Defining human cytochrome P450 7B1 structure and function to understand spastic paraplegia type 5 disease - proposal abstract human membrane cytochrome P450 7B1 (CYP7B1) is a steroidogenic…

Defining human cytochrome P450 7B1 structure and function to understand spastic paraplegia type 5 disease - proposal abstract human membrane cytochrome P450 7B1 (CYP7B1) is a steroidogenic heme-containing monooxygenase primarily expressed in liver and brain. CYP7B1 performs 7-hydroxylation of 25- and 27-hydroxycholesterol to ultimately generate the primary bile acid chenodeoxycholic acid. Biallelic, missense mutations in CYP7B1 can result in the loss of enzymatic function, causing accumulation of 25-hydroxycholesterol and 27-hydroxycholesterol in plasma and 27-hydroxycholesterol in cerebrospinal fluid. These mutations cause a neurological disorder called spastic paraplegia type 5 (SPG5). SPG5 patients suffer from severe progressive spasticity and weakness of the lower limbs due to the degeneration of their lower motor neurons. To date, there are no curative or disease-modifying treatments available for SPG5 patients, and little is known about how CYP7B1 mutations affect protein function and cause disease. This proposal takes a first step toward addressing this knowledge GAP by determining wild type CYP7B1 interactions with its substrates and functional defects produced by common SPG5-causing missense mutations. First, a CYP7B1 X-RAY structure will identify active site residues involved in binding 25-hydroxycholesterol and 27-hydroxycholesterol. Preliminary investment has yielded highly purified CYP7B1 protein and 2-dimensional plate crystals with 25- hydroxycholesterol, substantial progress towards an X-RAY structure. Second, common SPG5-causing missense mutations will be assessed to determine protein folding and heme incorporation, ligand binding capacity, and product formation to understand mutation impact and severity. Preliminary results reveal a variety of unpredicted effects, including issues with folding and heme incorporation and reduced substrate binding capacity. Overall, this information will provide the first human CYP7B1 protein structure, permitting identification of active site residues involved in normal substrate binding and will provide the biological roles of CYP7B1 mutants commonly known to cause SPG5. This characterization is a first step in advancing our molecular understanding of SPG5 defects, and has the potential to support future therapeutic treatments for SPG5 patients.