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Factors affecting protein recovery during Hsp40 affinity profiling.

Maureen R Montoya1, Guy M Quanrud1, Liangyong Mei2

  • 1Department of Chemistry, University of California, 501 Big Springs Rd, Riverside, CA, 92521, USA.

Analytical and Bioanalytical Chemistry
|June 8, 2024
PubMed
Summary

Hsp40 Affinity Profiling uses a modified Hsp40 protein to detect misfolded proteins under cellular stress. The DNAJB8H31Q variant effectively identifies destabilized proteins, aiding in disease diagnosis and biological characterization.

Keywords:
AP-MSDNAJB1DNAJB8Hsp40Misfolded proteinsProteomics

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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 FlagDNAJB8H31Q.
  • 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 DNAJB8H31Q and interacting proteins, indicating recognition of misfolded proteins.
  • Wild-type DNAJB8 (DNAJB8WT) 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 DNAJB8H31Q variant remains the most effective recognition element for recovering destabilized client proteins following cellular stress.
  • Hsp40 Affinity Profiling, particularly with the DNAJB8H31Q variant, offers a promising approach for bioanalytical characterization and disease diagnosis.
  • Understanding Hsp40-client interactions under stress provides insights into protein quality control mechanisms.