The expression and purification of human steroid hormone processing enzyme CYP7B1 and the characterization of its interactive network

Abstract

The cytochrome P450 monooxygenase CYP7B1 is an integral membrane protein at the endoplasmic reticulum (ER) membrane and catalyzes the hydroxylation of 7-alpha position of the steroid backbone, generating a series of important steroids such as 7α-hydroxy dehydroepiandrosterone and 7α-hydroxy pregnenolone. The disruption of CYP7B1 function could lead to abnormal oxysterols levels in neurons and the liver, and thus result in diseases such as hereditary spastic paraplegia and congenital bile acid synthesis defect. CYP7B1 was found to play a critical role in the biochemical network for biosynthesis/modification of steroids which involves hydrogenation/dehydrogenation, hydroxylation, oxygenation reaction at various carbon positions of the steroid backbone. Although a biologically-related enzyme, CYP7A1, has been successfully purified and structurally characterized, little was known about the structure and catalyzing mechanism for CYP7B1. To understand the structural basis of CYP7B1 functions, recombinant expression systems were screened for high-level expression and purification. Humans CYP7B1 was successfully expressed in the baculovirus/insect cell system and purified with its redox cofactor, indicating a natural folding for CYP7B1 samples. When challenged with detergents of various lengths of hydrophobic side chains, purified CYP7B1 proteins showed moderate stability but failed to maintain the natural folding upon the “harsh” detergent such as octyl glucoside. To explore the interactive proteins for CYP7B1, a pulldown-mass spectrometry assay using purified CYP7B1 was conducted and identified multiple ER-resident enzymes including cytochrome P450 reductase CYPOR as interactive proteins for CYP7B1, as well as other redox enzymes such as cytochrome b5 reductase and cytochrome b5 family members, indicating the existence of a physical interactive network at ER compromising cytochrome P450 and b5 family members and their reductases. Further efforts were conducted to confirm molecular interaction from mass-spectral assay and the in vitro macromolecular interaction assay showed the association between CYP7B1 and CYPOR. The CYP7B1-CYPOR protein complex was purified upon the optimization of co-expression system and in vitro assembly protocols, implying a catalytic complex might exist in ER membrane comprising CYP7B1, CYPOR, and other redox enzymes. Our results showed that human cytochrome P450 protein CYP7B1 could be purified in its cofactor-bound form and bind to cytochrome P450 reductase CYPOR to form a stable complex resistant to solubilization by detergent. Further studies should be conducted to map the components and interactions in the redox-enzyme complex at ER and determine the structural basis for the electron transfer therein.