Regulation of Retinal cGMP-Phosphodiesterases

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Project summary/Abstract The long-term goal of this research program is to elucidate the molecular mechanisms underlying the function and regulation of cGMP-phosphodiesterases (PDE6) in rod and cone photoreceptor cells. As a central effector enzyme in the phototransduction cascade, PDE6 is critical to photoreceptor cell physiology and survival. Severe retinal diseases including retinitis pigmentosa, achromatopsia, and Leber congenital amaurosis result from pathogenic mutations in genes that encode PDE6 subunits or aryl hydrocarbon receptor- interacting protein-like 1 (AIPL1), a specialized PDE6 chaperone. Despite our advanced understanding of the PDE6 regulation in phototransduction, little is known about the folding and maturation of this enzyme, and the proposed research aims to fill this gap. Our evidence suggests that AIPL1 forms a complex with ubiquitous chaperone HSP90 that is absolutely required for maturation of PDE6 into a functional enzyme. We will define the protein-protein interfaces and the structure of the HSP90/AIPL1 complex in solution to gain mechanistic insights into how this chaperone complex interacts with PDE6. Based on our preliminary studies, we hypothesize that nascent PDE6 assumes a closed nonfunctional state, and the HSP90/AIPL1 complex and the regulatory P-subunit of PDE6 induce and/or catalyze the transition of PDE6 from the closed state to the functional open state via an intermediate state. We propose a detailed model as to how HSP90, AIPL1 and P facilitate the maturation of PDE6 and will rigorously interrogate this model by leveraging our heterologous expression system for cone PDE6C in HEK293T cells, and rod PDEAB in the Pde6g-/-/Pde6ccpfl1 mouse model. In addition, we will use the HSP90 knockout mouse, which highlights the importance of HSP90 in photoreceptor cells because of its rapid retinal degeneration phenotype, to test our hypothesis that defects in PDE6 maturation are the key cause of the degenerative phenotype. Elucidation of the molecular mechanisms of PDE6 maturation will have important implications for retinal diseases and the design of new therapeutic strategies.