ABNORMAL BIGH3 AGGREGATIONS IN CORNEAL DYSTROPHIES

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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.