title: Transthyretin is a metallopeptidase with an inducible active site.
abstract: TTR (transthyretin) was found recently to possess proteolytic competency besides its well-known transport capabilities. It was described as a cryptic serine peptidase cleaving multiple natural substrates (including β-amyloid and apolipoprotein A-I) involved in diseases such as Alzheimer's disease and atherosclerosis. In the present study, we aimed to elucidate the catalytic machinery of TTR. All attempts to identify a catalytic serine residue were unsuccessful. However, metal chelators abolished TTR activity. Proteolytic inhibition by EDTA or 1,10-phenanthroline could be reversed with Zn2+ and Mn2+. These observations, supported by analysis of three-dimensional structures of TTR complexed with Zn2+, led to the hypothesis that TTR is a metallopeptidase. Site-directed mutagenesis of selected amino acids unambiguously confirmed this hypothesis. The TTR active site is inducible and constituted via a protein rearrangement resulting in ~7% of proteolytically active TTR at pH 7.4. The side chain of His88 is shifted near His90 and Glu92 establishing a Zn2+-chelating pattern HXHXE not found previously in any metallopeptidase and only conserved in TTR of humans and some other primates. Point mutations of these three residues yielded proteins devoid of proteolytic activity. Glu72 was identified as the general base involved in activation of the catalytic water. Our results unveil TTR as a metallopeptidase and define its catalytic machinery.
authors.affiliation: Nerve Regeneration Group, Instituto de Biologia Molecular e Celular-IBMC, 4150-180 Porto, Portugal.
authors.forename: Márcia A
authors.initials: MA
authors.lastname: Liz
authors.forename: Sérgio C
authors.initials: SC
authors.lastname: Leite
authors.forename: Luiz
authors.initials: L
authors.lastname: Juliano
authors.forename: Maria J
authors.initials: MJ
authors.lastname: Saraiva
authors.forename: Ana M
authors.initials: AM
authors.lastname: Damas
authors.forename: Daniel
authors.initials: D
authors.lastname: Bur
authors.forename: Mónica M
authors.initials: MM
authors.lastname: Sousa
mesh_terms: D020134:Catalytic Domain; D002850:Chromatography, Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D045726:Metalloproteases / Q000378:metabolism*; D011228:Prealbumin / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D059748:Proteolysis
chemical_list: D011228:Prealbumin; D045726:Metalloproteases