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Seongjin Seo, Poppy Datta, Chantal Allamargot, Joseph Hudson, Emily Andersen, Sajag Bhattarai, Arlene V Drack; Accumulation of non-outer segment proteins in the outer segment underlies photoreceptor degeneration in BBS. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4644.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations disrupting protein trafficking to the photoreceptor outer segment (OS) cause photoreceptor degeneration, and considerable research effort has been made to elucidate mechanisms of protein trafficking to the OS. Bardet-Biedl Syndrome (BBS) proteins are a group of proteins that are thought to be involved in this process. However, the precise role of BBS proteins in photoreceptors is poorly understood. In this study, we generated a new BBS mouse model (Lztfl1; aka Bbs17) and compared the OS proteome of BBS with that of normal OS to better understand molecular functions of BBS proteins and mechanisms of photoreceptor degeneration.
OSs from normal and Lztfl1 KO retinas were collected by sucrose gradient centrifugation at postnatal day 21 and submitted for label-free quantitative proteomics. Data were collected in 4 independent experiments and t-test was used to determine proteins with significant changes. Quantitative changes, localization, and OS ultrastructure were examined by Western blotting, immunofluorescence microscopy and transmission electron microscopy.
As in human BBS patients and other BBS mouse models, photoreceptors degenerate in the Lztfl1 KO retina. In quantitative proteomic analysis, most of the OS proteins (including Rho, Prph2, Rom1, Pde6a, and Pde6b) were found in normal amounts or only minimally affected. However, 140 proteins showed more than 3-fold enrichment in Lztfl1 mutant OS, while only 9 proteins showed more than 3-fold decrease. Among the enriched proteins, we focused on Stx3 and Munc18-1, which are involved in membrane fusion, for localization study and these proteins displayed striking accumulation in the mutant OS. Disruption of disks and accumulation of large vesicles were observed within the OS of Lztfl1 and Bbs1 mutant retinas.
Our study represents the first comprehensive analysis of OS proteome in ciliopathies and reveals that the main role of BBS proteins in rod photoreceptors is to export non-OS proteins from the OS rather than transporting from the IS to the OS as previously thought. Our data also suggest that accumulation of non-OS proteins and uncontrolled membrane fusion events in the BBS OS underlie photoreceptor cell death.
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