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Published on: March 24, 2012
Thermal Proteome Profiling Reveals Distinct Target Selectivity for Differentially Oxidized Oxysterols
Cecilia Rossetti1, Luca Laraia1
1Department of Chemistry, Technical University of Denmark, Kemitorvet 207, 2800, Kgs. Lyngby, Denmark.
Abstract:
Oxysterols are produced physiologically by many species; however, their distinct roles in regulating human physiology have not been studied systematically. The role of differing oxidation states and sites in mediating their biological functions is also unclear. As oxysterols have been associated with atherosclerosis, neurodegeneration, and cancer, a better understanding of their protein targets is desirable. To address this, we mapped the oxysterol interactome with three A- and B-ring oxidized sterols as well as 25-hydroxy cholesterol using thermal proteome profiling, validating selected targets with the cellular thermal shift assay and isothermal dose response fingerprinting. This revealed that the site of oxidation has a profound impact on target selectivity, with each oxysterol possessing an almost unique set of target proteins. Overall, targets clustered in pathways relating to vesicular transport and phosphoinositide metabolism, suggesting that while individual oxysterols bind to a unique set of proteins, the processes they modulate are highly interconnected.
Insights
Oxysterols, cholesterol metabolites, interact with unique protein targets based on oxidation site. These targets are involved in cellular transport and metabolism, revealing interconnected biological roles.
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.
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