Monohaloacetic Acids and Monohaloacetamides Attack Distinct Cellular Proteome Thiols

Abstract

Disinfection byproduct (DBP) exposure has been linked to multiple adverse health outcomes. However, the molecular initiating events by which DBPs induce their toxicities remain unclear. Herein, we combined reporter cell lines and activity-based protein profiling (ABPP) chemical proteomics to identify the protein targets of three monohaloacetic acids (mHAAs) and three monohaloacetamides (mHAMs), at the proteome-wide level. While mHAAs and mHAMs have similar potencies in reducing MTT activity, mHAMs induced greater Nrf2-mediated oxidative stress responses, demonstrating their distinct toxicity pathways. ABPP on crude cell lysates suggested that general proteome thiol reactivity correlates with cytotoxicity. Interestingly, live cell ABPP results revealed class-specific proteins attacked by mHAMs or mHAAs. Subsequent proteomic analysis identified >100 unique targets per DBP. mHAMs preferentially react with redox proteins including disulfide oxidoreductase enzymes, accounting for their stronger Nrf2 responses. To further probe alkylation mechanisms, we directly monitored protein adducts and identified 120 and 37 unique peptides with iodoacetamide and iodoacetic acid adducts, respectively. Of the latter, we confirmed glyceraldehyde-3-phosphate dehydrogenase as a key target of IAA, specifically attacking the catalytic Cys 152. This is the first study reporting the distinct cellular protein targets of mHAAs and mHAMs at the proteome-wide level, which highlights their different toxicity pathways despite their similar structures.

Quantitative Chemical Proteomics Reveals Interspecies Variations on Binding Schemes of L-FABP with Perfluorooctanesulfonate

“…Probe-based chemical proteomics, e.g. , activity-based protein profiling (ABPP), has made important progress in recent years to identify protein targets of small molecules on a proteome-wide level. − However, the application of probe-based chemical proteomics to quantitative chemical–protein interactions is limited by its intrinsic insensitivity to binding affinity. Methods for probe-free chemical proteomics have been recently developed based on protein thermal stability, conformation changes, or protease accessibility from our group and others. − Probe-free chemical proteomics is time-effective to evaluate many chemicals of interests, without the need for laborious chemical probe synthesis.…”

Comparison of Estrogenic, Spectroscopic, and Toxicological Analyses of Pilot-Scale Water, Wastewaters, and Processed Wastewaters at Select Military Installations

“…51,52 Recently, it was reported that DBPs attack cellular proteome thiols and are a molecular mechanism of toxicity. 55 Statistical Analyses. The cytotoxicity (LC 50 values), genotoxicity (50% tail DNA values), and thiol reactivity (EC 50 values) were calculated from the data using regression analysis.…”

“…A stoichiometric reaction between the thiol group and 5,5′-dithiobis-(2-nitrobenzoic acid) in which the disulfide bond is cleaved to produce 2-nitro-5-thiobenzoate was quantitatively measured at an absorbance of 412 nm . The metric for thiol reactivity was the EC 50 value, the CWS concentration factor that induced a reduction in the NAC thiol concentration by 50% as compared to concurrent negative controls. , Recently, it was reported that DBPs attack cellular proteome thiols and are a molecular mechanism of toxicity …”

Cellular Exposure to Chloroacetanilide Herbicides Induces Distinct Protein Destabilization Profiles

“…GAPDH is an enzyme that canonically uses a susceptible active-site cysteine to bind and reduce nicotinamide adenine dinucleotide (NAD) in glycolysis 72 , but consistent with its high abundance also engages in extensive moonlighting activities 73 . Due to its high abundance and pK-perturbed active site cysteines, GAPDH is a frequent conjugation target of electrophilic molecules 74,75 .…”