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Elavazhagan Murugan, Anandalakshmi Venkataraman, Roger W Beuerman, Rajamani Lakshminarayanan, Jodhbir S Mehta; TGFβIP model peptides with mutations reducing the overall charge forms cytotoxic amyloid fibrils.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3079.
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© ARVO (1962-2015); The Authors (2016-present)
Distinct mutations in transforming growth factor-β induced protein (TGFβIP) lead to different stromal corneal dystrophies. TGFβIP associated corneal dystrophy patient samples reveal smaller fragments of TGFβIP than the full-length protein. Since smaller peptides are known to exhibit greater aggregation propensities, using in silico methods, we identified a region (WT; 611aa-633aa) within TGFβIP that displayed high aggregation propensities. Mutations reducing the overall net charge of the polypeptides are known to increase their aggregation propensities. Hence we chose 5 pathological mutations (M619K, N622H, N622K, G623R, H626R) that reduced the overall net charge. We examined the biophysical properties and cytotoxicities of these model peptides.
The fibrillation of the peptides was followed by thioflavin T (ThT) binding and circular dichroism (CD) spectroscopy. The morphologies of the fibrils were examined using transmission electron microscopy (TEM). Thermal denaturation of the peptides were studied using CD while heating the peptides to 80°C and the integrity of the fibrils was studied using TEM. The cytotoxicities of the peptides and the fibrils on primary human corneal stromal fibroblast (HCSF) cells were studied using the xCELLigence system.
All the peptides formed fibrils, though at varying rates. While the G623R peptides formed fibrils fastest, the WT peptides were the slowest. The TEM images showed that N622H fibrils had the highest packing density. While, fully formed fibrils were visible in N622H, a population of smaller fragments were observed for WT. While the rest of the peptides disintegrated at 80°C, the G623R peptide did not undergo changes in its structure and TEM studies showed that the G623R peptide fibrils remained intact. All the fibrils were relatively more cytotoxic compared to the native peptides and amongst the fibrils the thermostable G623R peptide fibrils were the most cytotoxic.
Our results clearly show that charge reducing mutations increase the aggregation propensities of the polypeptides and different mutations lead to different modes of fibril formations. The substitution of uncharged, flexible glycine to postively charged arginine results to formation of thermostable fibrils by the G623R peptide. The amyloid fibrils of the peptides were more cytotoxic compared to the peptides themselves.
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