|Ph.D Student||Remez Lital|
|Subject||Functional Characterization of the PRCD Gene, Involved in|
Hereditary Retinal Degeneration
|Department||Department of Medicine||Supervisor||Professor Tamar Ben-Yosef|
|Full Thesis text|
Retinitis pigmentosa (RP) is a genetically heterogeneous group of inherited retinopathies and the most common form of hereditary retinal degeneration. Progressive rod-cone degeneration (PRCD) is one of several recessively inherited retinal diseases in dogs and serves as an animal model for human RP.
The PRCD protein encodes for a 54 aa long protein with unknown function. The ﬁrst 20 aa appear to encode for a signal peptide (SP), suggesting that PRCD is a secreted protein. Bioinformatic analysis predicted that PRCD's N-terminal α-helix serves as an SP and not as a transmembrane domain. Secretion experiments demonstrated that PRCD is secreted to the extracellular space and that its secretion is mediated by the N-terminal domain, which acts as an SP and is cleaved upon secretion.
To characterize the secretory pathway of PRCD, various pharmacological agents which interfere with transport of proteins through the ER and Golgi to the plasma membrane were used. PRCD secretion was signiﬁcantly inhibited by all tested pharmacological agents, conﬁrming that it is secreted through the classic ER/Golgi-dependent secretory pathway.
Two of the PRCD reported mutations (p.C2Y and p.P25T) are missense mutations. Both mutations affect highly conserved residues. The effect of the two mutations on the PRCD protein was tested, and it was found that p.C2Y, but not p.P25T, affects protein stability, and that neither mutation affects secretion. In addition, the p.C2Y mutation leads to a marked reduction in protein level. Bioinformatic analysis predicted that the p.P25T mutation has structural and functional implications on the PRCD protein.
PRCD location in mouse retina was characterized by immunostaining of retinal sections of WT mice. PRCD was mostly observed in rod photoreceptor outer segments. Since it was hard to define whether PRCD is expressed in cone photoreceptors as well, we used retinal section from Nrl−/− mice, in which all photoreceptors are cone-like. PRCD was significantly observed in these photoreceptors.
A potential interaction between PRCD and the TULP1 protein was previously identified in our lab by Ras-Recruitment System. TULP1 belongs to the tubby protein family, which share the highly conserved C-terminal region named ‘tubby domain’. Mutations in TULP1 and TUB cause RP in humans and retinal degeneration in mice. We verified the interaction between PRCD and TULP1 by co-immunoprecipitation in transfected cells. We also examined the interaction between PRCD and TUB, and found that they also interact with each other. The interaction between PRCD, TULP1 and TUB was mapped to the C-terminal region which includes the ‘tubby domain’.
Both TUB and TULP1 are secreted proteins with unconventional mechanism and have been previously characterized as MerTK ligands that facilitate phagocytosis by RPE cells. We therefore examined the possibility that PRCD may also be involved in RPE phagocytosis. For this purpose we performed serial phagocytosis tests using RPE19 cells. TUB-GST (positive control) stimulated phagocytosis by RPE19 cells, while PRCD_GST significantly inhibited phagocytosis by RPE19 cells. Our model suggests that bound and free PRCD can influence the rate of RPE phagocytosis. These ﬁndings shed a new light on PRCD function and the etiology of RP.