Choroideremia (CHM) is an X-linked eye disease which leads to degeneration of the choriocapillaris and adjoining retinal pigment epithelium (RPE) and photoreceptor layers. The responsible gene product, CHM, is ~Rab escort protein~ (REP1.). REP1. is the component A subunit of Rab geranylgerany1 transferase II (RabGG Tr II) which delivers newly synthesized small GTPases belonging to the rab gene family to the catalytic complex (component B) containing the alpha and beta subunits for post-translational geranylation. CHM-REP. is evolutionarily related to guanine nucleotide dissociation inhibitor or GDI, a ubiquitous protein involved in the recycling of Rab proteins required for membrane vesicular traffic through the exocytic and endocytic pathways. We have generated crystals of alpha-GDI which diffract to 1.8 A and have solved the structure of the protein using x- ray crystallography. The structure reveals that the conserved domains belonging to GDI and CHM-REP1 have nearly identical structural folds. These results suggest that regions responsible for GDI activity will have a similar role in CHM-REP1 in recognition of Rab proteins and receptors. This structural homology will be exploited to address the molecular basis for CHM- REP1 function in vitro and in vivo.
Specific Aim 1 will focus on solving the structure of CHM-REP1 and its complex with Rab using x-ray crystallography.
Specific Aim 2 will encompass a biochemical and molecular analysis of CHM-REP1 function in vitro. We will generate site-directed mutants in CHM-REP1 based on the effects of analogous substitutions to those already generated in GDI which have been found to block interaction with Rab. To establish the biochemical basis for the interaction of CHM-REP1 with the catalytic component B of RabGG Tr II, we will use in vitro assays which provide sensitive measures for complex formation and prenylation.
Specific Aim 3 will address the physiological role of CHM-REP1 and Rab in the retinal pigment epithelia RPE-J cell line by transfection with wild-type and mutant CHM-REP1. These studies will address the role of CHM-REP1 in vivo in the maintenance of Rab pools for membrane trafficking, and cell viability and differentiation, providing insight into the role of CHM- REP1 in the normal and diseased state in situ. Analysis of the structure of CHM-REP1 and its complex and Rab may lead to new pharmacological approaches to normalize the defect(s) in human disease.
