Genotype to Phenotype analysis for CYP1B1 variants implicated in POAG and PCG.

<p>Genotype to Phenotype analysis for CYP1B1 variants implicated in POAG and PCG.</p

CYP1B1 mutants assessed for 17β estradiol and retinol metabolizing activities.

<p><b><i>Panel A</i>: <i>Most CYP1B1 mutants have lower 17β-estradiol metabolizing activity compared to the wild type protein</i>.</b> 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.s003" target="_blank">S3A Fig</a>). Mutations reported to be associated with the type of glaucoma have been indicated by colored circles (POAG: red; PCG: blue; and POAG+PCG: green). <b><i>Panel B</i>: <i>CYP1B1 mutants reported in POAG and PCG cases show differential patterns for retinol metabolizing activity</i>.</b> 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 <i>Renilla</i> 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 <i>Renilla</i> 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 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.s003" target="_blank">S3B Fig</a>) Data represent the mean ± SEM for a triplicate per group. Data were tested with an unpaired <i>t</i> test. Differences in mean values were assessed for statistical significance (*, <i>p</i>< 0.01). Experiments were repeated three times.</p

MD simulation analysis of the R117P and wild type CYP1B1 structures.

<p><b><i>Panel A</i></b> 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. <b><i>Panel B</i></b> shows a similar distribution of first three major principal components of R117P and wild type (WT) CYP1B1 structures. <i>Panel B</i> shows that the R117P mutant possesses a significantly altered flexibility pattern within the B-C, G-H, and J-K block regions. <b><i>Panel C</i></b> 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. <b><i>Panels D</i> and <i>E</i></b> 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.</p

MD simulation analysis of the Q144H and WT CYP1B1 structures.

<p><b><i>Panel A</i></b> shows marginally similar distributions of the first three major principal components of the Q144H and wild type (WT) CYP1B1 structures. <b><i>Panel B</i></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. <b><i>Panel C</i></b> 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 <b>bar plot</b>) and length (in <b>black line</b>) 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. <b><i>Panel D</i></b> 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.</p

Summary of possible effects of mutations on CYP1B1 structure observed through MD simulation.

<p>Summary of possible effects of mutations on CYP1B1 structure observed through MD simulation.</p

Genotype to phenotype correlation for the role of <i>CYP1B1</i> in glaucoma pathogenesis (PCG vs POAG).

<p>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⁾[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref018" target="_blank">18</a>]; Ref.^(2,3)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref092" target="_blank">92</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref093" target="_blank">93</a>]; Ref.⁽⁴⁾[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref017" target="_blank">17</a>]; Ref.^(5,6)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref017" target="_blank">17</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156252#pone.0156252.ref083" target="_blank">83</a>].</p

MD simulation analysis of the F261L and wild type CYP1B1 structures.

<p><b><i>Panel A</i></b> 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. <b><i>Panel B</i></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. <b><i>Panel C</i></b> 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 <b>bar plot</b>) and length (in <b>black line</b>) 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. <b><i>Panel D</i></b> 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.</p

Mitochondrial Genome Analysis of Primary Open Angle Glaucoma Patients

<div><p>Primary open angle glaucoma (POAG) is a multi-factorial optic disc neuropathy characterized by accelerating damage of the retinal ganglion cells and atrophy of the optic nerve head. The vulnerability of the optic nerve damage leading to POAG has been postulated to result from oxidative stress and mitochondrial dysfunction. In this study, we investigated the possible involvement of the mitochondrial genomic variants in 101 patients and 71 controls by direct sequencing of the entire mitochondrial genome. The number of variable positions in the mtDNA with respect to the revised Cambridge Reference Sequence (rCRS), have been designated “Segregating Sites”. The segregating sites present only in the patients or controls have been designated “Unique Segregating Sites (USS)”. The population mutation rate (<i>θ = 4N_eμ</i>) as estimated by Watterson’s θ (θ_w), considering only the USS, was significantly higher among the patients (p = 9.8×10⁻¹⁵) compared to controls. The difference in θ_w and the number of USS were more pronounced when restricted to the coding region (p<1.31×10⁻²¹ and p = 0.006607, respectively). Further analysis of the region revealed non-synonymous variations were significantly higher in Complex I among the patients (p = 0.0053). Similar trends were retained when USS was considered only within complex I (frequency 0.49 vs 0.31 with p<0.0001 and mutation rate p-value <1.49×10⁻⁴³) and <i>ND5</i> within its gene cluster (frequency 0.47 vs 0.23 with p<0.0001 and mutation rate p-value <4.42×10⁻⁴⁷). <i>ND5</i> is involved in the proton pumping mechanism. Incidentally, glaucomatous trabecular meshwork cells have been reported to be more sensitive to inhibition of complex I activity. Thus mutations in <i>ND5</i>, expected to inhibit complex I activity, could lead to generation of oxidative stress and favor glaucomatous condition.</p></div

Efficient Delivery of Cell Impermeable Phosphopeptides by a Cyclic Peptide Amphiphile Containing Tryptophan and Arginine

Phosphopeptides are valuable reagent probes for studying protein–protein and protein–ligand interactions. The cellular delivery of phosphopeptides is challenging because of the presence of the negatively charged phosphate group. The cellular uptake of a number of fluorescent-labeled phosphopeptides, including F′-Gp­YLP­QTV, F′-NEp­YTA­RQ, F′-AE­EEI­YGE­FEA­KK­KK, F′-PEp­YLG­LD, F′-pY­VNV­QN-NH₂, and F′-GpYEEI (F′ = fluorescein), was evaluated in the presence or absence of a [WR]₄, a cyclic peptide containing alternative arginine (R) and tryptophan (W) residues, in human leukemia cells (CCRF-CEM) after 2 h incubation using flow cytometry. [WR]₄ improved significantly the cellular uptake of all phosphopeptides. PEpYLGLD is a sequence that mimics the pTyr1246 of ErbB2 that is responsible for binding to the Chk SH2 domain. The cellular uptake of F′-PEpYLGLD was enhanced dramatically by 27-fold in the presence of [WR]₄ and was found to be time-dependent. Confocal microscopy of a mixture of F′-PEp­YLG­LD and [WR]₄ in live cells exhibited intracellular localization and significantly higher cellular uptake compared to that of F′-PEpYLGLD alone. Transmission electron microscopy (TEM) and isothermal calorimetry (ITC) were used to study the interaction of PEp­YLG­LD and [WR]₄. TEM results showed that the mixture of PEp­YLG­LD and [WR]₄ formed noncircular nanosized structures with width and height of 125 and 60 nm, respectively. ITC binding studies confirmed the interaction between [WR]₄ and PEp­YLG­LD. The binding isotherm curves, derived from sequential binding models, showed an exothermic interaction driven by entropy. These studies suggest that amphiphilic peptide [WR]₄ can be used as a cellular delivery tool of cell-impermeable negatively charged phosphopeptides

Distribution of segregating sites in different regions of mtDNA.

<p>The frequency of segregating sites was marginally higher for coding regions in the patients relative to the controls (0.69 vs 0.62, p = 0.0347). No significant difference was observed in the RNA and the control region.</p