טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentNevet Yudith
SubjectCharacterization of CERKL and PRCD, Two Proteins Involved
in Human Retinal Degeneration, through
Identification of their Protein-Protein
Interactions
DepartmentDepartment of Medicine
Supervisor Professor Tamar Ben-Yosef
Full Thesis textFull thesis text - English Version


Abstract

Hereditary retinal dystrophies (HRDs) are a heterogeneous group of diseases, which cause visual loss due to the premature death of rod and cone photoreceptors in the retina. The most common form of HRD is retinitis pigmentosa (RP). The list of genes underlying HRD includes a large group of genes with unknown function, two of them are progressive rod-cone degeneration (PRCD) and ceramide kinase-like (CERKL).

Progressive rod-cone degeneration is a canine form of autosomal-recessive retinal degeneration, which serves as an animal model for human RP, and is caused by a missense mutation of the PRCD gene (p.C2Y). Until recently, the same mutation in a homozygous state in a single human RP patient, was the only mutation of PRCD reported in humans.

The cause for the high incidence of autosomal-recessive RP in an isolated Muslim Arab village in Northern Israel was investigated by haplotype analysis in affected families. This analysis showed linkage to the PRCD gene. The underlying mutation (p.R22X) was detected by direct sequencing. This founder mutation was found in a homozygous state in 18 patients, and its carrier frequency in the investigated village is 10%. The identification of a second pathogenic mutation of PRCD in multiple RP patients confirmed the role of PRCD in the etiology of RP in humans.

CERKL is a homolog of ceramide kinase (CERK). Ceramide is the central metabolite in the pathway of sphingolipid metabolism, and is involved in survival and apoptosis in the retina. However, despite vast research, no kinase activity could be demonstrated for CERKL.

In an effort to reveal the roles of PRCD and CERKL in the retina, we used the Ras Recruitment System (RRS) to identify CERKL and PRCD-binding proteins. The screen of a retinal cDNA library with CERKL-derived baits led to the identification of an interaction between CERKL and several neuronal calcium sensor (NCS) proteins, including GCAP1, GCAP2, and recoverin. These interactions were confirmed by co-immunoprecipitation (co-IP) experiments in transfected mammalian cells. Moreover, the interaction between endogenous CERKL and GCAP2 was confirmed by co-IP in both light and dark adapted photoreceptor outer segments. We found that CERKL-GCAP interaction is cation-dependent, and mediated by CERKL’s N-terminal region and by GCAPs cation-binding domains (EF-hands 2-4). We have preliminary results showing that CERKL has kinase activity. This study, which is the first to describe the interactions of CERKL with other retinal proteins, links CERKL to the photoresponse and provides important clues regarding CERKL’s retinal function.