Docosahexaenoic acid (22:6n-3, DHA), a long chain essential polyunsaturated fatty acid of the linolenic acid (18:3n-3) family, is the most abundant fatty acid in retina. Reductions in the retinal levels of 22:6n-3 can lead to changes in retinal function. Recent studies have shown that blood and retinal levels of 22:6n-3 are reduced in some inherited retinal degenerations. Under ordinary circumstances, the retina tenaciously conserves 22:6n-3 during through recycling between the retina and retinal pigment epithelium. Although we have learned how the retina conserves 22:6n-3, the mechanism of accretion of 22:6n-3 is still not known.
Specific Aim #1 is to determine the mechanism of enrichment of 22:6n-3 in the retina. We will test the hypothesis that specific proteins in the RPE, inter photoreceptor matrix (IPM), and/or retina promote selective release, transport, and uptake of 22:6n-3 and incorporation into ROS membrane phospholipids. It is now clear that the lower blood level of 22:6n-3 in humans with RP is found in several different mutations. Likewise, lower levels of 22:6n-3 are present in the ROS of animals with unrelated retina-specific mutations. Therefore, it seems likely that the 22:6n-3 phenotype in the retina is the result of some common convergent pathway, rather than the primary defect in each mutation.
Specific Aim #2 is to determine the role of 22:6n-3 in retinal degenerations. A variety of in vivo and in vitro experiments are proposed to test two hypothesis. Ho #1-The reduction of 22:6n-3 in ROS leads to death of photoreceptor cells; Ho #2-The reduction of 22:6n-3 in ROS is an adaptive response to metabolic stress. Other experiments are designed to identify endogenous factor(s) in the retinas of animals that provide protection against light-induced oxidant stress and determine if their effects can be enhanced by certain neuroprotective drugs. Two unique unsaturated fatty acids (14:1n-9 and 14:2n-6) are found N- terminally acylated to retinal proteins, but not to similar proteins in any other tissue.
Specific Aim #3 is to determine the mechanism of the selective N-terminal acylation of retinal proteins by these fatty acids. We will use molecular biology techniques to identify, clone, and sequence N- myristoyl transferases in the retina; express the proteins in a bacterial expression system; and study the kinetics of N-terminal acylation using authentic substrates 14:1n-9 and 14:2n-6.
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