...mentioning

confidence: 99%

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

Quanrud

Balamurugan

Canizal

et al. 2022

Preprint

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Herbicides in the popular chloroacetanilide class harbor a potent electrophilic moiety, which can damage proteins through nucleophilic substitution. In general, damaged proteins are subject to misfolding. Accumulation of misfolded proteins compromises cellular integrity by disrupting cellular proteostasis networks, which can further destabilize the cellular proteome. While direct conjugation targets can be discovered through affinity-based protein profiling, there are few approaches to probe how cellular exposure to toxicants impacts the stability of the proteome. We apply a quantitative proteomics methodology to identify chloroacetanilide-destabilized proteins in HEK293T cells based on their binding to the H31Q mutant of the human Hsp40 chaperone DNAJB8. We find that brief cellular exposure to the chloroacetanilides acetochlor, alachlor, and propachlor induces misfolding of dozens of cellular proteins. These herbicides feature distinct but overlapping profiles of protein destabilization, highly concentrated in proteins with reactive cysteine residues. Propachlor induces a general increase in protein aggregation, and selectively targets GAPDH and PARK7, leading to a decrease in their cellular activities. GAPDH is primarily modified by direct conjugation of propachlor at a catalytic cysteine residue, leading to global destabilization of the protein. The Hsp40 affinity strategy is an effective technique to profile cellular proteins that are destabilized by cellular toxin exposure. Raw proteomics data is available through the PRIDE Archive at PXD030635.

show abstract

Discovery of Non-Cysteine-Targeting Covalent Inhibitors by Activity-Based Proteomic Screening with a Cysteine-Reactive Probe

Cited by 11 publications

References 52 publications

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

Oxidant-Induced Bioconjugation for Protein Labeling in Live Cells

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

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