title: Characterization of Tiki, a New Family of Wnt-specific Metalloproteases.
abstract: The Wnt family of secreted glycolipoproteins plays pivotal roles in development and human diseases. Tiki family proteins were identified as novel Wnt inhibitors that act by cleaving the Wnt amino-terminal region to inactivate specific Wnt ligands. Tiki represents a new metalloprotease family that is dependent on Mn(2+)/Co(2+) but lacks known metalloprotease motifs. The Tiki extracellular domain shares homology with bacterial TraB/PrgY proteins, known for their roles in the inhibition of mating pheromones. The TIKI/TraB fold is predicted to be distantly related to structures of additional bacterial proteins and may use a core β-sheet within an α+β-fold to coordinate conserved residues for catalysis. In this study, using assays for Wnt3a cleavage and signaling inhibition, we performed mutagenesis analyses of human TIKI2 to examine the structural prediction and identify the active site residues. We also established an in vitro assay for TIKI2 protease activity using FRET peptide substrates derived from the cleavage motifs of Wnt3a and Xenopus wnt8 (Xwnt8). We further identified two pairs of potential disulfide bonds that reside outside the β-sheet catalytic core but likely assist the folding of the TIKI domain. Finally, we systematically analyzed TIKI2 cleavage of the 19 human WNT proteins, of which we identified 10 as potential TIKI2 substrates, revealing the hydrophobic nature of Tiki cleavage sites. Our study provides insights into the Tiki family of proteases and its Wnt substrates.
authors.affiliation: From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115 and.
authors.forename: Xinjun
authors.initials: X
authors.lastname: Zhang
authors.affiliation: From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115 and.
authors.forename: Bryan T
authors.initials: BT
authors.lastname: MacDonald
authors.affiliation: the Centers for Therapeutic Innovation, Pfizer, Boston, Massachusetts 02115.
authors.forename: Huilan
authors.initials: H
authors.lastname: Gao
authors.affiliation: the Centers for Therapeutic Innovation, Pfizer, Boston, Massachusetts 02115.
authors.forename: Michael
authors.initials: M
authors.lastname: Shamashkin
authors.affiliation: the Centers for Therapeutic Innovation, Pfizer, Boston, Massachusetts 02115.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Coyle
authors.affiliation: the Centers for Therapeutic Innovation, Pfizer, Boston, Massachusetts 02115.
authors.forename: Robert V
authors.initials: RV
authors.lastname: Martinez
authors.affiliation: From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115 and xi.he@childrens.harvard.edu.
authors.forename: Xi
authors.initials: X
authors.lastname: He
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D015971:Gene Expression Regulation, Enzymologic; D057809:HEK293 Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D008666:Metalloendopeptidases / Q000737:chemistry*; D045726:Metalloproteases / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D049049:Pheromones, Human / Q000378:metabolism; D017510:Protein Folding; D017433:Protein Structure, Secondary; D015398:Signal Transduction; D051153:Wnt Proteins / Q000737:chemistry*; D060509:Wnt3A Protein / Q000737:chemistry; D014981:Xenopus
chemical_list: D004220:Disulfides; D008024:Ligands; D008565:Membrane Proteins; D010455:Peptides; D049049:Pheromones, Human; C555510:WNT3A protein, human; D051153:Wnt Proteins; D060509:Wnt3A Protein; D008156:Luciferases; D045726:Metalloproteases; C000709687:TRABD2A protein, human; D008666:Metalloendopeptidases; C000595448:TRABD2B protein, human; D003545:Cysteine
keywords: PrgY; Tiki; TraB; Wnt signaling; metalloprotease; pheromone; protease; β-catenin