ABNORMAL BIGH3 AGGREGATIONS IN CORNEAL DYSTROPHIES

描述（由申请人提供）：bIGH3（角膜上皮蛋白）是负责细胞粘附的细胞外基质（ECM）的组成部分。几种常染色体显性角膜营养不良症可归因于人类5q31中超过30个bIGH3基因错义突变。这些营养不良患者发现有异常的间质沉积和相关的上皮粘连不良，导致疼痛的角膜糜烂。通常需要角膜移植来恢复角膜清晰度。该建议的工作假设是，这些不良蛋白聚集的形成是由错义突变和/或失调的bIGH3蛋白积累引起的bIGH3蛋白构象错误折叠引起的。本研究拟通过圆二色性（CD）光谱、本征荧光光谱、傅立叶变换红外共振（FTIR）光谱和有限蛋白水解研究天然和突变bIGH3蛋白的构象。淀粉样蛋白纤维的形成将通过刚果红和硫黄素T测定来评估。由不同降解的天然蛋白和突变蛋白形成的淀粉样原纤维也将在无血清系统中进行研究。合成肽的纤维形成将用于确定淀粉样蛋白的形成机制。各种ECM成分对淀粉样蛋白形成的调节也将被研究。已知bIGH3基因启动子受TGFb调控。为了进一步确定tgfb介导的基因激活，我们将使用体内UV光足迹和电泳迁移转移试验（EMSA）以及缺失突变来鉴定角膜中负责bIGH3基因激活的Sp1和Smads等转录因子。利用预先建立的角膜cDNA文库，将通过衣壳寻找方法（针对5-UTR中的变体）、cDNA末端的快速扩增（针对3-UTR中的变体）和外显子扫描PCR（针对剪接变体）确定角膜特异性转录变体。利用天然角膜基质和过表达bIGH3来模拟角膜营养不良的两种新的体外模型已经建立。将使用合成胶原蛋白海绵（作为基质替代品）进行比较。这些模型将监测igh3和相关淀粉样蛋白原纤维的沉积或随后这些蛋白质的溶解。新的治疗策略，如小干扰rna （siRNA）或甲基化肽（肽），以减轻不利的高3聚集也将被研究。本研究将有助于阐明bigh3相关角膜营养不良异常蛋白聚集的发病机制，并制定潜在的治疗策略。异常蛋白沉积与遗传性bGIH3（或角膜上皮蛋白）相关的角膜营养不良有关。通常需要角膜移植来恢复角膜清晰度和防止由不良蛋白沉积引起的痛苦的眼表破裂。本研究旨在探讨蛋白质异常聚集的分子机制，并设计新的治疗策略，如肽和sirna，以防止蛋白质沉积在角膜上。

DESCRIPTION (provided by applicant): bIGH3 (keratoepithelin) is a constituent of the extracellular matrix (ECM) responsible for cell adhesion. Several autosomal dominant corneal dystrophies are attributed to more than 30 missense mutations of the bIGH3 gene in 5q31 in humans. These dystrophies are found to have abnormal stromal deposits and related poor epithelial adhesions with resultant painful corneal erosions. Corneal transplantation is often needed to restore corneal clarity. The working hypothesis of this proposal is that formation of those untoward protein aggregates is caused by either conformational misfolding of bIGH3 proteins due to missense mutations and/or accumulation of dysregulated bIGH3 proteins. The proposal intends to investigate the conformations of native and mutant bIGH3 proteins by circular dichroism (CD) spectroscopy, intrinsic fluorescence spectroscopy, fourier transformed infrared resonance (FTIR) spectroscopy, and limited proteolysis. Amyloid fibril formation will be evaluated by Congo red and Thioflavin T assays. Amyloid fibrils formed by differentially degraded native and mutant proteins will also be studied in a serum-free system. Fibrillogenesis of synthetic peptides will be used to identify the amyloidogenic mechanisms. Modulation of amyloid formation by various ECM components will also be investigated. bIGH3 gene promoter is known to be regulated by TGFb. To further determine the TGFb-mediated gene activation, transcriptional factors such as Sp1 and Smads responsible for bIGH3 gene activation in corneas will be characterized using in vivo UV photofootprinting and electrophoresis mobility shift assay (EMSA) along with deletion mutagenesis for confirmation. Using pre-established corneal cDNA libraries, cornea-specific transcription variants by the capsite hunting method (for variants in the 5-UTR), rapid amplification of cDNA ends (for variants in 3-UTR), and exon scanning PCR (for splice variants) will be determined. Two new in vitro models using native corneal stroma and overexpression of bIGH3 to simulate corneal dystrophies have been established. A synthetic collagen sponge (as a stromal substitute) will be used for comparison. Depositions of bIGH3 and related amyloid fibrils or subsequent dissolution of those proteins will be monitored by these models. Novel therapeutic strategies, such as small interfering RNAs (siRNA) or methylated peptides (meptide), to mitigate the untoward bIGH3 aggregation will also be investigated. This research should help to elucidate the pathogenesis of abnormal protein aggregations in bIGH3-related corneal dystrophies and to formulate potential therapeutic strategies. PUBLIC HEALTH RELEVANCE Abnormal protein deposits are associated with hereditary bGIH3(or keratoepithelin)-related corneal dystrophies. Corneal transplantation is often needed to restore corneal clarity and prevent painful ocular surface breakdown caused by those untoward protein deposits. This study is undertaken to investigate the molecular mechanism of abnormal protein aggregation and to devise novel therapeutic strategies such as meptides and siRNAs to prevent the protein deposits on cornea.