Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

Abstract

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.

Reversible Covalent Inhibition─Desired Covalent Adduct Formation by Mass Action

“…Furthermore, covalent inhibitors may induce misfolding of proteins which could lead to a global protein misfolding profiles and responses. 57,58 Additionally, T-cell activation due to chemically reactive intermediates are not necessarily identifiable through MS methods. 49 These can occur via the formation of drug haptens (covalent adducts of proteins) which subsequently induce Tcell-mediated drug-hypersensitivity reactions.…”

“…Covalent inhibition of an enzyme may lead to misfolding of the protein. 57,58 If this is nonspecific, this may lead to global protein misfolding stress responses.…”

Profiling protein targets of cellular toxicant exposure

“…171 We have applied our assay to several major agents of exposure. 146,172 Just 15 min of cellular exposure to arsenic (sodium arsenite; 100 mM) is adequate to induce destabilization of several proteins, primarily RNA binding proteins such as TDP43 and HNRNPA0 and including the pyruvate dehydrogenase component PDHA1 146 (Fig. 7).…”

“…We also used this approach to profile several herbicides from the electrophilic chloroacetanilide class: propachlor, alachlor, and acetochlor. 172 These each share a highly reactive chloroacetamide motif, differing only slightly in the distribution of methyl/ethyl groups. Despite the profound methodological difference between this assay (Hsp40 affinity in HEK239T cells) and the previous ABPP study (conjugation to liver proteins in living mice), 85 the targets of acetochlor show a high degree of overlap.…”

“…175 Similarly, we used PRM with limited proteolysis to validate metal and chloroacetanilide targets first identified by the Hsp40 affinity assay. 146,172 The ready adaptation of mass spectrometry-based protein profiling technologies to targeted experiments has another potential but unrealized benefit. Laboratory toxicity studies frequently employ toxin concentrations much higher than natural exposures.…”