ABNORMAL BIGH3 AGGREGATIONS IN CORNEAL DYSTROPHIES

角膜营养不良中异常的 BIGH3 聚集

• 批准号: 7675983
• 负责人: Andrew J.W. Huang
• 金额: $ 34.2万
• 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/7675983
• 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; 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; public health relevance; 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（由申请人提供）：bIGH 3（角膜上皮素）是负责细胞粘附的细胞外基质（ECM）的成分。几种常染色体显性角膜营养不良归因于人类5 q31中bIGH 3基因的30多个错义突变。发现这些营养不良具有异常的基质沉积和相关的上皮粘附不良，导致疼痛的角膜糜烂。通常需要角膜移植来恢复角膜透明度。该建议的工作假设是，这些不利的蛋白质聚集体的形成是由bIGH 3蛋白的构象错误折叠引起的，所述构象错误折叠是由于错义突变和/或失调的bIGH 3蛋白的积累。该提案旨在通过圆二色性（CD）光谱，固有荧光光谱，傅立叶变换红外共振（FTIR）光谱和有限的蛋白水解研究天然和突变体bIGH 3蛋白的构象。将通过刚果红和硫磺素T试验评价淀粉样原纤维形成。淀粉样原纤维形成的差异降解的天然和突变蛋白质也将在无血清系统中进行研究。合成肽的纤维形成将用于鉴定淀粉样蛋白形成机制。还将研究各种ECM组分对淀粉样蛋白形成的调节。已知bIGH 3基因启动子受TGF β调节。为了进一步确定TGF β介导的基因激活，将使用体内UV光足迹法和电泳迁移率变动分析（EMSA）以及沿着缺失诱变来表征角膜中负责bIGH 3基因激活的转录因子如Sp1和Smads，以进行确认。使用预先建立的角膜cDNA文库，将通过帽位点狩猎法（对于5-UTR中的变体）、cDNA末端的快速扩增（对于3-UTR中的变体）和外显子扫描PCR（对于剪接变体）确定角膜特异性转录变体。已经建立了两种新的使用天然角膜基质和bIGH 3过表达来模拟角膜营养不良的体外模型。将使用合成胶原海绵（作为基质替代物）进行比较。通过这些模型监测bIGH 3和相关淀粉样蛋白原纤维的沉积或随后这些蛋白的溶解。还将研究新的治疗策略，如小干扰RNA（siRNA）或甲基化肽（meptide），以减轻bIGH 3的不良聚集。这项研究有助于阐明bIGH 3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。异常蛋白质沉积与遗传性bGIH 3（或角膜上皮素）相关的角膜营养不良有关。通常需要角膜移植来恢复角膜透明度并防止由这些不良蛋白质沉积物引起的疼痛性眼表破裂。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，如美肽和siRNA，以防止蛋白质沉积在角膜上。