Interphotoreceptor retinoid-binding protein (IRBP), the major soluble protein component of the interphotoreceptor matrix (IPM), has access to M?ller cells, photoreceptors, and RPE. The mechanism by which IRBP protects retinoids from isomeric and oxidative degradation while targeting their delivery/release between the above cells during the visual cycle is poorly understood. Our long-term goal is to understand at the molecular level how IRBP accomplishes its remarkable functions. The mechanism underlying IRBP's function or role in disease remains unknown because little is understood about its structure-function relationships. Its structure is unusual being composed of tandem homologous """"""""modules"""""""" each ~300 residues in length. Although the individual modules have some functional activity, they are not equivalent, and important interactions take place between them. A critical gap is that little is known about the structure of the full-length protein and quaternary association of the """"""""modules"""""""". However, obtaining IRBP at the concentrations needed for X-ray crystallography has been problematic as the protein denatures and precipitates when concentrated above 3 mgs/ml. In the current funding period, we purified to homogeneity full-length bovine, xenopus, human and zebrafish IRBPs in stable and functionally active pristine forms, devoid of fusion tags. These protein solutions can now be readily concentrated without denaturation or precipitation. We have optimized conditions for growing diffraction-quality crystals of these full-length IRBPs. Preliminary structure elucidation analysis for Xenopus IRBP suggests that the single module structure may be substantially modified in the full-length functional protein. Analyses of the expression, purification, stability, crystallization, ligand-binding, anti-oxidant activity, and homology-modeling data on these IRBPs have led to our hypothesis that quaternary association of the """"""""modules"""""""" contributes to the structural scaffold(s) that bind and protect retinoids from degradation, and that the """"""""modules"""""""" contribute unequally in these roles as well as in target retinoid delivery and release at the cell surface. This hypothesis will be evaluated through the following complementary aims.
Aim 1 : To determine the crystal structures of IRBPs composed of four modules (human, bovine, Xenopus), and two modules (zebrafish).
Aim 2 : To define binding-interactions of physiologically relevant ligands with IRBP and elucidate the molecular basis of IRBP's protective/anti-oxidant roles.
Aim 3 : To determine how IRBP efficiently targets retinoid removal/delivery.
Aim 4 : To determine the structural and functional """"""""hot-spots"""""""" in IRBP. ? ?
The mechanism by which IRBP protects retinoids from isomeric and oxidative degradation while targeting their delivery/release between photoreceptors, retinal pigmented epithelium and M?ller cells during the visual cycle is poorly understood. A critical gap is that little is known about the structure of the full-length protein and quaternary association of the """"""""modules"""""""" that comprise the structure of IRBP. Analyses of the expression, purification, stability, crystallization, ligand-binding, anti-oxidant activity, and homology-modeling data on these IRBPs have led to our hypothesis that quaternary association of the """"""""modules"""""""" contributes to the structural scaffold(s) that bind and protect retinoids from degradation, and that the """"""""modules"""""""" contribute unequally in these roles as well as in targeting retinoid delivery and release at the cell surface. ? ? ? ? ?
| Gonzalez-Fernandez, Federico; Fornalik, Mark; Garlipp, Mary Alice et al. (2018) Pericellular interphotoreceptor matrix dictates outer retina critical surface tension. Exp Eye Res 167:163-173 |
| Chen, Chunhe; Adler 4th, Leopold; Goletz, Patrice et al. (2017) Interphotoreceptor retinoid-binding protein removes all-trans-retinol and retinal from rod outer segments, preventing lipofuscin precursor formation. J Biol Chem 292:19356-19365 |
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| Patel, Sangita P; Schaefer, Jamie L; Jaber, Ryan et al. (2016) The Value of Cytology Smears for Acanthamoeba Keratitis. Case Rep Ophthalmol Med 2016:4148968 |
| Gonzalez-Fernandez, Federico; Betts-Obregon, Brandi; Tsin, Andrew T et al. (2016) Technical brief: Pump-probe paradigm in an integrating cavity to study photodecomposition processes. Mol Vis 22:953-8 |
| Ghosh, Debashis; Haswell, Karen M; Sprada, Molly et al. (2015) Structure of zebrafish IRBP reveals fatty acid binding. Exp Eye Res 140:149-158 |
| Gonzalez-Fernandez, Federico; Betts-Obregon, Brandi; Yust, Brian et al. (2015) Interphotoreceptor retinoid-binding protein protects retinoids from photodegradation. Photochem Photobiol 91:371-8 |
| Gonzalez-Fernandez, Federico; Sung, Dongjin; Haswell, Karen M et al. (2014) Thiol-dependent antioxidant activity of interphotoreceptor retinoid-binding protein. Exp Eye Res 120:167-74 |
| Sane, M; Selvadurai, A; Reidy, J et al. (2014) Transient visual loss due to reversible 'pending' central retinal artery occlusion in occult giant cell arteritis. Eye (Lond) 28:1387-90 |
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