PERIPHERIN/RDS IN PHOTORECEPTOR RENEWAL AND DISEASE

点击翻译按钮获取中文摘要

DESCRIPTION (Applicant's Description): Defects in the human RDS gene result in nearly a dozen clinically distinct retinal diseases - all of which impair central and/or peripheral vision progressively. The RDS gene product, peripherin/rds, is an integral membrane protein required for the formation of the photoreceptor outer segment (OS); however, neither its function at the molecular level, nor its role in retinal pathophysiology is currently understood. We hypothesize that inherited defects in peripherin/rds can affect particular protein domains to preferentially inhibit photoreceptor OS disk morphogenesis, stacking, or shedding - and thereby generate heterogeneous disease phenotypes. Specific aim 1 will test the hypothesis that polytopic protein domains in peripherin/rds contribute differentially to its biosynthesis, subcellular localization, and ability to promote in vitro membrane fusion (fusogenicity), by using an insertional mutagenesis approach combined with cell-free and heterologous expression systems. These studies will determine if, and how, mutations in individual regions of peripherin/rds selectively affect particular aspects of protein structure and/or in vitro function. Specific aim 2 will test the hypothesis that peripherin/rds fusogenicity is required in vivo for OS disk shedding. A heterologous expression system will be used to design a partial loss-of-function mutant that is defective only in its ability to catalyze membrane fusion. Light and electron microscopic analyses will determine whether disk shedding is inhibited in mouse rod photoreceptors expressing this transgene for "non-fusogenic" peripherin/rds. Specific aim 3 will test the hypothesis that peripherin/rds plays a role in maintaining the integrity of outer segment disk stacks. A heterologous expression system will be used to design an otherwise wild-type peripherin/rds variant that contains a highly-specific Factor Xa protease cleavage site; this variant will be expressed as a transgene to rescue photoreceptors in the retinal degeneration slow (rds) mouse (rds does not express peripherin/rds). In vitro Factor Xa proteolysis combined with electron microscopic and Western blot analyses will be used to determine whether specific cleavage of this peripherin/rds variant disrupts the disk stack organization of transgenic mouse photoreceptor OSs. In sum, this research program is directed towards establishing the normal function(s) of peripherin/rds in OS renewal, to begin a molecular description of the etiologies behind a wide variety of progressive blinding diseases that result from defects in RDS.