Molecular Biology Of Outer Retina-specific Proteins

视网膜色素上皮（RPE）在外视网膜的发育和功能中起着关键作用。我们对RPE特异性机制感兴趣，无论是在调控水平还是功能水平，我们一直在研究RPE65的功能和调控，RPE65是一个基因，其表达仅限于RPE，以及导致人类严重失明的突变。Rpe65基因敲除小鼠的表型是由于基于rpe的维生素A视觉循环被破坏，该循环的作用是通过维生素A的异构化再生视觉色素色团。我们已经发现，在Rpe65基因敲除小鼠中，全反式视黄酰基酯过度积累，11-顺式视网膜完全缺失，这表明其作用与视黄醇异构体水解酶有关，视黄醇异构体水解酶是视觉色素色团再生的关键酶。最近，我们已经确定了RPE65在合成11-顺式视黄醇中的催化作用，并将其确定为长期寻找的视觉循环异构水解酶。我们还继续进行了β -胡萝卜素15,15'-单加氧酶（BCMO1）的研究。BCMO1与RPE65密切相关，两者都是新出现的类胡萝卜素裂解酶家族的成员。我们假设BCMO1和RPE65具有相似的作用机制。由于它们具有相同的结构特征，包括催化组合中的相同残基，我们发现BCMO1是我们研究RPE65机制的有用模型

The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels, and we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE, and mutations in which cause severe blindness in humans. The phenotype of the Rpe65 knockout mouse is due to disruption of the RPE-based vitamin A visual cycle whose role is to regenerate visual pigment chromophore by isomerization of vitamin A. We have shown that in the Rpe65 knockout mouse there is overaccumulation of all-trans-retinyl esters and total absence of 11-cis-retinal, suggesting a role related to that of retinol isomerohydrolase, the crucial enzyme in visual pigment chromophore regeneration. Recently, we have established a catalytic role for RPE65 in the synthesis of 11-cis retinol, identifying it as the long-sought visual cycle isomerohydrolase. We have also continued studies on beta-carotene 15,15'-monooxygenase (BCMO1). BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that BCMO1 and RPE65 share a similar mechanism of action. Because they share structural features, including identical residues in the catalytic assemblage, we have found BCMO1 to be a useful model for our mechanistic studies addressing RPE65 In the past year we have made the following progress: a) The best characterized RPE65 variant is the methionine (M) variant of mouse RPE65 residue 450 (normally L). This is associated with additionally lowered light sensitivity and with resistance to light damage in C57Bl/6 mice, consistent with lowered total activity and has contributed greatly to our understanding of the role of RPE65 in retinal physiology and retinal sensitivity to light-induced retinal damage. We determined how this variant affects RPE65 and how it is modulated by other rodent-specific variations. We found that wildtype dog RPE65 is more active than wildtype mouse RPE65, perhaps partially explaining the slower regeneration rate in the mouse. We also saw that the effect of Met at aa450 is more severe in mouse RPE65, reducing activity to 45% of wildtype, than in dog, where activity was reduced to 70% of wildtype. Interestingly, the effects of variation at residues 446 (K in rodents or R in all other species) modulated variation at aa450. The sensitivity of aa450 to change was underscored by the abolition of activity in the pathogenic human L450R mutation. These results suggest that subtle species-specific residue changes may be involved in tuning of RPE65 activity to required evolutionary criteria. b) While half of human RPE65 mutations are missense mutations, and many of these do not completely abolish isomerase activity (hypomorphic mutations), we do not have a clear understanding of why residual (not zero but less than 10% of wildtype activity) RPE65 activity allows usable vision early in life, while delaying but not preventing eventual retinal degeneration. We investigated the effect of missense mutations in RPE65 associated with less severe, but progressive, degenerations to understand their effects on protein structure and stability. We have found that many of these mutants have low isomerase activity (5-10%), but their carriers can retain usable vision into 3rd or 4th decade of life. The ultimate goal of this aspect of our research is to determine if the activity of such hypomorphic RPE65 mutants can be augmented by modulation of their stability by pharmacologic agents. c) To complement the work reported in (b), we are generating a panel of hypomorphic knock-in mice in the mouse Rpe65 gene by homologous recombination. It is anticipated that these will provide important insight into the variability of RPE65-deficient phenotypes, in comparison with the extreme case of the knockout. In particular, they are anticipated to provide insight into the slower progression of the retinal degeneration such as seen in less severe cases of human RPE65 mutations. They also will provide animal models to test pharmacologic strategies developed in (b). The first of these constructs have been taken to the stage of chimeric progeny and will be bred to generate the knock-in line. The second has successfully undergone homologous recombination, and is awaiting injection into embryos. The third is at stage of construction of targeting vector. d) Previously, we demonstrated a crucial role in enzymatic activity for histidine and acidic residues in BCMO1 (and RPE65) that we hypothesized to be involved in metal coordination. These observations, in conjunction with the predicted structure of a related bacterial enzyme, Synechocystis apocarotenal oxygenase (ACO) confirmed a catalytic role for iron in this family of proteins, but other crucial aspects of the mechanism (substrate binding, electron transfer, etc.) remain unclear. In light of the ACO structure we are investigating how BCMO1, BCMO2 and RPE65 have evolved to fulfill their functions utilizing the basic structure of the carotenoid oxygenase family. All 3 mammalian members of the family are expressed in retina and/or RPE, but RPE65 is specific to the RPE. In the past year, we have shown that mouse BCMO1 and mouse BCMO2 exhibit cleavage activity toward variety of carotenoids produced in E.coli (Beta-carotene, zeaxanthin, lycopene). Previously, activity of BCMO1 was shown only toward Beta-cryptoxanthin but not zeaxanthin in vitro. We observed degradation of zeaxanthin in E.coli by BCMO1 and BCMO2 and its attenuation by various introduced mutations. We demonstrated that the residues E405 (required for fixing of catalytic histidine #4) and Y326 (a residue modulating size of substrate tunnel) influence catalytic activity of BCMO1 more than the paralogous E417 and Q328 residues in BCMO2. The activity of BCMO1 and BCMO2 towards zeaxanthin opens up further questions regarding their role in retina, especially the human/primate retina where zeaxanthin is specifically concentrated in the macula.