TY - JOUR
T1 - Synonymous variants in HTRA1 implicated in AMD susceptibility impair its capacity to regulate TGF-βsignaling
AU - Friedrich, Ulrike
AU - Datta, Shyamtanu
AU - Schubert, Thomas
AU - Plössl, Karolina
AU - Schneider, Magdalena
AU - Grassmann, Felix
AU - Fuchshofer, Rudolf
AU - Tiefenbach, Klaus Jürgen
AU - Längst, Gernot
AU - Weber, Bernhard H.F.
N1 - 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.
PY - 2015/11/15
Y1 - 2015/11/15
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84949035607&partnerID=8YFLogxK
U2 - 10.1093/hmg/ddv346
DO - 10.1093/hmg/ddv346
M3 - Article
C2 - 26310622
AN - SCOPUS:84949035607
VL - 24
SP - 6361
EP - 6373
JO - Human Molecular Genetics
JF - Human Molecular Genetics
SN - 0964-6906
IS - 22
ER -