Thermal Proteome Profiling Reveals Distinct Target Selectivity for Differentially Oxidized Oxysterols

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Oxysterols are physiological cholesterol metabolites with unclear roles in human health.
• Their distinct functions are poorly understood, especially concerning varying oxidation states and sites.
• Oxysterols are implicated in diseases like atherosclerosis, neurodegeneration, and cancer.

Purpose of the Study:

• To systematically map the protein targets of different oxysterols.
• To understand how oxidation site and state influence oxysterol-protein interactions.
• To elucidate the biological pathways modulated by oxysterols.

Main Methods:

• Thermal proteome profiling was used to identify oxysterol-binding proteins.
• Selected targets were validated using cellular thermal shift assay and isothermal dose response fingerprinting.
• Three A- and B-ring oxidized sterols and 25-hydroxy cholesterol were analyzed.

Main Results:

• Each oxysterol exhibited unique target protein selectivity based on its oxidation site.
• Identified targets were predominantly involved in vesicular transport and phosphoinositide metabolism.
• The study revealed a significant impact of oxidation site on protein binding.

Conclusions:

• Oxysterol-protein interactions are highly specific, dictated by the site of oxidation.
• Despite unique targets, oxysterols modulate interconnected cellular processes.
• Understanding these interactions is crucial for deciphering oxysterol roles in physiology and disease.