Profiling Chloroacetanilide Herbicide Protein Targets Using Hsp40 Affinity Profiling Proteomics

Description:
Environmental exposures can damage proteins, presenting a threat to cellular function and survival. Chemicals that modify the active site of an enzyme directly inhibit its catalytic function. If modifications change a protein’s stability, the protein can misfold or aggregate into proteotoxic species, causing a gain of toxic function. Because protein reactivity is idiosyncratic, cannot be predicted from primary sequence, and covers a large dynamic range, we cannot readily predict which proteins are targeted by individual toxicants, nor how they impact protein homeostasis. To identify the proteins targeted by cellular exposure, we have developed an Hsp40 affinity assay that profiles the cellular misfolded proteome. A mutant DNAJB8 binds to misfolded proteins, which are then isolated, labeled with isobaric tags, and identified/quantified by shotgun proteomics. We applied this assay to characterize the protein targets of a series of chloroacetanilide herbicides, which are widely used worldwide and are also alkylating agents. HEK293T cells were exposed to 1 mM of each herbicide for 30 min. We find 560 proteins significantly destabilized by propachlor exposure, 435 by alachlor, and 25 by acetochlor. About a quarter of targeted proteins are not iodoacetamide reactive, suggesting destabilization could proceed by mechanisms beyond cysteine conjugation. Despite structural similarities, acetochlor, propachlor, and alachlor target distinct proteomes, with only thymidylate synthase and acetyl-CoA acetyltransferase 1 being strong targets of all three. GAPDH and DJ-1 are strongly and uniquely targeted by propachlor, with loss of activity for both proteins. Damage to either of these proteins is associated with Parkinson's Disease, suggesting that propachlor might contribute to the well-established relationship between occupational herbicide exposure and Parkinson's Disease risk.

Speaker: Joseph Genereux - University of California, Riverside Department of Chemistry
Dr. Genereux received his B.A. in Chemistry from Swarthmore College, a B.S. in Physics at UC Irvine, and a PhD in Chemistry from Caltech characterizing the contribution of single-step transfer to DNA-mediated charge transport with Prof. Jackie Barton. Dr. Genereux worked with Profs. Jeffery Kelly and Prof. Luke Wiseman at TSRI as an NIH NRSA and AHA Postdoctoral Fellow, characterizing how the ER stress-responsive transcription factor ATF6 remodels proteostasis in the extracellular space. At UC Riverside, the Genereux lab applies its expertise in mass spectrometry-based proteomics to probe molecular aspects of proteostasis. To enable these studies they have developed methodology for profiling misfolded and mislocalized proteins in the cell.