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Structure of the RPE65-CCT folding intermediate


EMSL Project ID
50626

Abstract

The retinal pigment epithelium 65 kDa protein (RPE65) plays an essential role in human vision by catalyzing the conversion of all-trans retinol esters to 11-cis retinol. This reaction is a key step in the visual cycle that takes the all-trans retinal byproduct of light activation and converts it back to 11-cis retinal to regenerate the rhodopsin chromophore. Mutations in RPE65 are a major cause of retinal degenerative diseases, including Leber congenital amaurosis (LCA), the most common form of childhood blindness. Many RPE65 mutations disrupt its folding, thereby interrupting the supply of 11-cis retinal to rhodopsin. Unfortunately, little is known about the means by which RPE65 is folded in the cell. We seek to fill this gap in knowledge by investigating the molecular mechanism of RPE65 folding and the defects caused by RPE65 mutations.
We have found that RPE65 interacts robustly with the cytosolic chaperonin CCT, suggesting that CCT contributes to RPE65 folding. We seek to test this hypothesis by pursuing the following specific aims: 1) Define the role of CCT in RPE65 folding by measuring the effect of loss of CCT on RPE65 function, 2) Determine the atomic structure of the RPE65-CCT complex by cryo-electron microscopy (cryo-EM) to understand at the molecular level how CCT folds RPE65, and 3) Solve the structure with an RPE65 folding mutant bound to CCT to understand how mutations disrupt folding.
These studies will allow for structure-based drug design to develop therapeutics to treat the retinal diseases caused by misfolded RPE65.

Project Details

Start Date
2018-12-01
End Date
2019-05-31
Status
Closed

Team

Principal Investigator

Peter Shen
Institution
University of Utah

Co-Investigator(s)

Barry Willardson
Institution
Brigham Young University

Team Members

Harry Scott
Institution
Oregon Health & Science University

Theresa Smith
Institution
Brigham Young University

Helen Donelick
Institution
University of Utah