The visual cycle is a series of enzymatic reactions essential for regenerating 11-cis retinal (11cRAL), which functions as a molecular switch for activating opsin in response to light stimulation. 11cRAL keeps opsin inactive. When light hits the visual pigments, its energy converts the pigments'11cRAL into all-trans isomer, thus activating opsin. The activated opsin then triggers phototransduction, which converts the light-activated biochemical signal to an electrical energy by closing the cGMP-gated ion channels in the photoreceptors. Since the opsin that lost 11cRAL is no longer responsive to light, 11cRAL must be regenerated via the visual cycle to re-form the light-sensitive visual pigments. The key enzymatic reaction in the visual cycle is the isomerization of the all-trans retinoid to an 11-cis isomer. We have identified RPE65, a retinal pigment epithelium (RPE) specific membrane-associated protein, as the retinoid isomerase. Mutations in the human RPE65 gene have been associated with an early-onset retinal degenerative disease known as Leber congenital amaurosis (LCA). Despite the importance of this protein, the molecular mechanisms that regulate the function of RPE65 are largely unknown. The long-term goals of our research are (1) to define the mechanisms that regulate the enzymatic activity of normal and LCA-associated (LCAA) RPE65s and (2) to develop a novel and effective strategy for rescuing LCAA RPE65s. Identification and characterization of proteins that regulates RPE65 function is the key to defining the mechanisms. By screening of the bovine RPE expression library, we have identified three negative regulators of RPE65. The goal of Specific Aim 1 of this proposal is to elucidate the action mechanisms of the negative regulators in vitro and determine their functional role in the visual cycle in vivo. As a step toward defining the inhibitory mechanisms of RPE65 by the regulators, we will first test if the new regulators bind with RPE65. Next we will test if the new regulator can compete with RPE65 for binding to the substrate of RPE65. We will then determine the role of the new regulator in vision by analyzing the visual cycle-related phenotypes of the mutant mice that lack the new regulator. The goal of Specific Aim 2 of this project is to determine whether the new regulators are involved in the pathological mechanism of the LCAA RPE65 mutations and to define the molecular basis for the pathogenicity of LCAA RPE65s with non-active site missense mutations. The proposed research is innovative because it can establish a new research direction in regulation of isomerase activities of normal and LCAA RPE65s and can lead to the development of a novel rescue strategy for delaying or preventing vision loss and photoreceptor degeneration in patients with LCA that is caused by mutations in the RPE65 gene.

Public Health Relevance

Relevance: Mutations in the human RPE65 gene cause early onset childhood blindness known as Leber's congenital amaurosis (LCA). The proposed research project will increase our understanding of how the function of the normal and LCA-associated mutant RPE65s is regulated. The knowledge gained from this research will facilitate the development of a novel and effective therapeutic intervention to delay or prevent vision loss in patients with LCA.

National Institute of Health (NIH)
National Eye Institute (NEI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-BDPE-J (09))
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Shen, Grace L
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Louisiana State Univ Hsc New Orleans
Schools of Medicine
New Orleans
United States
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Jin, Minghao; Li, Songhua; Hu, Jane et al. (2016) Functional Rescue of Retinal Degeneration-Associated Mutant RPE65 Proteins. Adv Exp Med Biol 854:525-32
Li, Songhua; Samardzija, Marijana; Yang, Zhihui et al. (2016) Pharmacological Amelioration of Cone Survival and Vision in a Mouse Model for Leber Congenital Amaurosis. J Neurosci 36:5808-19
Lee, Minsup; Li, Songhua; Sato, Kota et al. (2016) Interphotoreceptor Retinoid-Binding Protein Mitigates Cellular Oxidative Stress and Mitochondrial Dysfunction Induced by All-trans-Retinal. Invest Ophthalmol Vis Sci 57:1553-62
Rice, Dennis S; Calandria, Jorgelina M; Gordon, William C et al. (2015) Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival. Nat Commun 6:6228
Rice, Dennis S; Calandria, Jorgelina M; Gordon, William C et al. (2015) Corrigendum: Adiponectin receptor 1 conserves docosahexaenoic acid and promotes photoreceptor cell survival. Nat Commun 6:7225
Li, Songhua; Hu, Jane; Jin, Robin J et al. (2015) Temperature-sensitive retinoid isomerase activity of RPE65 mutants associated with Leber Congenital Amaurosis. J Biochem 158:115-25
Li, Songhua; Izumi, Tadahide; Hu, Jane et al. (2014) Rescue of enzymatic function for disease-associated RPE65 proteins containing various missense mutations in non-active sites. J Biol Chem 289:18943-56
Li, Songhua; Lee, Jungsoo; Zhou, Yongdong et al. (2013) Fatty acid transport protein 4 (FATP4) prevents light-induced degeneration of cone and rod photoreceptors by inhibiting RPE65 isomerase. J Neurosci 33:3178-89
Sato, Kota; Li, Songhua; Gordon, William C et al. (2013) Receptor interacting protein kinase-mediated necrosis contributes to cone and rod photoreceptor degeneration in the retina lacking interphotoreceptor retinoid-binding protein. J Neurosci 33:17458-68
Li, Songhua; Yang, Zhihui; Hu, Jane et al. (2013) Secretory defect and cytotoxicity: the potential disease mechanisms for the retinitis pigmentosa (RP)-associated interphotoreceptor retinoid-binding protein (IRBP). J Biol Chem 288:11395-406

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