The complex role of phosphodiesterase 6 in rod photoreceptor health and function

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ABSTRACT Retinal photoreceptor cells can respond to light throughout an individual’s life due to continuous resetting of the light sensing molecules (visual pigments) and their associated signaling elements. Defects in almost all proteins involved in these processes cause photoreceptor degeneration, which could result not only from a deficiency in enzymatic or other functional activity, but also from a loss of structural elements that maintain the complex topology of the rod outer segment (ROS). Our long-term goal is to elucidate the molecular mechanisms of phototransduction and retinal degeneration to promote the discovery of therapeutics for inherited blinding diseases in humans caused by mutations in phototransduction genes. Mutations in the gene encoding phosphodiesterase 6 (PDE6) are among the main causes of such diseases. Accordingly, we propose two thematically and experimentally linked specific aims. Aim 1: Determine the high- resolution structure of rod outer segments (ROS) and rim region of individual disks derived from three- dimensional (3D) cryo-electron tomograms. This aim has been subdivided into three sub-aims that will focus on determining the molecular identity of the spacers that hold disks precisely arranged, determining the structure of ROS with reduced levels of PDE6 and its impact on ROS organization and the number of pillars, and determining the structure of ROS disk rims and the role of ABCA4 on ROS structural integrity. This research will advance our understanding of the function of PDE6 not only as an enzyme but also as a structural protein. Aim 2: Define the role of the N-terminal pony-tail helical region and delineate the allosteric regulation of PDE6. This aim has been subdivided into three sub-aims that will focus on the membrane anchoring role of the Pt-motif, elucidating the allosteric activation mechanism of the PDE6αβγ2 complex, and determining the structure of the Gtα-GTP-PDE6αβγ2 complex. Overall, the findings from this proposal will facilitate the development of a rational approach to alleviate retinal dystrophies related to mutations in the PDE6 gene.