Biological and Medicinal Chemistry

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles



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.


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Supplementary material

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Supplemental Information
Supplemental Figures
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Table S1
CVs for PRM peptides.
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Table S2
Hsp40 affinity TMT-AP-MS results for acetochlor treatment.
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Table S3
Hsp40 affinity TMT-AP-MS results for alachlor treatment.
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Table S4
Hsp40 affinity TMT-AP-MS results for propachlor treatment.
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Table S5
Identified chloroacetanilide peptide conjugates.
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Table S6
Whole cell and insoluble TMT-MS results following propachlor treatment.