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

Hum Mol Genet. 2015 Nov 15;24(22):6361-73. doi: 10.1093/hmg/ddv346. Epub 2015 Aug 26.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Down-Regulation
  • Exons
  • Genetic Predisposition to Disease
  • HEK293 Cells
  • High-Temperature Requirement A Serine Peptidase 1
  • Humans
  • Macular Degeneration / enzymology
  • Macular Degeneration / genetics*
  • Macular Degeneration / metabolism*
  • Plasminogen Activator Inhibitor 1 / genetics
  • Polymorphism, Single Nucleotide
  • Promoter Regions, Genetic
  • RNA, Messenger / genetics
  • Risk Factors
  • Serine Endopeptidases / genetics*
  • Serine Endopeptidases / metabolism*
  • Signal Transduction
  • Silent Mutation*
  • Transforming Growth Factor beta1 / metabolism*

Substances

  • Plasminogen Activator Inhibitor 1
  • RNA, Messenger
  • SERPINE1 protein, human
  • Transforming Growth Factor beta1
  • High-Temperature Requirement A Serine Peptidase 1
  • HTRA1 protein, human
  • Serine Endopeptidases