Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

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.