Prohibitin as the Molecular Binding Switch in the Retinal Pigment Epithelium

Abstract

Previously, our molecular binding study showed that prohibitin interacts with phospholipids, including phosphatidylinositide and cardiolipin. Under stress conditions, prohibitin interacts with cardiolipin as a retrograde response to activate mitochondrial proliferation. The lipid-binding switch mechanism of prohibitin with phosphatidylinositol-3,4,5-triphosphate and cardiolipin may suggest the role of prohibitin effects on energy metabolism and age-related diseases. The current study examined the region-specific expressions of prohibitin with respect to the retina and retinal pigment epithelium (RPE) in age-related macular degeneration (AMD). A detailed understanding of prohibitin binding with lipids, nucleotides, and proteins shown in the current study may suggest how molecular interactions control apoptosis and how we can intervene against the apoptotic pathway in AMD. Our data imply that decreased prohibitin in the peripheral RPE is a significant step leading to mitochondrial dysfunction that may promote AMD progression.

Keywords: Macular degeneration; Mitochondria; Oxidative stress; Prohibitin; Retinal pigment epithelium.

Figure 1. Multiple Sequence Alignment of Prohibitin and p47

(A) Sequence alignment of prohibitin and PX domain containing p47 Phox (A). The conserved basic amino acid residues (R 41 and R72) in prohibitin are homologous to the cationic residues in the putative PIP3 binding pocket (R43, R90) of p47^phox. (B) The sequence alignment SH3 domain containing PI3K and prohibitin is also shown to compare the presence of PX and SH3 domains.

Figure 2. Predicted Lipid Binding Pocket of Prohibitin

Based on sequence alignment, a 3D model of prohibitin was built using the 3D structure 2DYB chain ‘A’ as template. (A) 2DYB chain A is the Protein Databank structure for p47 Phox and it is based on the putative binding pocket residues that alignment with amino acids in the prohibitin sequence that can serve as a binding pocket in prohibitin-PIP3 binding (B). The p47 Phox template shares 17% identities with the query sequence using the ALIGN program.

Figure 3. Lipid binding and lipid-dependent prohibitin regulation

A. Western blotting using anti-prohibitin primary antibody demonstrates that prohibitin has different interactions with phospholipids, including cardiolipin and phosphatidylserine. Prohibitin in the retina has high affinity to cardiolipin, while prohibitin in the RPE shows weaker affinity. Microsomal prohibitin in the retina shows high affinity to phosphotidylserine, cardiolipin, and cholesterol. Microsomal fraction applied on the PVDF membrane is considered as a positive control. B. Cholesterol-dependent prohibitin expression in the RPE. ARPE-19 cells were treated with cholesterol (16 hours). Increased cholesterol levels significantly downregulate PHB1 expression whereas PHB2 levels show less sensitivity toward the elevated cholesterol levels. C. Graphical representation shows that cardiolipin levels decreased in human RPE under elevated oxidative stress.

Figure 4. Prohibitin-DNA binding analysis

A. Mitochondrial and nuclear PHB fractions from bovine retina were mixed with gel-purified mitochondrial (mt) and nuclear (nu) DNA extracted from bovine retina. DNA-protein complexes were analyzed by native-PAGE and prohibitin was quantitated by Western blotting analysis. B. Ten μg of total proteins in mitochondria (mt) were mixed with 5μg of mitochondrial DNA or TE buffer (negative control) followed by 40 minutes incubation at RT and centrifugation (16,000 × g, 30 minutes). Protein-DNA mixtures from upper half supernatant vs. bottom portion were subjected to SDS-PAGE (8–16%), and prohibitin expression was visualized by Western blotting analysis. Data showed mtPHB and mtDNA formed complexes and precipitated down to the bottom of the sample. Bands were quantified by QuantityOne software.

Figure 5

A. Downregulated prohibitin is shown by siRNA knockdown. ARPE-19 cells were transfected by prohibitin specific siRNA in serum free medium in a time-dependent manner (36 to 72 h). Prohibitin expression and depleted levels were analyzed by Western blotting analysis from cell extracts. Prohibitin levels were diminished during siRNA knockdown. β-actin was used as a loading control. B. Mitochondrial morphological changes during prohibitin siRNA knockdown analysis. ARPE-19 cells were incubated using prohibitin specific siRNA (175 ng for 48 h) and random sequence control. Prohibitin and organelles were visualized by immunocytochemical analysis using DAPI (blue, nucleus), MitoTracker Orange (red, mitochondria), and Alexa-Fluor 488 (green, prohibitin). Disrupted mitochondrial morphological changes were observed under prohibitin depleted levels. The scale bar represents 5 μm. C, D. ARPE19 cells were incubated using 100μM DMSO-dissolved melatonin for 30 mintures, followed by incubation in untreated medium (12 hours). Cells were visualized using prohibitin antibody and Alexa-Fluor-488 secondary antibody. Mitochondria and nucleus were labeled using MitoTracker Orange and DAPI respectively. Cell morphology was tracked at initial, half-hour, and 12-hour time points. Cells incubated for 12 hours are shown.

Figure 5

A. Downregulated prohibitin is shown by siRNA knockdown. ARPE-19 cells were transfected by prohibitin specific siRNA in serum free medium in a time-dependent manner (36 to 72 h). Prohibitin expression and depleted levels were analyzed by Western blotting analysis from cell extracts. Prohibitin levels were diminished during siRNA knockdown. β-actin was used as a loading control. B. Mitochondrial morphological changes during prohibitin siRNA knockdown analysis. ARPE-19 cells were incubated using prohibitin specific siRNA (175 ng for 48 h) and random sequence control. Prohibitin and organelles were visualized by immunocytochemical analysis using DAPI (blue, nucleus), MitoTracker Orange (red, mitochondria), and Alexa-Fluor 488 (green, prohibitin). Disrupted mitochondrial morphological changes were observed under prohibitin depleted levels. The scale bar represents 5 μm. C, D. ARPE19 cells were incubated using 100μM DMSO-dissolved melatonin for 30 mintures, followed by incubation in untreated medium (12 hours). Cells were visualized using prohibitin antibody and Alexa-Fluor-488 secondary antibody. Mitochondria and nucleus were labeled using MitoTracker Orange and DAPI respectively. Cell morphology was tracked at initial, half-hour, and 12-hour time points. Cells incubated for 12 hours are shown.

Figure 6

Prohibitin expressions in human AMD retina (8mm macular, peripheral retina) and RPE (8mm central and peripheral region, n=6 for two biological sample for triplicate experiments)) were analyzed by Western blotting. Retinal tissue and RPE cells were homogenized in RIPA buffer followed by sonication. As a loading control, β-tubulin was used. Prohibitin was analyzed quantitatively based on pixel size and intensity. A. Prohibitin in the macular and peripheral region from AMD retina. B. Prohibitin in the central and peripheral region from AMD RPE. Statistical comparisons between means were performed by 2-tailed t test. A p value of ≤0.05 was considered as statistically significant (P > 0.05 not significant; P ≤ 0.05 *; P ≤ 0.01 **; P 0.001 ***).

Figure 7

Prohibitin in control, diabetic retinopathy (DR) and aged retina (n=9 for biological triplicate and technical triplicate) were analyzed. Retinal tissue was homogenized in RIPA buffer followed by sonication. β-actin blot was used as a loading control. A. Prohibitin from diabetic retinopathy (DR) and aged mouse retina was compared to control. B. Prohibitin from diabetic retinopathy (DR) and aged rat retina was compared to control. C. Prohibitin from diabetic retinopathy (DR) and aged human retina was compared to control. Statistical comparisons between means were performed by 2-tailed t test. A p value of ≤ 0.05 was considered as statistically significant (P > 0.05 ns (not significant); P ≤ 0.05 *; P ≤ 0.01 **; P ≤ 0.001 ***).

Figure 8. Up- and downstream regulation of prohibitin network

Prohibitin bindings and up/down stream regulations are shown. Basic amino acids of N-terminus may catalyze PI3K pathway by PIP3 binging, whereas C-terminus cardiolipin binding may have a role as an anti-apoptotic signaling by mtDNA binding.