The long-term goal of this research program is to elucidate the molecular mechanisms underlying the function and regulation of cGMP-phosphodiesterases (PDE6) in rods and cones. PDE6 is a key effector enzyme in the phototransduction cascade. The clinical importance of PDE6 is evident from the fact that its malfunction due to mutations in genes encoding it or the specialized chaperone, aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1), lead to severe retinal diseases such as retinitis pigmentosa, achromatopsia, and Leber congenital amaurosis (LCA). For many years progress in understanding the structure and function of PDE6 has been hampered by a lack of an effective heterologous expression system. The current proposal takes advantage of a novel, robust system based on the co-expression of cone PDE6C with AIPL1 and the inhibitory ?-subunit (P?) in HEK293T cells. This system offers a unique opportunity to gain mechanistic insights into the interaction between PDE6 and AIPL1 and their client/chaperone relationship. Mutational analysis of PDE6C will be performed to identify the structural determinants of the catalytic efficiency of PDE6 and to elucidate the mechanisms whereby mutations lead to disease. We will test the hypothesis that many pathogenic mutations disrupt the interaction of PDE6 with P???leading to markedly diminished expression of functional PDE6. Using enzymatic activity as readout for the AIPL1 chaperone function, we will examine the roles of AIPL1 domains and specific partner interactions in the folding of PDE6, and delineate the mechanisms whereby AIPL1 mutations cause LCA. We propose and will test a model in which P? augments expression of functional PDE6 by acting as an affinity adaptor for the AIPL1-PDE6 interaction during maturation of the enzyme. Our biochemical studies will be complemented by structural analyses (X-ray crystallography and NMR) aimed at obtaining a high-resolution structure of AIPL1 in complex with the PDE6 prenyl moiety and P?. This structure will signify an unprecedented advance in understanding AIPL1 and its interaction with PDE6. The ultimate goal of the proposed studies is to generate critical insights into the mechanisms that underlie PDE6- and AIPL1-linked retinal diseases, and to thereby contribute to the development of new therapeutic strategies.
. Rod and cone cGMP phosphodiesterases (PDE6 family) are the key effector enzymes in vision that depend on a specialized chaperone aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) for their folding and activity. Mutations in PDE6 and AIPL1 lead to severe retinal diseases in humans, such as retinitis pigmentosa, achromatopsia and Leber congenital amaurosis. Our goals are to markedly advance the understanding of the AIPL1-assisted PDE6 folding and elucidate the mechanisms of PDE6 and AIPL1 mutations in retinal disease to facilitate development of new therapeutic strategies for the PDE6-linked visual disorders.
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