Abstract
Purpose :
Cytochrome P450 1B1 (CYP1B1) mutations are the most frequent cause of Primary Congenital Glaucoma (PCG) globally; however, the mechanism/s by which the mutations cause glaucoma has not been yet elucidated. The purpose of this work was to investigate the binding ability of CYP1B1 to all trans retinal (t-RAL) and its effect on p53 protein levels and downstream pathways in human trabecular meshwork (HTM) cells.
Methods :
Protein-ligand binding studies were performed using molecular docking where Alpha naphthoflavone and 17β estradiol were used as positive controls, and microscale thermophoresis (MST) using wild type and G61E mutant CYP1B1. Expression of p53 and GADD45 was assessed by western blot in triplicate using protein extracted from primary HTM cells (from an 11-month-old donor) treated with different concentrations of t-RAL. Relative band densities were determined by densitometry and differences between control and each of the treatment groups were determined by using ANOVA. p≤0.05 was considered significant.
Results :
Molecular docking showed a binding affinity of CYP1B1 for t-RAL that was 1.5 and 2-fold higher than Alpha naphthoflavone and 17β estradiol respectively. With MST, the binding affinity of CYP1B1 to t-RAL was 3.5-fold greater in the wild type CYP1B1 compared to the mutant. t-RAL bound to wild type CYP1B1 with an average KD of 3338.75 ± 115.18 nM and to the mutant CYP1B1 with an average KD of 11608.05 ± 2289.5 nM (p=0.036). The average EC50 was 2764.55 ±139.5 and 4313 ±41.5 for wild type and mutant respectively. Treatment of HTM cells with t-RAL increased p53 and GADD45 expression in a dose-dependent manner. When compared to the control, p53 increased significantly by 1.92 and 3.3-fold in the 1 µM (p=0.03) and 5 µM (p=0.01) of t-RAL treatments respectively, whereas GADD45 increased by 1.25 and 3.48-fold in the 1 µM (p=0.03) and 5 µM (p=0.001) of t-RAL treatments respectively.
Conclusions :
These results suggest that CYP1B1 binds to t-RAL and may be involved in retinoic acid synthesis, and that mutations in CYP1B1 lead to t-RAL accumulation in the trabecular meshwork. The latter induces cellular stress and activates p53 resulting in the subsequent growth arrest phenotype like that seen in PCG. This study will help in shedding light on the pathophysiology of PCG and other diseases caused by mutations in CYP1B1.
This is a 2021 ARVO Annual Meeting abstract.