Hsp40 Affinity to Identify Proteins Destabilized by Cellular Toxicant Exposure

Area of Science:

• Biochemistry
• Cellular Biology
• Toxicology

Background:

• Environmental toxins pose a threat to cellular proteostasis by damaging proteins.
• Current methods for identifying misfolded proteins often involve broad proteome screening.
• Understanding how toxins affect protein stability is crucial for cellular health.

Purpose of the Study:

• To develop and apply a quantitative proteomics methodology for identifying destabilized proteins.
• To investigate the impact of environmental toxicants on cellular proteostasis.
• To identify specific proteins destabilized by toxic exposure.

Main Methods:

• Utilized a quantitative proteomics approach based on protein binding to the human Hsp40 chaperone DNAJB8.
• Validated identified protein targets using an orthogonal limited proteolysis assay with parallel reaction monitoring.
• Exposed HEK293T cells to meta-arsenite to assess toxin-induced protein destabilization.

Main Results:

• Identified over two dozen proteins exhibiting increased binding affinity to DNAJB8 after meta-arsenite exposure.
• Confirmed that arsenite treatment destabilizes known arsenite-sensitive proteins.
• Specifically found destabilization of the pyruvate dehydrogenase complex E1 subunit and several RNA-binding proteins.

Conclusions:

• The developed proteomics platform effectively identifies proteins destabilized by environmental toxins.
• This method provides insights into how toxicants disrupt cellular proteostasis.
• The platform can be used to map the susceptible proteome to various environmental insults.