Characterization of Tiki, a New Family of Wnt-specific Metalloproteases
Authors: Xi He
Bryan T MacDonald
Anthony J Coyle
Xinjun Zhang
Robert V Martinez
Huilan Gao
Michael Shamashkin
Journal: Journal of Biological Chemistry (2016)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: PrgY; Tiki; TraB; Wnt signaling; metalloprotease
Affiliations: From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115 and., the Centers for Therapeutic Innovation, Pfizer, Boston, Massachusetts 02115., From the F. M. Kirby Neurobiology Center, Boston Children's Hospital, Department of Neurology, Harvard Medical School, Boston, Massachusetts 02115 and .

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. 

Retracted: No

PubPeer comments:
 Figure 5A: Bands look unexpectedly similar. ![file] , 
 Regarding same figure as above, I could spot other great similarity between the FLAG bands. Could the authors check it provide the original gel? ![file] , 
 We appreciate comments by the two readers on the FLAG panel of Fig. 5a of our JBC article. We have investigated the issue carefully since the comments by the first reader was posted. We have reexamined original data and lab notebooks to try to reconstruct, to our best recollection, the process by which Fig. 5a was assembled before submission to JBC. We now recognize that two mistakes occurred inadvertently during the assembly of the FLAG panel of Fig. 5a. The interpretation of Fig. 5a was unaffected. Below we provide a detailed explanation in hindsight on how the two mistakes might have happened: The experiment in Fig.5a was to examine which of the 19 human Wnt proteins can be cleaved by TIKI2, a Wnt protease. The experiment was done by co-transfection of an expression vector of each Wnt (harboring HA and V5 tags) with or without an expression vector for TIKI2N (harboring a FLAG tag). Thus the FLAG panel indicates TIKI2N overexpression and control (without TIKI2N expression). When we performed this experiment, we grouped the most commonly studied Wnts including Wnt1, 3, 3A, 5A, 5B, and 8A in one SDS-PAGE gel, and other Wnts in two additional SDS-PAGE gels. Earlier versions of Fig. 5a that we prepared showed19 Wnts arranged as in these gels, out of the numerical order. During the final preparation of Fig. 5a, we decided to make the figure more reader-friendly by rearranging Wnts numerically, i.e., from Wnt1, Wnt2, to Wnt16. We felt that this would make Fig. 5a easier to follow. Thus we digitally cut the western blot pictures into small pieces and pasted and reassembled them into the current Fig. 5a. Because the key information was about how each Wnt behaved with or without TIKI2N, we cut and pasted each Wnt with and without TIKI2N as a unit, or together with other Wnts if they were already in the numerical order (such as Wnt3 and Wnt3A). We rearranged 19 Wnts, from Wnt1 to Wnt16 this way in Fig.5a. Unfortunately this rearrangement introduced two mistakes inadvertently in the FLAG panel during figure assembly: the FLAG panels from these SDS-PAGE gels were similar, each showing the presence and absence of a FLAG band in alternating lanes (i.e., with and without TIKI2N). We probably opened multiple windows gel photos at the same time, causing one FLAG panel (for Wnt 8B, 9A, 9B, 10A, 10B, 11) to be used twice, and the FLAG blot for Wnt9B was used twice (see the illustration), resulting in issues pointed out by the two readers. We appreciate the effort by the two readers for their careful examination of Fig. 5a. We sincerely apologize for the mistakes in FLAG panel assembly. Please be assured that the interpretation of Fig. 5a was unaffected. We will submit a correction to JBC for Fig. 5a. ![file] Xinjun Zhang and Xi He , 
 2020 correction. https: www.jbc.org content 295 5 1367.short In the FLAG panel of Fig. 5A, the TIKI2N-FLAG bands corresponding to Wnt2, -2B, -4, -6, -7A, -7B, and -16 were incorrect. Additionally, the figure did not indicate that the images were a composite from different immunoblots. These errors have now been corrected and do not affect the results or conclusions of this work. ,