Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

Abstract

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.

Factors Affecting Protein Recovery During Hsp40 Affinity Profiling

“…Every experiment involving DNAJB8 used one 10 cm plate per condition. 4-plex experiments involving DNAJB1 used two 10 cm plates per condition to account for its lower overexpression. Where heat shock was applied, cells were placed in an incubator at the indicated temperature for 30 min.…”

“…A variety of exposure agents can damage proteins, threatening this maintenance of proteome integrity [2]. To evaluate these threats, we recently introduced Hsp40 Affinity Profiling as a technology for identifying proteins that are destabilized by cellular toxicant exposure [3][4][5]. It exploits the capacity of Hsp40 family proteins (also called J-domain proteins) to recognize and bind to misfolded proteins [6,7].…”

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.…”

“…Performing competition reactions of the protein target and glutathione with RCIs in addition to monitoring for glutathione depletion in cells , can provide helpful information on the effect of RCIs on electrophilic stress in cells. Furthermore, covalent inhibitors may induce misfolding of proteins which could lead to a global protein misfolding profiles and responses. , …”