Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles.

Area of Science:

• Proteomics
• Toxicology
• Molecular Biology

Background:

• Chloroacetanilide herbicides can damage cellular proteins via nucleophilic substitution, leading to misfolding and proteostasis disruption.
• Existing methods for identifying toxicant-protein interactions primarily focus on direct conjugation, with limited approaches to assess broader proteome destabilization.

Purpose of the Study:

• To apply a quantitative proteomics methodology using a human Hsp40 chaperone mutant (DNAJB8 H31Q) to identify proteins destabilized by chloroacetanilide herbicides.
• To compare the efficacy of Hsp40 affinity profiling with activity-based protein profiling (ABPP) in identifying herbicide targets.

Main Methods:

• Quantitative proteomics was employed to profile chloroacetanilide-induced protein destabilization in HEK293T cells.
• Hsp40 affinity profiling using DNAJB8 H31Q was used to capture misfolded proteins.
• Activity-based protein profiling (ABPP) was utilized for comparison.

Main Results:

• Exposure to acetochlor, alachlor, and propachlor induced misfolding of numerous cellular proteins, with distinct yet overlapping profiles.
• Protein destabilization was concentrated in proteins containing reactive cysteine residues, with reactivity being idiosyncratic.
• Propachlor selectively targeted GAPDH and PARK7, decreasing their cellular activities and causing general protein aggregation.

Conclusions:

• Hsp40 affinity profiling is an effective strategy for identifying cellular proteins destabilized by toxicant exposure.
• This method identified a broader range of herbicide targets compared to ABPP.
• Propachlor's modification of GAPDH at a catalytic cysteine residue exemplifies direct conjugation leading to protein destabilization.