Factors affecting protein recovery during Hsp40 affinity profiling

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Identifying misfolded proteins in complex biological samples is crucial for understanding cellular stress responses and diagnosing diseases.
• Current bioanalytical methods face challenges in accurately quantifying misfolded proteins.

Purpose of the Study:

• To evaluate and improve the performance of Hsp40 Affinity Profiling, a novel bioanalytical assay for profiling protein stability under cellular stress.
• To assess the efficacy of different strategies, including intracellular crosslinking and J-domain ablation, in enhancing protein interactor recovery.

Main Methods:

• Development of Hsp40 Affinity Profiling using ectopically introduced Hsp40 ^FlagDNAJB8^H31Q.
• Application of quantitative proteomics to measure changes in protein affinity for DNAJB8 under cellular stress.
• Evaluation of intracellular crosslinking and J-domain ablation modifications.

Main Results:

• Cellular thermal stress increases the affinity between DNAJB8^H31Q and interacting proteins, indicating recognition of misfolded proteins.
• Wild-type DNAJB8 (DNAJB8^WT) did not show this stress-induced affinity increase, suggesting a role in handing off misfolded proteins to Hsp70.
• While modifications like crosslinking and J-domain ablation improved interactor recovery for DNAJB1, they were offset by lower DNAJB1 yield.

Conclusions:

• The DNAJB8^H31Q variant remains the most effective recognition element for recovering destabilized client proteins following cellular stress.
• Hsp40 Affinity Profiling, particularly with the DNAJB8^H31Q variant, offers a promising approach for bioanalytical characterization and disease diagnosis.
• Understanding Hsp40-client interactions under stress provides insights into protein quality control mechanisms.