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Charlotte Skovgaard Sørensen, Kasper Runager, Carsten Scavenius, Kristian W Sanggaard, Jan J Enghild; Identification of a fibril core-based inhibitor of TGFBIp fibrillation in relation to lattice corneal dystrophy. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1004.
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© ARVO (1962-2015); The Authors (2016-present)
The Ala546Thr substitution in the transforming growth factor β-induced protein (TGFBIp) results in amyloid type aggregates in the corneal stroma also known as lattice corneal dystrophy. We have previously shown that the FAS1-4 domain may be used as a model system for TGFBIp in regards of stability and fibrillation propensity. To shed light on the fibrillation mechanisms of TGFBIp and to identify potential inhibitors of TGFBIp fibrillation we characterized the structure of FAS1-4 fibrils.
In order to probe the structure and core region of FAS1-4 A546T fibrils, fibrils were subjected to hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses. Furthermore we utilized limited proteolysis to identify the fibril core regions. Two synthetic peptides corresponding to the fibril core regions were probed for fibrillating propensity using ThT fluorescence and finally the inhibitory effect of two modified fibril core peptides was evaluated.
Comparison of the HDX-MS data from soluble and fibrillated Ala546Thr FAS1-4 reveals that a large part of FAS1-4 becomes highly protected against hydrogen exchange after fibrillation, suggesting that it is involved in a rigid fibril structure. The protected regions are separated in two by a small region with less protection against hydrogen exchange. Limited digestion of FAS-1-4 fibrils identified the same two core regions and we have shown that both of them contain an intrinsic fibrillating propensity. Finally, we show that a modified version of one of the core peptides is able to inhibit the fibrillation process of the unmodified peptide and Ala546Thr FAS1-4.
Using HDX-MS and limited proteolysis we have identified the fibril cores of FAS1-4 Ala546Thr fibrils and we have shown that both core regions possess an intrinsic fibrillating propensity. Furthermore, we have succeeded in inhibiting the fibrillation process of the core peptide, using a modified peptide, which is also shown to have an effect on the fibrillation of FAS1-4. Knowledge of the core regions could be crucial in elucidating the disease mechanism and in the present study we have shown that this knowledge facilitates design of peptide inhibitors of fibrillation.
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