Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-βsignaling

Ulrike Friedrich, Shyamtanu Datta, Thomas Schubert, Karolina Plössl, Magdalena Schneider, Felix Grassmann, Rudolf Fuchshofer, Klaus Jürgen Tiefenbach, Gernot Längst, Bernhard H.F. Weber*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

42 Citations (Scopus)

Abstract

High-temperature requirement A1 (HTRA1) is a secreted serine protease reported to play a role in the development of several cancers and neurodegenerative diseases. Still, the mechanism underlying the disease processes largely remains undetermined. In age-related macular degeneration (AMD), a common cause of vision impairment and blindness in industrialized societies, two synonymous polymorphisms in exon 1 of the htra1 gene were associated with a high risk to develop disease. Here, we show that the two polymorphisms result in a protein with altered thermophoretic properties upon heat-induced unfolding, trypsin accessibility and secretion behavior, suggesting unique structural features of the AMDrisk-associated HTRA1 protein. Applying MicroScale Thermophoresis and protease digestion analysis, we demonstrate direct binding and proteolysis of transforming growth factor ß1 (TGF-β1) by normal HTRA1 but not the AMD-risk-associated isoform. As a consequence, both HTRA1 isoforms strongly differed in their ability to control TGF-β mediated signaling, as revealed by reporter assays targeting the TGF-β1-induced serpin peptidase inhibitor (SERPINE1, alias PAI-1) promoter. In addition, structurally altered HTRA1 led to an impaired autocrine TGF-β signaling in microglia, as measured by a strong down-regulation of downstream effectors of the TGF-β cascade such as phosphorylated SMAD2 and PAI-1 expression. Taken together, our findings demonstrate the effects of two synonymous HTRA1 variants on protein structure and protein interaction with TGF-β1. As a consequence, this leads to an impairment of TGF-β signaling and microglial regulation. Functional implications of the altered properties on AMD pathogenesis remain to be clarified.

Original languageEnglish
Pages (from-to)6361-6373
Number of pages13
JournalHuman Molecular Genetics
Volume24
Issue number22
Early online date26 Aug 2015
DOIs
Publication statusPublished - 15 Nov 2015

Bibliographical note

Funding: This work was supported in part by grants from the Deutsche Forschungsgemeinschaft (DFG) (WE1259/19-1 and WE1259/19-2 to B.H.F.W.).

Acknowledgements: We thank Thomas Langmann and Marcus Karlstetter (Laboratory for Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Germany) for providing BV-2 cells. We also thank Raphael Lange (Institute of Human Genetics, University of Regensburg, Germany) for excellent technical assistance.

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