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Anil Kumar Chekuri, Katarzyna Zientara-Rytter, Shyamanga Borooah, Marina voronchikhina, Angel Soto-Hermida, Chloe Stanton, Hiroko Matsui, Matteo D'Antonio, Peter Shaw, David S Goodsell, Kelly Frazer, Caroline Hayward, Suresh Subramani, Radha Ayyagari; Regulation of HTRA1 activity by CTRP5 may underlie the pathology of Late Onset Retinal Degeneration (L-ORD).. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4896.
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© ARVO (1962-2015); The Authors (2016-present)
Late Onset Retinal Degeneration (L-ORD) is an autosomal dominant, monogenic macular dystrophy resulting from mutations in C1QTNF5/CTRP5. A mouse model (Ctrp5+/-) of the most common mutation S163R has previously been described. In this study, we screen and validate interacting partners of the protein CTRP5 to understand mechanism underlying L-ORD.
Yeast two hybrid (Y2H) analysis was performed using a normalized human cDNA library to identify the interacting partners of CTRP5, which were further confirmed by co-immunoprecipitation. Interactions between CTRP5 and High Temperature Requirement factor A1 (HTRA1) were established using targeted Y2H constructs with and without the PDZ-binding motif of CTRP5. The impact of wild type (WT) and the CTRP5(S163R) mutant on the protease activity of HTRA1 was measured using an elastin degradation assay. Cleavage of WT and mutant CTRP5 by HTRA1 was analyzed using purified HTRA1 and CTRP5 proteins. CTRP5 expression in the RPE-choroidal tissue of Ctrp5+/- mice was detected using immunohistochemistry and western blot. The potential influence of the CTRP5(S163R) mutation on extracellular matrix (ECM) components was studied by analyzing the RPE-choroidal tissue of Ctrp5+/- mice by western blot.
Y2H analysis identified HTRA1 as an interacting partner of CTRP5. This interaction was mediated through the PDZ-binding motif of CTRP5. Both CTRP5(WT) and CTRP5(S163R) activated the enzymatic activity of HTRA1. However, the activation of HTRA1 was significantly lower when it was pre-incubated with CTRP5(S163R) than with CTRP5(WT) (p<0.038). In addition, HTRA1 cleave CTRP5(WT) while CTRP5(S163R) was resistant to cleavage. Consistent with this, an accumulation of CTRP5 was found in the sub-RPE of Ctrp5+/- mice. Levels of known HTRA1 ECM substrates were decreased in Ctrp5+/- mice compared with WT mice.
The present study identifies a novel interaction between CTRP5 and HTRA1, resulting in a functional modification of HTRA1 activity. The findings suggest that the CTRP5(S163R) mutation also results in a resistance to HTRA1 mediated degradation. Modified HTRA1 activity as a result of S163R-CTRP5 mutation may locally modulate ECM remodeling which potentially underlies the pathology of L-ORD.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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