The vertebrate visual cycle is comprised of biochemical reactions involved in processing all-trans retinal produced by the photoisomerisation of 11-cis retinal in rhodopsin. The visual cycle is essential for vision and visual adaptation. The goal of this project is to characterize on a molecular level essential components of the visual cycle and to learn how the cycle is regulated. Two of the key reactions in the visual cycle are catalyzed by the retinal pigment epithelium membrane bound enzymes, lecithin retinol acyl transferase (LRAT)and the isomerohydrolase. LRAT transfers an acyl group from lecithin to vitamin A to generate all-trans retinyl esters and the omerohydrolase processes the esters to produce 11-cis retinol. Both enzymes are essential for vision. An understanding of LRAT at the molecular level is of major interest in this grant proposal. LRAT has a unique sequence which does not reveal the mechanistic class to which it belongs. Biochemical studies including chemical mapping studies using the novel technique of biotin affinity labeling and site-specific mutagenic studies are proposed to both map elements of the active-site structure of LRAT and define its molecular mechanism of action. Chemical mapping studies are also proposed to begin to elucidate the structure of LRAT in the membrane and to reveal nearest neighbor proteins in RPE membranes. One of the LRAT associated proteins may be the isomerohydrolase. Identification and characterization of isomerohydrolase is another important aspect of this proposal. Approaches to the identification of this enzyme system will involve both exploiting interactions with LRAT to either affinity purify or cross-link isomerohydrolase and photoaffinity labeling approaches to label the enzyme. When isomerohydrolase is identified it will be coned sequenced and expressed in LRAT transfected HEK cells. The structure, mechanism of action and regulation of the isomerohydrolase will be explored as will possible relationships to diseases of vision caused by mutations in the enzyme.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY004096-19
Application #
6126656
Study Section
Special Emphasis Panel (ZRG1-VISB (01))
Program Officer
Mariani, Andrew P
Project Start
1982-04-01
Project End
2005-08-31
Budget Start
2000-09-01
Budget End
2001-08-31
Support Year
19
Fiscal Year
2000
Total Cost
$488,919
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Fishkin, Nathan; Yefidoff, Revital; Gollipalli, Deviprasad R et al. (2005) On the mechanism of isomerization of all-trans-retinol esters to 11-cis-retinol in retinal pigment epithelial cells: 11-fluoro-all-trans-retinol as substrate/inhibitor in the visual cycle. Bioorg Med Chem 13:5189-94
Xue, Linlong; Rando, Robert R (2004) Roles of cysteine 161 and tyrosine 154 in the lecithin-retinol acyltransferase mechanism. Biochemistry 43:6120-6
Xue, Linlong; Gollapalli, Deviprasad R; Maiti, Pranab et al. (2004) A palmitoylation switch mechanism in the regulation of the visual cycle. Cell 117:761-71
Gollapalli, Deviprasad R; Rando, Robert R (2004) The specific binding of retinoic acid to RPE65 and approaches to the treatment of macular degeneration. Proc Natl Acad Sci U S A 101:10030-5
Krosky, Paula M; Baek, Moon-Chang; Jahng, Wan Jin et al. (2003) The human cytomegalovirus UL44 protein is a substrate for the UL97 protein kinase. J Virol 77:7720-7
Jahng, Wan Jin; Xue, Linlong; Rando, Robert R (2003) Lecithin retinol acyltransferase is a founder member of a novel family of enzymes. Biochemistry 42:12805-12
Gollapalli, Deviprasad R; Maiti, Pranab; Rando, Robert R (2003) RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters. Biochemistry 42:11824-30
Gollapalli, Deviprasad R; Rando, Robert R (2003) All-trans-retinyl esters are the substrates for isomerization in the vertebrate visual cycle. Biochemistry 42:5809-18
Bok, Dean; Ruiz, Alberto; Yaron, Orna et al. (2003) Purification and characterization of a transmembrane domain-deleted form of lecithin retinol acyltransferase. Biochemistry 42:6090-8
Gollapalli, Deviprasad R; Rando, Robert R (2003) Specific inactivation of isomerohydrolase activity by 11-cis-retinoids. Biochim Biophys Acta 1651:93-101

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