title: Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-β signaling.
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 (rs1049331:C>T, and rs2293870:G>T) 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 AMD-risk-associated HTRA1 protein. Applying MicroScale Thermophoresis and protease digestion analysis, we demonstrate direct binding and proteolysis of transforming growth factor β1 (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.
authors: Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
authors: Ulrike
authors: U
authors: Friedrich
authors: Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
authors: Shyamtanu
authors: S
authors: Datta
authors: Department of Biochemistry, University of Regensburg, 2bind GmbH, Josef Engert Straße 13, 93053 Regensburg, Germany.
authors: Thomas
authors: T
authors: Schubert
authors: Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
authors: Karolina
authors: K
authors: Plössl
authors: Institute of Human Anatomy and Embryology, University of Regensburg.
authors: Magdalena
authors: M
authors: Schneider
authors: Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
authors: Felix
authors: F
authors: Grassmann
authors: Institute of Human Anatomy and Embryology, University of Regensburg.
authors: Rudolf
authors: R
authors: Fuchshofer
authors: Institute of Biophysics and Physical Biochemistry, University of Regensburg, Universitätsstraße 31, 93053 Regensburg, Germany and.
authors: Klaus-Jürgen
authors: KJ
authors: Tiefenbach
authors: Department of Biochemistry, University of Regensburg.
authors: Gernot
authors: G
authors: Längst
authors: Institute of Human Genetics, University of Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany, bweb@klinik.uni-regensburg.de.
authors: Bernhard H F
authors: BH
authors: Weber
mesh_terms: D015536:Down-Regulation; D005091:Exons; D020022:Genetic Predisposition to Disease; D057809:HEK293 Cells; D000074289:High-Temperature Requirement A Serine Peptidase 1; D006801:Humans; D008268:Macular Degeneration / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism*; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D012307:Risk Factors; D012697:Serine Endopeptidases / Q000235:genetics* / Q000378:metabolism*; D015398:Signal Transduction; D000069456:Silent Mutation; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; C051321:SERPINE1 protein, human; D053773:Transforming Growth Factor beta1; D000074289:High-Temperature Requirement A Serine Peptidase 1; C474012:HTRA1 protein, human; D012697:Serine Endopeptidases