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C. Yuan, M. R. Little, H. L. Berscheit, A. J. W. Huang; Mechanisms of Amyloid Fibril Formation for Keratoepithelin. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1011. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the mechanisms of amyloid fibril formation for keratoepithelin (KE).
Mammalian cell-expressed recombinant wildtype KE and two dystrophy-related mutants, R124C and R555W, were used for characterization of molecular properties. The conformational changes and thermostability were evaluated by circular dichroism spectroscopy, intrinsic tryptophan fluorescence, fluorescence quenching and 1-anilino-8-naphthalene sulfonate (ANS) fluorescence assay. Formation of oligomers was monitored by Western blots. Protofibrils and matured fibrils were measured by bis-ANS and Thioflavin T (ThT) fluorescence assays, respectively. The oligomerization and aggregations of amyloidogenic KE were further characterized utilizing size-exclusion chromatography and transmission electron microscopy (TEM).
Compared to wildtype KE, R124C and R555W displayed altered conformations based on intrinsic tryptophan, ANS fluorescence spectroscopies and quenching experiments. Results from guanidine hydrochloride denaturation or high temperature incubation also revealed reduced thermostability of mutants. Circular dichroism and ThT fluorescence studies showed that KE aggregated and readily formed amyloid fibrils under sub-denaturation conditions, such as 10% trifluoroethanol. Although wildtype and mutant proteins all form amyloid fibrils in vitro when incubated for extended periods of time, mutant proteins tend to produce more cytotoxic oligomers and prominent aggregations as judged from the size-exclusion chromatography. The fibril morphology, as determined by TEM study, also differed from that of wildtype.
Our study demonstrates that diseased-related KE mutants have altered protein conformations and reduced thermostability. These pertubations from missense mutations likely contribute to the pathological amyloid fibril formation in KE.
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