Table 1.
Genotype to Phenotype analysis for CYP1B1 variants implicated in POAG and PCG.
Fig 1.
CYP1B1 mutants assessed for 17β estradiol and retinol metabolizing activities.
Panel A: Most CYP1B1 mutants have lower 17β-estradiol metabolizing activity compared to the wild type protein. Forty-six hours post-transfection, the steroid metabolism activity of CYP1B1 was measured using the CYP450-GLO™ Assay kit. Protein expression of the wild type and mutant CYP1B1 at the time of enzyme assays was estimated by western blot (S3A Fig). Mutations reported to be associated with the type of glaucoma have been indicated by colored circles (POAG: red; PCG: blue; and POAG+PCG: green). Panel B: CYP1B1 mutants reported in POAG and PCG cases show differential patterns for retinol metabolizing activity. To assay retinoic acid metabolism activity, HEK 293T cells were transiently transfected with different CYP1B1 variant clones and CYP1B1 expression was allowed for 12h. Next, cells were transfected with an inducible RARE-responsive firefly luciferase construct mixed with a constitutively expressing Renilla luciferase construct available in the SA-Bioscience Kit. After another 16h, retinol was added to each well at 2μM concentration. Six hours post retinol treatment, cells were washed and lysed with luciferase cell lysis buffer. Firefly (FF) and Renilla Luciferase (RL) luminescence was measured using the Dual Luciferase kit from Promega. Each assay was performed in technical triplicates and repeated three times. The FF–RLU value was normalized by dividing with RL–RLU value. Cells expressing wild type and mutant CYP1B1 proteins convert retinol into RA, which binds the inducible-RARE construct and luminescence is generated. The enzyme activity of the mutant proteins was expressed as a percentage of the activity retained as compared to the native (wild type) enzyme. Protein expression of the wild type and mutant CYP1B1 at the time of enzyme assays was estimated by western blot (S3B Fig) Data represent the mean ± SEM for a triplicate per group. Data were tested with an unpaired t test. Differences in mean values were assessed for statistical significance (*, p< 0.01). Experiments were repeated three times.
Fig 2.
Protein turnover rate of CYP1B1 constructs.
Transfected HEK 293T cells were treated with CHX for 12h to inhibit protein synthesis. Twenty μg cell extracts were probed sequentially, by western blot analysis, with appropriate antibodies: Myc (to detect recombinant CYP1B1) Cell Signaling Technology, USA] and β-actin (to serve as a loading control) (Sigma, USA). Immunoblots were scanned, and net pixel intensities of the bands were measured with Image J software. CYP1B1 values were normalized to β-actin, the mean values were taken for three separate transfections, and the relative amounts of CYP1B1 were expressed as a percentage of levels of WT at respective time points. Representative lanes from western blots are furnished on the right side of the panels. Panel A: Mutations found in only POAG cases; Panel B: Mutations found in both POAG and PCG cases; Panel C: Mutations found in only PCG cases. Level of WT protein under similar conditions is shown in panel A. Data represent the mean ± SEM for a triplicate per group. Data were tested by an unpaired t test. Differences in mean were assessed for statistical significance (p< 0.01). Experiments were repeated three times.
Fig 3.
MD simulation analysis of the F261L and wild type CYP1B1 structures.
Panel A shows a similar distribution of the first three major principal components of F261L and wild type (WT) CYP1B1 structures, suggesting relatively unchanged functional motions in the F261L mutant as compared to the WT structure. Panel B shows the altered flexibility pattern of the F261L mutant as compared to wild type. The log2ratio is calculated as . Therefore a positive value indicates an increase in flexibility in the F261L mutant and a negative value indicates a decrease in flexibility as compared to WT CYP1B1. F261L mutant has a significantly altered flexibility pattern within the C-D, F and G'-H block regions, shown separately in the right hand side of the panel. Panel C shows the altered tunnels in two different orientations (top and bottom view) of the F261L and WT CYP1B1 structures. The upper panel of "Tunnel properties" shows the radius (in bar plot) and length (in black line) of the tunnels (Top view orientation) in the mutant (orange) and WT (green) structures while the lower panel shows the similar properties of tunnels observed in the bottom view orientation. Panel D shows docked retinol in the WT and F261L mutant CYP1B1 structures. The panel also shows an overall increase in binding energy in F261L mutant retinol binding observed through MD simulation.
Fig 4.
MD simulation analysis of the R117P and wild type CYP1B1 structures.
Panel A shows a multiple sequence alignment of CYP1B1 homologs. The red color indicates the conserved Arginine 117 position in other homologs. Thelower panel shows the structural importance of Arginine 117in their interaction with the O1A/O2A atom of the heme ligand. Panel B shows a similar distribution of first three major principal components of R117P and wild type (WT) CYP1B1 structures. Panel B shows that the R117P mutant possesses a significantly altered flexibility pattern within the B-C, G-H, and J-K block regions. Panel C shows the altered flexibility pattern in the R117P mutant as compared to WT. The log2 ratio is calculated as . Therefore, a positive value indicates an increase in flexibility in the R117P mutant and a negative value indicates a decrease in flexibility as compared to WT CYP1B1. The R117P mutant has a significantly altered flexibility pattern within the B-C, G-H and J-K block regions, shown separately in the right hand side of the panel. Panels D and E illustrate RMSD deviation and average bond angle deviation of the heme ligand in ΔRMSD and Δdegrees matrices, respectively. The difference matrices were calculated by subtracting RMSD and average bond angle values of mutant CYP1B1 from that of WT CYP1B1. The fluctuations and bond angle deviations in the initial stages of the MD simulation indicate a potential instability in the heme ligand binding affinity within the mutant protein.
Fig 5.
MD simulation analysis of the Q144R and wild type CYP1B1 structure.
Panel A shows the distribution of the first three major principal components of the Q144R and wild type (WT) structures. The distributions are observed to be different, suggesting an altered functional motion in the Q144R mutant. Panel B shows the altered flexibility pattern of the Q144R mutant as compared to WT. The log2 ratio is calculated as . Therefore a positive value indicates an increase in flexibility in the Q144R mutant and a negative value indicates a decrease in flexibility as compared to WT CYP1B1. The Q144R mutant has a significantly altered flexibility pattern within the C-D, F and G'-H block region, shown separately in the right hand side of the panel. Panel C shows the altered tunnels in two different orientations (top and bottom view) of the Q144R and WT CYP1B1 structures. The upper panel of "Tunnel properties" shows the radius (in bar plot) and length (in black line) of the tunnels (top view orientation) in the mutant (blue) and WT (green) structures. The lower panel shows the similar properties of the tunnels observed in the bottom view orientation. Panel D shows docked retinol in the WT and mutant CYP1B1 structures. The panel also shows an overall decrease in binding energy in retinol binding for the mutant protein, observed through MD simulation.
Fig 6.
MD simulation analysis of the Q144H and WT CYP1B1 structures.
Panel A shows marginally similar distributions of the first three major principal components of the Q144H and wild type (WT) CYP1B1 structures. Panel B shows that the Q144H mutant possesses a significantly altered flexibility pattern within the B-C, F-G and H block regions. The log2 ratio is calculated as . Therefore, a positive value indicates an increase in flexibility in the Q144H mutant and a negative value indicates a decrease in flexibility as compared to WT CYP1B1. The significantly altered flexible regions are shown separately in the right hand side of the panel. Panel C shows the altered tunnels in two different orientations (top and bottom view) of the Q144H and WT CYP1B1 structures. Interestingly no tunnel was observed in the top orientation of the Q144H structure. The upper panel of "Tunnel properties" shows the radius (in bar plot) and length (in black line) of the tunnels (Top view orientation) in the mutant (pink) and WT (green) structures. The lower panel shows similar properties of tunnels observed in the bottom view orientation. Panel D shows docked retinol in the WT and mutant CYP1B1 structures. The panel also shows an overall decrease in binding energy in retinol binding for the mutant protein, observed through MD simulation.
Fig 7.
Genotype to phenotype correlation for the role of CYP1B1 in glaucoma pathogenesis (PCG vs POAG).
The flowchart shows the potential activity of CYP1B1 variants for two different substrates (estradiol and retinol), as estimated by an in vitro cell based assay in HEK293T cells and attempted correlation of the biochemical activities (based on genotype) with potential glaucoma pathogenesis. Ref.(1)[18]; Ref.(2,3)[92, 93]; Ref.(4)[17]; Ref.(5,6)[17, 83].
Fig 8.
Summary of possible effects of mutations on CYP1B1 structure observed through MD simulation.