MOLECULAR BIOLOGY OF OUTER RETINA-SPECIFIC PROTEINS

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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. To this end 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. To better understand the function of RPE65 and its role in RPE/photoreceptor interactions we have made an Rpe65 knockout mouse. Disruption of the RPE-based metabolism of all-trans-retinyl esters to 11-cis-retinal appears to underlie its phenotype. The function of RPE65 thus appears to be closely related to that of the retinol isomerase, the crucial enzyme in visual pigment regeneration. In the past year we have made the following progress: a) Analysis of visual cycle proteins downstream of the isomerization step shows that all are present in normal or close to normal amounts in the Rpe65 knockout. Gas chromatography/mass-spectroscopy of the retinyl esters in the RPE of the knockout and wildtype mice reveals that there is no chemical difference between the two. This implies that in the Rpe65 knockout mouse, lack of 11-cis-retinal is not due to the absence of subsequent processing of the isomerization product but rather to lack of the isomerization product, or due to the presence of inappropriate retinyl esters (isomerase substrate). b) Adenoviral and lentiviral constructs are being made and tested for gene transfer rescue of the knockout mouse. c) Further analysis of the RPE65 promoter was targeted at identifying factors binding to the putative control region. We have made further transgenic animals to investigate possible positive and negative elements outside these regions. d) Progress in purification of the RPE65 protein has provided material for biochemical and structural analyses (e.g., mass- spectroscopy and atomic absorption spectroscopy) and for generation of an antibody to the native protein. - Retinal pigment epithelium; retinoids; Transgenic/knockout mice; gene regulation; retinal dystrophies; protein chemistry