A major goal of our laboratory is to understand the molecular details of vitamin A (retinol) processing within the retinoid cycle as well as the consequences of side reactions that occur between retinal and various biological molecules. A variety of serious diseases result from defects in the retinoid (visual) cycle or abnormalities in retinal clearance. Understanding of the fundamental biochemical processes underlying these diseases is essential for the development of effective therapeutics. Photoisomerization of the 11-cis-retinal chromophore of rhodopsin triggers a complex set of molecular events leading to light perception. Continuity of vision depends on constant regeneration of all-trans-retinal back to its cis isomer in a process known as the retinoid cycle. This series of reactions takes place in photoreceptor and RPE cells. Retinal, the initial substrate and final product of this cycle, is highly chemically reactive and can form toxic conjugates with proteins and lipids. Many visual disorders are caused by malfunctions of the retinoid cycle. Thus, much experimental effort has been devoted to elucidating enzymatic steps comprising the retinoid cycle and all-trans-retinal-mediated retinal degeneration. These studies have delineated key steps of the retinoid cycle. Nevertheless, many important details regarding chemical transformations of retinal and its derivatives are not well understood and many proteins involved in 11-cis-retinal regeneration still await structural, biochemical and functional characterization. This proposal aims to significantly improve our understanding of retinoid cycle transformations at the molecular level as well as to delineate all-trans-retinal-mediated retinal degeneration and the mechanisms that exist in vivo to prevent it. First, we will use a number of biophysical and biochemical techniques to characterize the structure and catalytic mechanism of RPE65, the enzyme responsible for transforming all-trans-retinyl esters to 11-cis-retinol. Second, we will determine the structural and functional properties of LRAT, a crucial enzyme of the retinoid cycle that converts all-trans-retinol to all-trans-retinyl esters. Third, we will purify ABCA4, the photoreceptor-specific ABC transporter that prevents accumulation of the toxic compounds, all- trans-retinal and its derivatives, including N-retinylidene-PE, inside rod outer segment disks. We then will investigate its structure, posttranslational modifications and mechanisms that regulate its activity. Finally, we will identify the mechanisms of all-trans-retinal-induced retinal degeneration by dissecting out those signaling events that lead to the death of retinal cells.

Public Health Relevance

The visual cycle consists of a sequence of enzymatic reactions occurring in photoreceptor and retinal pigment epithelium cells that continuously regenerate the visual chromophore, 11-cis- retinal. Insights derived from analysis of critical protein components of this cycle will significantly improve our understanding of its principal reactions and the consequences of its toxic byproducts that accumulate in disease states.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
5R01EY009339-25
Application #
8625303
Study Section
Special Emphasis Panel (ZRG1-BDPE-J (09))
Program Officer
Neuhold, Lisa
Project Start
1992-08-01
Project End
2016-02-29
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
25
Fiscal Year
2014
Total Cost
$465,861
Indirect Cost
$169,134
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Hofmann, Lukas; Alexander, Nathan S; Sun, Wenyu et al. (2017) Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors. Biochemistry 56:2338-2348
Sui, Xuewu; Weitz, Andrew C; Farquhar, Erik R et al. (2017) Structure and Spectroscopy of Alkene-Cleaving Dioxygenases Containing an Atypically Coordinated Non-Heme Iron Center. Biochemistry 56:2836-2852
Alexander, Nathan S; Katayama, Kota; Sun, Wenyu et al. (2017) Complex binding pathways determine the regeneration of mammalian green cone opsin with a locked retinal analogue. J Biol Chem 292:10983-10997
Alexander, Nathan S; Palczewski, Krzysztof (2017) Crowd sourcing difficult problems in protein science. Protein Sci 26:2118-2125
Jastrzebska, Beata; Comar, William D; Kaliszewski, Megan J et al. (2017) A G Protein-Coupled Receptor Dimerization Interface in Human Cone Opsins. Biochemistry 56:61-72
Sears, Avery E; Bernstein, Paul S; Cideciyan, Artur V et al. (2017) Towards Treatment of Stargardt Disease: Workshop Organized and Sponsored by the Foundation Fighting Blindness. Transl Vis Sci Technol 6:6
Chiang, Cheng-Kang; Tworak, Aleksander; Kevany, Brian M et al. (2017) Quantitative phosphoproteomics reveals involvement of multiple signaling pathways in early phagocytosis by the retinal pigmented epithelium. J Biol Chem 292:19826-19839
Kiser, Philip D; Zhang, Jianye; Badiee, Mohsen et al. (2017) Rational Tuning of Visual Cycle Modulator Pharmacodynamics. J Pharmacol Exp Ther 362:131-145
Palczewski, Krzysztof (2017) Skunkworks project for Big Pharma. Pharmacol Res 124:167-168
Hofmann, Lukas; Tsybovsky, Yaroslav; Alexander, Nathan S et al. (2016) Structural Insights into the Drosophila melanogaster Retinol Dehydrogenase, a Member of the Short-Chain Dehydrogenase/Reductase Family. Biochemistry 55:6545-6557

Showing the most recent 10 out of 196 publications