Biochemical Characterization of Human Cytochrome P450 7B1 Missense Mutations to Understand Spastic Paraplegia Type 5 Disease

Abstract ID 94281 Poster Board 036 Human cytochrome P450 7B1 (CYP7B1) is a steroidogenic heme-containing monooxygenase primarily expressed in liver and brain. In liver, CYP7B1 7a-hydroxylates substrates 25-hydroxycholesterol and 27-hydroxycholesterol to generate 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 accumulation in cerebrospinal fluid. These mutations cause a neurological disorder called spastic paraplegia type 5 (SPG5), in which patients suffer from severe progressive spasticity and weakness of the lower limbs due to the degeneration of their lower motor neurons. To date, little is known about how CYP7B1 mutations affect protein function to cause disease. Site-directed mutagenesis was performed to generate a number of CYP7B1 mutations causing SPG5. These mutated proteins are being expressed, purified, and analyzed with respect to protein folding/stability, heme incorporation, substrate binding, and substrate metabolism. The H285L, R417C, and R417H mutants yielded little or no folded protein after purification and did not have spectra indicating proper heme incorporation, suggesting significant protein instability. The R486C mutant has much lower yield than the wild type enzyme and has significant loss of heme during purification, suggesting that protein stability and folding are impacted; however, the remaining protein has spectra consistent with normal heme incorporation. In contrast, the T297A mutation has both yield and spectral characteristics similar to wild type, suggesting normal folding and heme incorporation. However, ligand binding assays indicate reduced substrate binding capacity and increased dissociation constants for both 25-hydroxycholesterol and 27-hydroxycholesterol, suggesting that the mutation interferes with CYP7B1 substrate interaction. Collectively, the functional information gathered from CYP7B1 mutations should facilitate the molecular understanding of SPG5 defects and potentially support future therapeutic treatments for SPG5 patients.