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Kasper Runager, Jarl Underhaug, Charlotte Sørensen, Henrik Karring, Gordon Klintworth, Niels Nielsen, Jan Enghild; Insights into the molecular mechanisms of TGFBIp aggregation in corneal dystrophies. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4738.
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© ARVO (1962-2015); The Authors (2016-present)
Transforming growth factor-β induced protein (TGFBIp) is one of the most abundant proteins in the human cornea. With more than 40 different disease-causing mutations it is an extremely fascinating protein from a structural and biophysical point of view. The TGFBI-linked corneal dystrophies are characterized by an accumulation of misfolded TGFBIp in the sub-epithelial and stromal regions of the cornea. These aggregates have been found to be of either amorphous (granular corneal dystrophies) or amyloid (lattice corneal dystrophies) phenotypes, suggesting that different mechanisms of aggregation are involved. In order to obtain insight into these mechanisms, we undertook a biophysical and structural study of native TGFBIp as well as of a number of disease-causing mutants.
Using transverse urea-gradient gel electrophoresis and circular dichroism spectroscopy we determined the relative stabilities of WT and mutant TGFBIp and of the fourth FAS1 domain (FAS1-4) of TGFBIp. Limited proteolysis was used to identify the core region of A546T FAS1-4 fibrils. Nuclear magnetic resonance spectroscopy was used to determine the three-dimensional structures of WT and mutant FAS1-4.
Our results demonstrate that mutations in the FAS1-4 domain lead to significant alterations in the stability of native TGFBIp. Significantly, we find that this behavior is mirrored when studying the FAS1-4 domain in isolation suggesting that the FAS1-4 domain can be used as a model for TGFBIp stability and aggregation. We observe a decreased susceptibility towards proteolytic degradation in the R555W mutant compared to WT. A solution structure of WT and R555W FAS1-4 shows that this is likely due to decreased flexibility in the region around W555. We further show that conditions promoting electrostatic interactions increase the aggregation propensity of the R555W mutant. For the amyloid fibril-forming mutant A546T we have identified a protease resistant core (the fibril core), which is essential for the dense assembly of protein material characteristic for amyloid fibers.
Our newly gained insight into the structures of mutant FAS1-4 gives us important clues to the mechanisms of aggregation responsible for TGFBIp deposition in the cornea. In turn, we believe that this will prove valuable in rational design of compounds to inhibit TGFBIp aggregation in vivo.
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