ABNORMAL BIGH3 AGGREGATIONS IN CORNEAL DYSTROPHIES

角膜营养不良中异常的 BIGH3 聚集

• 批准号: 7904039
• 负责人: Andrew J.W. Huang
• 金额: $ 33.86万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7904039
• 关键词: 5q31; Adhesions; Amyloid; Amyloid Fibrils; Animal Model; Binding; Binding Sites; Biological Assay; Cell Adhesion; Characteristics; Circular Dichroism Spectroscopy; Coculture Techniques; Collagen; Complementary DNA; Congo Red; Cornea; Corneal Stroma; Corneal dystrophy; Deletion Mutagenesis; Deposition; Electrophoresis; Electrophoretic Mobility Shift Assay; Elements; Epithelial; Epithelial Cells; Exons; Extracellular Matrix; Fluorescence; Fluorescence Spectroscopy; Fourier Transform; Gene Activation; Genes; Genetic Transcription; Goals; Human; Inherited; Keratoplasty; Knowledge; Length; Mediating; Methods; Missense Mutation; Modeling; Molecular; Molecular Conformation; Monitor; Pain; Pathogenesis; Peptide Hydrolases; Peptides; Phenotype; Porifera; Production; Property; Proteins; Proteolysis; RNA Splicing; Race; Recombinant Proteins; Recombinants; Reporting; Research; Role; Scanning; Screening procedure; Serum; Simulate; Small Interfering RNA; Solutions; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis; Staining method; Stains; System; Therapeutic; Thioflavin T; Transforming Growth Factor-Beta Induced Protein IGH3; Untranslated Regions; Variant; Work; abstracting; amyloid fibril formation; amyloid formation; cDNA Library; fibrillogenesis; genetic regulatory protein; in vitro Model; in vivo; mutant; novel; novel strategies; novel therapeutics; ocular surface; overexpression; prevent; promoter; protein aggregate; protein aggregation; protein expression; synthetic peptide; transcription factor

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.

描述(申请人提供)：bIGH3(角化上皮蛋白)是细胞外基质(ECM)的一种成分，负责细胞黏附。一些常染色体显性遗传性角膜营养不良是由于人类bIGH3基因5q31的30多个错义突变所致。这些营养不良症被发现有异常的基质沉积和相关的上皮粘连不良，从而导致疼痛的角膜侵蚀。通常需要角膜移植来恢复角膜的清晰度。这一建议的工作假设是，这些不利于蛋白质聚集的形成要么是由于错义突变导致的bIGH3蛋白质构象错误折叠和/或是bIGH3蛋白质的异常积累。本研究拟通过圆二色谱(CD)、本征荧光光谱、傅里叶变换红外光谱(FTIR)和有限蛋白分解等方法研究天然和突变的bIGH3蛋白的构象。淀粉样原纤维的形成将通过刚果红和硫代黄素T分析进行评估。由不同降解的天然蛋白和突变蛋白形成的淀粉样纤维也将在无血清系统中进行研究。合成肽的纤维形成作用将被用来确定淀粉样蛋白的形成机制。我们还将研究各种ECM成分对淀粉样蛋白形成的调节作用。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，将利用体内紫外光足迹和凝胶迁移率改变分析(EMSA)以及缺失突变来鉴定与角膜中bIGH3基因激活有关的转录因子Sp1和Smads。使用预先建立的角膜cDNA文库，将确定通过Capite Hunting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)的角膜特异性转录变体。利用天然角膜基质和bIGH3的过表达来模拟角膜营养不良，建立了两种新的体外模型。一种合成的胶原蛋白海绵(作为基质替代品)将用于比较。这些模型将监测bIGH3和相关的淀粉样纤维的沉积或这些蛋白质随后的溶解。新的治疗策略，如小干扰RNA(SiRNA)或甲基化多肽(Meptie)，以减轻不利的bIGH3聚集也将被研究。这项研究将有助于阐明bIGH3相关角膜营养不良中异常蛋白聚集的发病机制，并制定潜在的治疗策略。公共卫生相关性异常蛋白沉积与遗传性bGIH3(或角化上皮蛋白)相关的角膜营养不良有关。通常需要角膜移植来恢复角膜的清晰度，并防止因这些不愉快的蛋白质沉积而导致的眼表疼痛。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，如多肽和siRNAs，以防止蛋白质沉积在角膜上。