Ghaffari Sharaf Mehdi, Cetinel Sibel, Semenchenko Valentyna, Damji Karim F, Unsworth Larry D, Montemagno Carlo
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB, Canada; Ingenuity Lab, University of Alberta, Edmonton, AB, Canada.
National Institute of Nanotechnology, National Research Council, Edmonton, AB, Canada.
Exp Eye Res. 2017 Dec;165:109-117. doi: 10.1016/j.exer.2017.10.001. Epub 2017 Oct 3.
Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. βB2-crystallin, a member of β-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human βB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.
晶状体蛋白是位于眼球晶状体中的主要蛋白质家族。白内障被认为是由于不溶性纤维状聚集体的形成，这些聚集体主要由晶状体蛋白家族的蛋白质组成。如今，现有的唯一白内障治疗方法是手术，而发展中国家的个人可能难以获得这种治疗。开发治疗白内障的新型药物治疗方法取决于对这些结构的特异性靶向。βB2-晶状体蛋白是β-晶状体蛋白家族的成员，是晶状体中晶状体蛋白的主要成分，因此被用于在体外形成模型纤维。使用噬菌体展示技术鉴定出与这些纤维具有高亲和力的肽。重组表达的人βB2-晶状体蛋白在不同温度下于10%（v/v）三氟乙醇（TFE）溶液（pH 2.0）中进行纤维化，并使用硫黄素-T（ThT）测定、透射电子显微镜（TEM）和X射线纤维衍射（XRFD）分析确认其淀粉样结构。使用酶联免疫吸附测定（ELISA）分析鉴定出的噬菌体展示肽的亲和力。一种环肽（CKQFKDTTC）的特异性结合显示出最高亲和力，这通过竞争性抑制测定得到证实。
