Characterization of Tiki, a New Family of Wnt-specific Metalloproteases
| Autor: | Bryan T. MacDonald, Xi He, Huilan Gao, Robert V. Martinez, Michael Shamashkin, Anthony J. Coyle, Xinjun Zhang |
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| Rok vydání: | 2015 |
| Předmět: |
0301 basic medicine
Proteases Protein Folding Xenopus Amino Acid Motifs Pheromones Human Biology Cleavage (embryo) Ligands Biochemistry Gene Expression Regulation Enzymologic Protein Structure Secondary 03 medical and health sciences Catalytic Domain Wnt3A Protein Fluorescence Resonance Energy Transfer Animals Humans Cysteine Disulfides Luciferases Molecular Biology Metalloproteinase Wnt signaling pathway Membrane Proteins Metalloendopeptidases Cell Biology biology.organism_classification Wnt Proteins 030104 developmental biology HEK293 Cells Membrane protein Metalloproteases Mutagenesis Site-Directed Protein folding Additions and Corrections Peptides Hydrophobic and Hydrophilic Interactions Signal Transduction |
| Zdroj: | The Journal of biological chemistry. 291(5) |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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