Destruction of Amyloid Fibrils of Keratoepithelin Peptides by Laser Irradiation Coupled with Amyloid-specific Thioflavin T

Daisaku Ozawa,Yuichi Kaji,Hisashi Yagi,Kazumasa Sakurai,Toru Kawakami,Hironobu Naiki,Yuji Goto

doi:10.1074/jbc.m111.222901

Abstract

Mutations in keratoepithelin are associated with blinding ocular diseases, including lattice corneal dystrophy type 1 and granular corneal dystrophy type 2. These diseases are characterized by deposits of amyloid fibrils and/or granular non-amyloid aggregates in the cornea. Removing the deposits in the cornea is important for treatment. Previously, we reported the destruction of amyloid fibrils of β(2)-microglobulin K3 fragments and amyloid β by laser irradiation coupled with the binding of an amyloid-specific thioflavin T. Here, we studied the effects of this combination on the amyloid fibrils of two 22-residue fragments of keratoepithelin. The direct observation of individual amyloid fibrils was performed in real time using total internal reflection fluorescence microscopy. Both types of amyloid fibrils were broken up by the laser irradiation, dependent on the laser power. The results suggest the laser-induced destruction of amyloid fibrils to be a useful strategy for the treatment of these corneal dystrophies.

Highlights

Fluorescence dye thioflavin T (ThT)3, resulting in a characteristic fluorescence emission at 482– 490 nm with an excitation maximum at 446 – 455 nm [10, 11]
We developed a unique technique for their direct observation, which combines total internal reflection fluorescence microscopy (TIRFM) with an amyloidspecific ThT (18 –22)
To observe individual amyloid fibrils with TIRFM, we searched for the best conditions for the spontaneous formation of fibrils of C110 –131 and H110 –131 peptides

Summary

Introduction

Fluorescence dye thioflavin T (ThT), resulting in a characteristic fluorescence emission at 482– 490 nm with an excitation maximum at 446 – 455 nm [10, 11]. We developed a unique technique for their direct observation, which combines total internal reflection fluorescence microscopy (TIRFM) with an amyloidspecific ThT (18 –22). The approach can provide important information about the morphology, growth rate, and direction of extension of individual fibrils in real time. Using this method, we previously observed the laser irradiation-dependent inhibition of ␤2-microglobulin fibril growth and the destruction of preformed fibrils of K3, a 22-residue peptide of ␤2-microglobulin [23]. Irradiation during the formation of fibrils resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils, leading to a bell-. We propose that the effects of the irradiation are determined by a balance between the laser-induced acceleration of propagation and the destruction

Results

Discussion

Conclusion