Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure

Environmental toxins and toxicants can damage proteins and threaten cellular proteostasis. Most current methodologies to identify misfolded proteins in cells survey the entire proteome for sites of changed reactivity. We describe and apply a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. These protein targets are validated by an orthogonal limited proteolysis assay using parallel reaction monitoring. We find that a brief exposure of HEK293T cells to meta-arsenite increases the af inity of two dozen proteins to DNAJB8, including known arsenite-sensitive proteins. In particular, arsenite treatment destabilizes both the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. This platform can be used to explore how environmental toxins impact cellular proteostasis and to identify the susceptible proteome.

Automated summary

This study developed a quantitative proteomics methodology to identify destabilized proteins based on their binding to the human Hsp40 chaperone DNAJB8. Exposure of HEK293T cells to meta-arsenite increased the affinity of certain proteins to DNAJB8, including the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins. The platform can be used to investigate the impact of environmental toxins on cellular proteostasis and identify susceptible proteins.