Systematic identification of the HSP90 candidate regulated proteome

Area of Science:

• Proteomics
• Molecular Biology
• Cancer Research

Background:

• Heat shock protein 90 (HSP90) is crucial for protein folding and maturation, with over 200 identified clients relevant to cancer.
• HSP90 inhibition is a promising therapeutic strategy, with multiple inhibitors in clinical trials.
• Identifying all HSP90 clients and regulated proteins is vital for understanding its biological and medical significance.

Purpose of the Study:

• To comprehensively identify HSP90 clients and regulated proteins using a global chemical proteomic approach.
• To investigate the impact of HSP90 inhibition on protein turnover and kinase activity.
• To compare the molecular mechanisms of different HSP90 inhibitors.

Main Methods:

• Global chemical proteomics using stable isotope labeling with amino acids in cell culture (SILAC) and quantitative mass spectrometry.
• Gene ontology and pathway/network analysis.
• Protein turnover measurements using pulsed SILAC.
• HSP90 immunoprecipitation for client validation.

Main Results:

• Identification of >6200 proteins across four human cell lines, with ~1600 significantly regulated upon geldanamycin treatment.
• Discovery of 98 regulated protein kinases, including >50 previously unknown HSP90 targets.
• Correlation between protein down-regulation and protein half-life upon HSP90 inhibition, with kinases exhibiting shorter half-lives.
• Validation of novel HSP90 clients including kinases (AXL, DDR1, TRIO) and signaling proteins (BIRC6, ISG15, FLII).
• Highly similar proteomic responses observed for geldanamycin and PU-H71, suggesting shared molecular mechanisms.

Conclusions:

• HSP90 inhibition broadly impacts the cellular proteome, affecting protein folding, kinase activity, and protein turnover.
• Protein kinases are particularly sensitive to HSP90 inhibition, presenting both challenges and opportunities for cancer therapy.
• This study provides a valuable resource for understanding HSP90 function and developing targeted cancer treatments.