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 #
5R01EY009339-24
Application #
8436225
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
2013-03-01
Budget End
2014-02-28
Support Year
24
Fiscal Year
2013
Total Cost
$451,601
Indirect Cost
$163,957
Name
Case Western Reserve University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
077758407
City
Cleveland
State
OH
Country
United States
Zip Code
44106
Kiser, Philip D; Palczewski, Krzysztof (2016) Retinoids and Retinal Diseases. Annu Rev Vis Sci 2:197-234
Chen, Yu; Palczewski, Krzysztof (2016) Systems Pharmacology Links GPCRs with Retinal Degenerative Disorders. Annu Rev Pharmacol Toxicol 56:273-98
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
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Hofmann, Lukas; Gulati, Sahil; Sears, Avery et al. (2016) An effective thiol-reactive probe for differential scanning fluorimetry with a standard real-time polymerase chain reaction device. Anal Biochem 499:63-5
Kiser, Philip D; Zhang, Jianye; Badiee, Mohsen et al. (2015) Catalytic mechanism of a retinoid isomerase essential for vertebrate vision. Nat Chem Biol 11:409-15
Salom, David; Cao, Pengxiu; Yuan, Yiyuan et al. (2015) Isotopic labeling of mammalian G protein-coupled receptors heterologously expressed in Caenorhabditis elegans. Anal Biochem 472:30-6
Sexton, Timothy J; Van Gelder, Russell N (2015) G-Protein Coupled Receptor Kinase 2 Minimally Regulates Melanopsin Activity in Intrinsically Photosensitive Retinal Ganglion Cells. PLoS One 10:e0128690
Kolesnikov, Alexander V; Maeda, Akiko; Tang, Peter H et al. (2015) Retinol dehydrogenase 8 and ATP-binding cassette transporter 4 modulate dark adaptation of M-cones in mammalian retina. J Physiol 593:4923-41
Sui, Xuewu; Golczak, Marcin; Zhang, Jianye et al. (2015) Utilization of Dioxygen by Carotenoid Cleavage Oxygenases. J Biol Chem 290:30212-23

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