Thermal Proteome Profiling Reveals Distinct Target Selectivity for Differentially Oxidized Oxysterols

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.

Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting OSBP

“…However, our results clearly differentiate the effect of orpinolide from 25-OHC where the functional dependence of orpinolide on active PI4P signaling was not recapitulated by 25-OHC. The promiscuity of 25-OHC, which interacts with a plethora of sterol-sensing proteins 52,53 , could serve as an explanation for this difference, but definitive elucidation will require further investigation. Along similar lines, several bioactive NPs have previously been reported as OSBP/ORP4 modulators and have collectively been referred to as 'ORPphilins' 40 .…”

Orpinolide disrupts a leukemic dependency on cholesterol transport by inhibiting the oxysterol-binding protein OSBP

“…However, our results clearly differentiate the effect of orpinolide from 25-OHC where the functional dependence of orpinolide on active PIP4 signaling was not recapitulated by 25-OHC. The promiscuity of 25-OHC, which interacts with a plethora of sterol-sensing proteins 47, 48 could serve as an explanation for this difference, but definitive elucidation will require further investigation. Along similar lines, several bioactive NPs have previously been reported as OSBP/ORP4 modulators and have collectively been referred to as “ORPphilins”.…”

Protein‐Metabolite Interactions: Discovery and Significance

“…This study led to the identification of more than 50 protein targets of staurosporine in mammalian cells. TPP has been used by several research groups to discover the protein binding partners of various metabolites in cells and in cell lysates, including ATP, [32] nucleotides, [33] and oxysterols [34] …”

“…[25] metabolites in cells and in cell lysates, including ATP, [32] nucleotides, [33] and oxysterols. [34] Taken together, ligand binding-induced alterations in protein stability has been powerfully exploited by several techniques including SPROX, TPP-MS and LiP-SMap discussed above. Despite this tremendous success, it is important to note that metabolite binding may not always significantly influence protein stability.…”