Heterotrimeric and small G (ras-family) proteins are post- translationally modified by isoprenylation/methylation, a modification important for membrane association and function of these important signal transducing proteins. This grant request is focused on the molecular enzymology and functional importance of isoprenylation/methylation as it relates to signal transduction in the visual system. With respect to the enzymology, two novel endoproteases are essential for the maturation and degradation of transducin, the photoreceptor heterotrimeric G protein which interacts with activated rhodopsin. The two endoproteases are to be purified, chemically labeled, cloned, and sequenced. The biochemical roles of these proteases with respect to transducin and the ras family of G proteins will be explored. Deleterious mutations in either of these proteases would be expected to have serious consequences in vision. Inhibitors of one of the proteases blocks oncogenic ras function, suggesting the possibility that these inhlbitors may be candidate anti-cancer agents. While isoprenylation/methylation operates by a hydrophobic mechanism in allowing transducin to associate with photoreceptor disk membranes, the situation with respect to the small G protein family is unclear. Here specific ligand-receptor interactions may be mediated via the isoprenylated/methylated cysteine moiety. Functional studies on isoprenylation/methylation will be extended to the ras family of small G proteins where the importance of the individual enzymatic steps of isoprenylation/methylation will be determined. The specific effects of small molecule farnesylcysteine analogs on small G protein function will be addressed in this context. Biochemical assays will include determining the effects of isoprenylation/methylation and the farnesylcysteine analogs on small G protein membrane docking, and on activation of effectors, such as the PAK and MAP kinases. These studies will be extended to an investigation of the nature of the small O proteins in photoreceptors, and their possible roles in visual signal transduction.A subset of farnesylcysteine analogs have been found to activate protein kinase C (PKC) specifically, an important signal transducing enzyme found in photoreceptors and many other cell types. Structure-activity studies on these analogs have uncovered novel specific purine analogs which are effector-site inhibitors of PKC. These new antagonists will be used to uncover the physiological importance of protein PKC in visual signal transduction. The possibility that endogenous purines are antagonists of PKC will be explored.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Research Project (R01)
Project #
2R01EY003624-17
Application #
2407946
Study Section
Visual Sciences C Study Section (VISC)
Project Start
1980-12-01
Project End
2002-06-30
Budget Start
1997-07-01
Budget End
1998-06-30
Support Year
17
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
082359691
City
Boston
State
MA
Country
United States
Zip Code
02115
Mondal, M S; Ruiz, A; Hu, J et al. (2001) Two histidine residues are essential for catalysis by lecithin retinol acyl transferase. FEBS Lett 489:14-8
Mondal, M S; Ruiz, A; Bok, D et al. (2000) Lecithin retinol acyltransferase contains cysteine residues essential for catalysis. Biochemistry 39:5215-20
Parish, C A; Rando, R R (2000) Isoprenylation/methylation and transducin function. Methods Enzymol 316:451-64
Tok, J B; Cho, J; Rando, R R (1999) Aminoglycoside antibiotics are able to specifically bind the 5'-untranslated region of thymidylate synthase messenger RNA. Biochemistry 38:199-206
Hamasaki, K; Killian, J; Cho, J et al. (1998) Minimal RNA constructs that specifically bind aminoglycoside antibiotics with high affinities. Biochemistry 37:656-63
Perez-Sala, D; Gilbert, B A; Rando, R R et al. (1998) Analogs of farnesylcysteine induce apoptosis in HL-60 cells. FEBS Lett 426:319-24
Cho, J; Hamasaki, K; Rando, R R (1998) The binding site of a specific aminoglycoside binding RNA molecule. Biochemistry 37:4985-92
Hamasaki, K; Rando, R R (1998) A high-throughput fluorescence screen to monitor the specific binding of antagonists to RNA targets. Anal Biochem 261:183-90
Wang, Y; Hamasaki, K; Rando, R R (1997) Specificity of aminoglycoside binding to RNA constructs derived from the 16S rRNA decoding region and the HIV-RRE activator region. Biochemistry 36:768-79
Hamasaki, K; Rando, R R (1997) Specific binding of aminoglycosides to a human rRNA construct based on a DNA polymorphism which causes aminoglycoside-induced deafness. Biochemistry 36:12323-8

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