Cholesterol is essential for development, growth, and maintenance of organisms. Mutations in cholesterol biosynthetic genes are embryonic lethal and few polymorphisms have been so far associated with pathologies in humans. Previous analyses show that lanosterol 14?-demethylase (CYP51A1) from the late part of cholesterol biosynthesis has only a few missense mutations with low minor allele frequencies and low association with pathologies in humans. The aim of this study is to evaluate the role of amino acid changes in the natural missense mutations of the hCYP51A1 protein. We searched SNP databases for existing polymorphisms of CYP51A1 and evaluated their effect on protein function. We found rare variants causing detrimental missense mutations of CYP51A1. Some missense variants were also associated with a phenotype in humans. Two missense variants have been prepared for testing enzymatic activity in vitro but failed to produce a P450 spectrum. We performed molecular modeling of three selected missense variants to evaluate the effect of the amino acid substitution on potential interaction with its substrate and the obligatory redox partner POR. We show that two of the variants, R277L and especially D152G, have possibly lower binding potential toward obligatory redox partner POR. D152G and R431H have also potentially lower affinity toward the substrate lanosterol. We evaluated the potential effect of damaging variants also using data from other in vitro CYP51A1 mutants. In conclusion, we propose to include damaging CYP51A1 variants into personalized diagnostics to improve genetic counseling for certain rare disease phenotypes.
B.06 Other
COBISS.SI-ID: 6312730co-mentor for master thesis Lanosterol 14?-demethylase (CYP51A1), a member of the cytochrome P450 superfamily, is an essential enzyme in cholesterol biosynthesis. The catalytic activity and structure of CYP51 is evolutionarily conserved and the gene has a low number of polymorphisms. In a database of 1000 human genomes we identified natural single-nucleotide polymorphisms (SNP) that cause missense aminoacid substitutions with damaging SIFT and PolyPhen-2 predictions. We chose the mutations R271L and R425H (Q16850, UniProt) for the experimental analysis and prepared them with a site-directed mutagenesis. We expressed the native and mutant proteins in Esherichia coli and isolated them from the spheroplasts fractions. We isolated a catalytically active native protein with an absorbance peak in CO-difference spectra at 447 nm, but failed to isolate the active mutants hCYP51-R271L and hCYP51-R425H, which had an absorbance peak at 417 nm. To evaluate the substitution effect of the aminoacid on enzyme substrate binding and interaction with POR (cytochrome P450 oxireductase), we also performed molecular dynamics simulations with polymorphism D146G. The results show that the mutations R425H and D146D have damaging effects on the interaction between hCYP51 and the substrate, whereas the mutations R271L and D146G prevent the interaction between hCYP51 and the redox partner POR. However, we were unable to produce active mutants and were therefore unable to test the catalytic efficiency of the mutants in vitro. We presume that the changes R271L and R425H have a damaging effect on conserved structure of hCYP51. The mutations effect the protein structure in such a way that the heme-thiolate bond cannot form, thus making the mutants inactive.
D.10 Educational activities
COBISS.SI-ID: 1537489347