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

University Of California-Irvine, Irvine CA

Investigators

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Abstract

PROJECT SUMMARY/ABSTRACT The rod outer segment (ROS) houses all the protein components necessary for visual phototransduction. Genetic defects in these proteins have been linked to human retinal-degenerative diseases. Insights obtained from detailed structural and functional analyses, in vitro and in vivo, have advanced our understanding of the etiology of related retinopathies, but several fundamental questions remain. Accordingly, we propose two thematically and experimentally linked specific aims that fill critical gaps pertaining to the subunit assembly of rod phosphodiesterase 6 (PDE6) and its role in ROS organization. PDE6 is a key enzyme of our vision, and it is implicated in retinopathies; thus, an advanced understanding of its subunit structures, particularly in vivo, is essential to elucidate the consequences of its dysfunction. Specific Aim 1. Through enzymatic inactivation and N-terminal truncation, determine the relative contributions of the PDE6α and PDE6β subunits to the function and structural organization of the rod photoreceptors. Aim 1A. Via selective enzymatic inactivation, determine the relative contributions of the PDE6α and PDE6β subunits to the stability and activity of tetrameric PDE6αβγγ. Based on structures of the PDE6αβγγ complex and other related members of the PDE family, the H599A mutation in PDE6? and the H597A mutation in PDE6β are expected to render these PDE6 subunits catalytically inactive by disabling hydrolysis. We have developed single heterozygote knock-in mouse bearing the respective mutant PDE6 catalytic subunits. Moreover, we generated novel mouse models featuring N-terminal truncations (2-48 of PDE6α and 2-46 in PDE6β). Single heterozygote knock-in mice with deletions in the respective N-termini of the PDE6 subunits will enable us to thoroughly characterize the resultant effects on the structure of the rod photoreceptor cells. Aim 1B. Characterize the PDE6 architecture in complexes with lipids and small molecules, using native mass spectrometry. Using this highly innovative technology we will quantify PDE6 subunit arrangements in different activation states with Gt and small ligands (cGMP, GDP/GTP, PDE6 inhibitors). Specific Aim 2. Delineate the impact of PDE mutations on the structural integrity of the ROS at nanometer resolution. Cryo-electron tomography (cryo-ET) provides the least destructive and highest resolution method to investigate cellular structures at the molecular level in an environment similar to the native state. Aim 2A. Identify changes in the structure and frequency of interdiscal spacers in the ROS of mutant-PDE6 mice compared to WT. Aim 2B. Assign the physical locations and light-dependent dynamics of key proteins of the ROS, including membrane-bound rhodopsin. We intend to take advantage of recent technological advances to investigate other individual proteins in the ROS, including rhodopsin. By elucidating the molecular details of normal and aberrant ROS, we will better understand the ensuing pathology resulting from mutations in PDE6 and other key components of phototransduction. Furthermore, the findings from this project could facilitate the development of a rational approach to alleviate retinal dystrophies affecting ROS structure.