Using Hsp40 Affinity to Profile Destabilized Proteomes
Proteins in their native or optimal conformational states can perform essential cellularfunctions to assist in the stability of the proteome and the overall health of the cell. However, if a protein misfolds, it instead can gain a new toxic function. Misfolded proteins can aggregate into more stable toxic precursors that can cause cellular degradation and ultimately induce health disorders such as Parkinson’s Disease, Alzheimer’s Disease, and Huntington’s Disease. Cellular stresses are significant factors in protein misfolding. These stimuli can change protein structure through several mechanisms such as covalent modification of cysteines or oxidation of methionine residues. The immediate consequence of posttranslational modifications of a protein from stress is difficulty in refolding. The long term consequence is that these altered proteins can instead gain new toxic functions as they proceed to aggregate. Therefore, changes to protein structures induced by stresses can create cellular havoc as the cell scrambles to recover from the affected misfolded proteins. Several analytical assays can measure the effects of stress on protein stability. viii Flourescence assays show direct changes to a protein based on gain or loss of signal. Mass Spectrometry surveys entire proteomes by measuring a protein’s ability to react after exposure to a cellular stress. Herein, we describe a quantitative proteomics approach that measures proteins binding to Hsp40 chaperone DNAJB8, a protein quality control factor designed to recognize misfolded proteins. We aimed to use the chaperone to identify destabilized proteins and deduce toxic cellular mechanisms arising from environmental stresses. We found several ribosomal proteins such as TAR DNA-Binding protein (TDP-43) and Pyruvate Dehydrogenase E1 subunit (PDHA1) misfolded after arsenite exposure. Later, we found potential biomarkers in GAPDH and PARK7 involved in driving the cellular toxicity of propachlor. Led by limited proteolysis, we conducted several validation experiments to show that DNAJ8 recognized significantly destabilized proteins after exposure to environmental toxins. In total, we hope to show the effectiveness of this approach in exploring how environmental toxins can impact cellular proteostasis and identifying the resulting susceptible proteome.