Quantification of cholesterol-metabolizing P450s CYP27A1 and CYP46A1 in neural tissues reveals a lack of enzyme-product correlations in human retina but not human brain.
Cytochrome P450 enzymes (CYP or P450) 46A1 and 27A1 play important roles in cholesterol elimination from the brain and retina, respectively, yet they have not been quantified in human organs because of their low abundance and association with membrane. On the basis of our previous development of a multiple reaction monitoring (MRM) workflow for measurements of low-abundance membrane proteins, we quantified CYP46A1 and CYP27A1 in human brain and retina samples from four donors. These enzymes were quantified in the total membrane pellet, a fraction of the whole tissue homogenate, using ¹⁵N-labled recombinant P450s as internal standards. The average P450 concentrations/mg of total tissue protein were 345 fmol of CYP46A1 and 110 fmol of CYP27A1 in the temporal lobe, and 60 fmol of CYP46A1 and 490 fmol of CYP27A1 in the retina. The corresponding P450 metabolites were then measured in the same tissue samples and compared to the P450 enzyme concentrations. Investigation of the enzyme-product relationships and analysis of the P450 measurements based on different signature peptides revealed a possibility of retina-specific post-translational modification of CYP27A1. The data obtained provide important insights into the mechanisms of cholesterol elimination from different neural tissues.
Institute for Bioscience and Biotechnology Research, Rockville, Maryland 20850, USA.
Wei-Li
WL
Liao
Gun-Young
GY
Heo
Nathan G
NG
Dodder
Rachel E
RE
Reem
Natalia
N
Mast
Suber
S
Huang
Pier Luigi
PL
Dipatre
Illarion V
IV
Turko
Irina A
IA
Pikuleva
D002462:Cell Membrane / Q000737:chemistry; D053493:Cholestanetriol 26-Monooxygenase / Q000032:analysis* / Q000378:metabolism; D002782:Cholestenes / Q000032:analysis / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D000072556:Cholesterol 24-Hydroxylase; D006801:Humans; D006888:Hydroxycholesterols / Q000032:analysis / Q000378:metabolism; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D009587:Nitrogen Isotopes; D015203:Reproducibility of Results; D012160:Retina / Q000737:chemistry*; D013250:Steroid Hydroxylases / Q000032:analysis* / Q000378:metabolism; D013702:Temporal Lobe / Q000737:chemistry*
D002782:Cholestenes; D006888:Hydroxycholesterols; D009587:Nitrogen Isotopes; C514718:cholestenoic acid; C044563:24-hydroxycholesterol; C076996:27-hydroxycholesterol; D002784:Cholesterol; D013250:Steroid Hydroxylases; D000072556:Cholesterol 24-Hydroxylase; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase