Defects in CYB5A and CYB5B impact sterol-C4 oxidation in cholesterol biosynthesis and demonstrate regulatory roles of dimethyl sterols

Previous studies have shown that the microsomal form of CYB5 (CYB5A) is required for sterol C4-demethylation in vitro, but the in vivo experiments suggest that cholesterol biosynthesis remains intact in Cyb5a knockout mice. Here, we employed CRISPR-mediated knockout of CYB5A and the mitochondrial form of CYB5 (CYB5B) cells, along with AAV-mediated knockdown of Cyb5a in a liver-specific Cyb5b knockout mouse model, and used LC-MS/MS analysis to demonstrate the compensatory roles of CYB5A and CYB5B in SC4MOL-catalyzed sterol-C4 demethylation. Additionally, the analysis of mouse metabolic phenotypes combined with RNA-seq data indicates that the sterol intermediates, T-MAS and dihydro-T-MAS, can inhibit the SREBP pathway and activate the PPARγ pathway.