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The serine protease HtrA1 cleaves misfolded transforming growth factor ?-induced protein (TGFBIp) and induces amyloid formation.

ABSTRACT: The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor ?-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in normal TGFBIp turnover and in corneal amyloid formation. We show that HtrA1 can cleave WT TGFBIp but prefers amyloidogenic variants. Corneal TGFBIp is extensively processed in healthy people, resulting in C-terminal degradation products spanning the FAS1-4 domain of TGFBIp. We show here that HtrA1 cleaves the WT FAS1-4 domain only inefficiently, whereas the amyloidogenic FAS1-4 mutations transform this domain into a considerably better HTRA1 substrate. Moreover, HtrA1 cleavage of the mutant FAS1-4 domains generated peptides capable of forming in vitro amyloid aggregates. Significantly, these peptides have been previously identified in amyloid deposits in vivo, supporting the idea that HtrA1 is a causative agent for TGFBIp-associated amyloidosis in corneal dystrophy. In summary, our results indicate that TGFBIp is an HtrA1 substrate and that some mutations in the gene encoding TGFBIp cause aberrant HtrA1-mediated processing that results in amyloidogenesis in corneal dystrophies.

SUBMITTER: Poulsen ET

PROVIDER: S-EPMC6682723 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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The serine protease HtrA1 cleaves misfolded transforming growth factor β-induced protein (TGFBIp) and induces amyloid formation.

Poulsen Ebbe Toftgaard ET Nielsen Nadia Sukusu NS Scavenius Carsten C Mogensen Emilie Hage EH Risør Michael W MW Runager Kasper K Lukassen Marie V MV Rasmussen Casper B CB Christiansen Gunna G Richner Mette M Vorum Henrik H Enghild Jan J JJ

The Journal of biological chemistry 20190613 31

The serine protease high-temperature requirement protein A1 (HtrA1) is associated with protein-misfolding disorders such as Alzheimer's disease and transforming growth factor β-induced protein (TGFBIp)-linked corneal dystrophy. In this study, using several biochemical and biophysical approaches, including recombinant protein expression, LC-MS/MS and 2DE analyses, and thioflavin T (ThT) fluorescence assays for amyloid fibril detection, and FTIR assays, we investigated the role of HtrA1 both in no ...[more]

PMID: 31197037

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Project description:Specific mutations in the transforming growth factor beta induced (TGFBI) gene are associated with lattice corneal dystrophy (LCD) type 1 and its variants. In this study, we performed an in-depth proteomic analysis of human corneal amyloid deposits associated with the heterozygous A546D mutation in TGFBI.Corneal amyloid deposits and the surrounding corneal stroma were procured by laser capture microdissection from a patient with an A546D mutation in TGFBI. Proteins in the captured corneal samples and healthy corneal stroma were identified with liquid chromatography-tandem mass spectrometry and quantified by calculating exponentially modified Protein Abundance Index values. Mass spectrometry data were further compared for identifying enriched regions of transforming growth factor beta induced protein (TGFBIp/keratoepithelin/?ig-h3) and detecting proteolytic cleavage sites in TGFBIp.A C-terminal fragment of TGFBIp containing residues Y571-R588 derived from the fourth fasciclin 1 domain (FAS1-4), serum amyloid P-component, apolipoprotein A-IV, clusterin, and serine protease HtrA1 were significantly enriched in the amyloid deposits compared to the healthy cornea. The proteolytic cleavage sites in TGFBIp from the diseased cornea are in accordance with the activity of serine protease HtrA1. We also identified small amounts of the serine protease kallikrein-14 in the amyloid deposits.Corneal amyloid caused by the A546D mutation in TGFBI involves several proteins associated with other varieties of amyloidosis. The proteomic data suggest that the sequence 571-YHIGDEILVSGGIGALVR-588 contains the amyloid core of the FAS1-4 domain of TGFBIp and point at serine protease HtrA1 as the most likely candidate responsible for the proteolytic processing of amyloidogenic and aggregated TGFBIp, which explains the accumulation of HtrA1 in the amyloid deposits. With relevance to identifying serine proteases, we also found glia-derived nexin (protease-nexin 1) in the amyloid deposits, making this serine protease inhibitor a good candidate for the physiologically relevant inhibitor of one of the amyloid-associated serine proteases in the cornea and probably in other tissues. Noteworthy, the present results are in accordance with our findings from a previous study of corneal amyloid deposits caused by the V624M mutation in TGFBI, suggesting a common mechanism for lattice corneal dystrophies (LCDs) associated with mutations in the TGFBIp FAS1-4 domain.

Dataset Information

The serine protease HtrA1 cleaves misfolded transforming growth factor ?-induced protein (TGFBIp) and induces amyloid formation.

Publications

The serine protease HtrA1 cleaves misfolded transforming growth factor β-induced protein (TGFBIp) and induces amyloid formation.

Similar Datasets

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