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Lili Feng, Elod Kortvely, Andreas Vogt, Karsten Boldt, Marius Ueffing; Quantitative Proteomic Identification of Novel Interacting Partners for the Serine Protease Htra1. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4993.
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Genetic variations on chromosome 10q26 harboring three genes (PLEKHA1, ARMS2 and HTRA1) are associated with an increased risk of the development of age-related macular degeneration (AMD), the most common cause of blindness in the elderly. Htra1 is a secreted serine protease involved in several signaling pathways suggesting that its dysregulation is causally responsible for the impact of this locus on AMD. In spite of the growing knowledge about the molecular processes Htra1 is involved in, no direct pathomechanistic link has been established so far. The identification of the physiological substrates as well as the regulators of this protease may contribute to the understanding of disease mechanism.
A high-throughput quantitative proteomic approach was used to identify proteins interacting with Htra1. Human cell lines (HeLa and ARPE-19) were transfected with a plasmid coding for a tagged, protease-dead point mutant Htra1 protein followed by co-immunoprecipitation of protein complexes. Stable Isotope Labeling of Amino acids in Cell culture (SILAC) approach have been used to identify binding partners of Htra1. Besides, the proteolytically active Htra1 naturally expressed by a human ovarian adenocarcinoma cell line (SKOV3) was immunoprecipitated along with its interacting partners and subjected for labeling (isotope-coded protein label, ICPL) and subsequent protein identification. Finally, this latter high-throughput quantitative proteome profiling approach was used to compare the protein composition of retinas isolated from wild-type vs. Htra1 knock-out mice.
More than 25 proteins were identified as binding partners for Htra1, many of which are putative substrates or members of the serine protease inhibitor family (SERPIN). Importantly, using ARPE-19 cells, Htra1 was coprecipitated with components of the complement systems. Our data also revealed interactions between basement membrane-specific heparan sulfate containing proteins and Htra1.
These results suggest that the compromised regulation of Htra1 may contribute to AMD pathogenesis. The interaction with extracellular matrix proteins is of pivotal importance in spatially restricting the activity of this enzyme. The targeted regulation of Htra1 by using its specific inhibitors or activators might be an important therapeutic strategy for AMD in the future.
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