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John W. Crabb, Ph.D.Professor and Staff
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Research
Research in our laboratory seeks a better understanding of the biochemistry
of vision in two broad areas, namely, proteome changes associated with retinal
degenerative diseases and the mammalian visual cycle.
Proteomics of Age-related Macular Degeneration
Age-related macular degeneration (AMD) is the most common cause of legal blindness in the elderly population of developed countries. Both genetic and environmental factors contribute to the disease however the cause of AMD is unknown and presently there are no cures. We hypothesize that similar mechanisms of oxidative damage are involved in AMD and retinal light damage and are identifying proteins and protein chemical modifications associated with AMD and light damaged rat retina as an approach to defining these pathways. Major risk factors for developing AMD are extracellular deposits termed drusen, which accumulate with age beneath the retinal pigment epithelium on Bruch’s membrane. Methods used for proteomic characterization of drusen and retinal tissues include 1D and 2D chromatography and/or electrophoresis, bioinformatics and mass spectrometry. Oxidative protein modifications identified in AMD tissues include apparent crosslinks, carboxymethyl lysine and carboxyethyl pyrrole (CEP) protein adducts. CEP adducts are more abundant in AMD than in normal Bruch’s membrane, stimulate angiogenesis in vivo in chorioallantoic membrane and corneal implant assays and may contribute to choroidal neovascularization in late stage AMD. CEP immunoreactivity and CEP autoantibody titer are also significantly elevated in plasma from AMD donors relative to that from age-matched normal donors, and may be of diagnostic utility as biomarkers for predicting AMD susceptibility. Oxidative protein modifications identified in rat retina following intense in vivo light exposure include CEP adducts, argpyrimidine and nitrotyrosine. These data directly link oxidative injury with AMD and retinal light damage. We anticipate this protein chemical approach will provide insights into the etiology of AMD and new opportunities for finding cures for the disease.
Visual Cycle Studies
The process by which all-trans-retinal released from rhodopsin during bleaching is enzymatically isomerized to 11-cis-retinal in the retinal pigment epithelium (RPE), then shuttled back to the rod photoreceptor cells for visual pigment regeneration is known as the rod visual cycle. While the molecular details of this multicomponent process are not completely understood, the cellular retinaldehyde-binding protein (CRALBP) appears to serve multiple roles, a central function being as an 11-cis-retinol acceptor for the RPE isomerization of all-trans- to 11-cis-retinol. We are probing CRALBP structure-function relationships using a combination of protein chemical, nuclear magnetic resonance and molecular biological approaches. The structure of the CRALBP ligand binding pocket is of interest because it determines the specificity of ligand binding and influences the timely release of 11-cis-retinoid from CRALBP. CRALBP protein interactions are also critical in visual cycle processes and a functional interaction has now been unequivocally demonstrated with homogeneous recombinant 11-cis-retinol dehydrogenase. Other visual cycle protein-protein interactions have been sought in bovine RPE microsomes. Using reciprocal immunoprecipitations, immunoaffinity purification and mass spectrometry methods we have identified an RPE visual cycle protein complex. Current efforts focus upon identifying additional components of this RPE retinoid metabolizing protein complex and molecular details of the visual cycle.