Abstract
A complex interaction of signalling events, including the Wnt pathway, regulates sprouting of blood vessels from pre-existing vasculature during angiogenesis. Here we show that two distinct mutations in the (uro)chordate-specific gumby (also called Fam105b) gene cause an embryonic angiogenic phenotype in gumby mice. Gumby interacts with disheveled 2 (DVL2), is expressed in canonical Wnt-responsive endothelial cells and encodes an ovarian tumour domain class of deubiquitinase that specifically cleaves linear ubiquitin linkages. A crystal structure of gumby in complex with linear diubiquitin reveals how the identified mutations adversely affect substrate binding and catalytic function in line with the severity of their angiogenic phenotypes. Gumby interacts with HOIP (also called RNF31), a key component of the linear ubiquitin assembly complex, and decreases linear ubiquitination and activation of NF-κB-dependent transcription. This work provides support for the biological importance of linear (de)ubiquitination in angiogenesis, craniofacial and neural development and in modulating Wnt signalling.
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Acknowledgements
We thank T. Saunders and the Michigan University Transgenic Core facility for generating gumby BAC transgenic mice; K. Iwai for HOIL and HOIP constructs; K. Nakajima for NF-κB and AP2 reporter constructs; B. Alman and C. C. Hui for TOPGAL reporter mice; J. Woodgett for TOPFLASH, FOPFLASH and PRL vectors; T. Pawson for WNT3A expressing L-cells; M. Barrios-Ramos for help with fluorometry; J. Culotti and C. C. Hui for critical feedback; and I. Kourinov and staff at NE-CAT for collecting diffraction data set. B.R. holds the Canada research chair in proteomics and molecular medicine. A.-C.G. holds the Canada research chair in Functional Proteomics. F.S. holds the Canada research chair in Structural Principles of Signal Transduction. S.M.A. and T.S. were supported by CIHR predoctoral student fellowships. This work was supported by operating funds from the JSPS KAKENHI (grant numbers 15200032 to Y.G. and 21240043 to Y.G. and R.F.), and Canadian Institutes for Health Research to B.R. (MOP119289), A.-C.G. (MOP123433), F.S. (MOP57795) and S.P.C. (MOP 97966, IHO 94384 and MOP 111199).
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E.R. designed genetic experiments with S.P.C., identified and confirmed the gumby causative mutation, performed expression analyses and characterized the GumW96R angiogenic phenotype; S.M.A. performed protein interaction assays and analysed the gumby–LUBAC connection; D.F.C. and Y.-C.J. performed X-ray crystallographic analyses and DUB assays; T.A.M. analysed angiogenesis and Wnt signalling; T.S. performed DUB chain profiling assay; H.H. and Y.-C.J. performed ITC analyses; Y.G. supervised R.F.; R.F. identified the GumD336E mutant; W.H.D. ran the mass spectrometry; A.-C.G. supervised W.H.D.; G.X. helped with SNP mapping; B.R. supervised T.S. and F.S. supervised D.C., H.H., Y-C.J. and together they designed experiments and wrote the manuscript; S.P.C. conceived and coordinated the project, designed and performed experiments, supervised E.R., S.M.A. and T.A.M., and wrote the manuscript.
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This file contains Supplementary Figures 1–11 and Supplementary Tables 1–3. (PDF 8693 kb)
Supplementary Data
This file contains tabulated data for the enzymatic Gumby assays. (XLSX 43 kb)
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Rivkin, E., Almeida, S., Ceccarelli, D. et al. The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis. Nature 498, 318–324 (2013). https://doi.org/10.1038/nature12296
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DOI: https://doi.org/10.1038/nature12296
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