The linear ubiquitin-specific deubiquitinase gumby regulates angiogenesis

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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Identification of the gumby (GumW96R) causative mutation and the new GumD336E allele.
Figure 2: Analyses of vascular phenotypes and gumby expression.
Figure 3: Structural and biochemical analysis of gumby.
Figure 4: Gumby interacts with HOIP and counteracts LUBAC activity
Figure 5: Gumby interacts with DVL2 and can modulate Wnt signalling.

Accession codes

Accessions

Protein Data Bank

Data deposits

The coordinates and structure factors have been deposited in the Protein Data Bank under accession numbers 4KSJ (apo-gumby), 4KSK (gumby–ubiquitin complex) and 4KSL (gumby–linear diubiquitin complex).

References

  1. 1

    Risau, W. Mechanisms of angiogenesis. Nature 386, 671–674 (1997)

    CAS  ADS  Article  Google Scholar 

  2. 2

    Logan, C. Y. & Nusse, R. The Wnt signaling pathway in development and disease. Annu. Rev. Cell Dev. Biol. 20, 781–810 (2004)

    CAS  Article  Google Scholar 

  3. 3

    Liebner, S. & Plate, K. H. Differentiation of the brain vasculature: the answer came blowing by the Wnt. J. Angio. Res. 2, 1 (2010)

    Article  Google Scholar 

  4. 4

    Zerlin, M., Julius, M. A. & Kitajewski, J. Wnt/Frizzled signaling in angiogenesis. Angiogenesis 11, 63–69 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Daneman, R. et al. Wnt/β-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc. Natl Acad. Sci. USA 106, 641–646 (2009)

    CAS  ADS  Article  Google Scholar 

  6. 6

    Stenman, J. M. et al. Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science 322, 1247–1250 (2008)

    CAS  ADS  Article  Google Scholar 

  7. 7

    Cirone, P. et al. A role for planar cell polarity signaling in angiogenesis. Angiogenesis 11, 347–360 (2008)

    Article  Google Scholar 

  8. 8

    Wharton, K. A., Jr Runnin’ with the Dvl: proteins that associate with Dsh/Dvl and their significance to Wnt signal transduction. Dev. Biol. 253, 1–17 (2003)

    CAS  Article  Google Scholar 

  9. 9

    Kirisako, T. et al. A ubiquitin ligase complex assembles linear polyubiquitin chains. EMBO J. 25, 4877–4887 (2006)

    CAS  Article  Google Scholar 

  10. 10

    Behrends, C. & Harper, J. W. Constructing and decoding unconventional ubiquitin chains. Nature Struct. Mol. Biol. 18, 520–528 (2011)

    CAS  Article  Google Scholar 

  11. 11

    Walczak, H., Iwai, K. & Dikic, I. Generation and physiological roles of linear ubiquitin chains. BMC Biol. 10, 23 (2012)

    CAS  Article  Google Scholar 

  12. 12

    Iwai, K. Linear polyubiquitin chains: a new modifier involved in NFκB activation and chronic inflammation, including dermatitis. Cell Cycle 10, 3095–3104 (2011)

    CAS  Article  Google Scholar 

  13. 13

    Gerlach, B. et al. Linear ubiquitination prevents inflammation and regulates immune signalling. Nature 471, 591–596 (2011)

    CAS  ADS  Article  Google Scholar 

  14. 14

    Ikeda, F. et al. SHARPIN forms a linear ubiquitin ligase complex regulating NF-κB activity and apoptosis. Nature 471, 637–641 (2011)

    CAS  ADS  Article  Google Scholar 

  15. 15

    Tokunaga, F. et al. SHARPIN is a component of the NF-κB-activating linear ubiquitin chain assembly complex. Nature 471, 633–636 (2011)

    CAS  ADS  Article  Google Scholar 

  16. 16

    Niu, J., Shi, Y., Iwai, K. & Wu, Z. H. LUBAC regulates NF-κB activation upon genotoxic stress by promoting linear ubiquitination of NEMO. EMBO J. 30, 3741–3753 (2011)

    CAS  Article  Google Scholar 

  17. 17

    Nijman, S. M. et al. A genomic and functional inventory of deubiquitinating enzymes. Cell 123, 773–786 (2005)

    CAS  Article  Google Scholar 

  18. 18

    Mar, L., Rivkin, E., Kim, D. Y., Yu, J. Y. & Cordes, S. P. A genetic screen for mutations that affect cranial nerve development in the mouse. J. Neurosci. 25, 11787–11795 (2005)

    CAS  Article  Google Scholar 

  19. 19

    Hogan, K. A., Ambler, C. A., Chapman, D. L. & Bautch, V. L. The neural tube patterns vessels developmentally using the VEGF signaling pathway. Development 131, 1503–1513 (2004)

    CAS  Article  Google Scholar 

  20. 20

    Gondo, Y. Trends in large-scale mouse mutagenesis: from genetics to functional genomics. Nature Rev. Genet. 9, 803–810 (2008)

    CAS  Article  Google Scholar 

  21. 21

    Roca, C. & Adams, R. H. Regulation of vascular morphogenesis by Notch signaling. Genes Dev. 21, 2511–2524 (2007)

    CAS  Article  Google Scholar 

  22. 22

    Edelmann, M. J. et al. Structural basis and specificity of human otubain 1-mediated deubiquitination. Biochem. J. 418, 379–390 (2009)

    CAS  Article  Google Scholar 

  23. 23

    Juang, Y. C. et al. OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function. Mol. Cell 45, 384–397 (2012)

    CAS  Article  Google Scholar 

  24. 24

    Wiener, R., Zhang, X., Wang, T. & Wolberger, C. The mechanism of OTUB1-mediated inhibition of ubiquitination. Nature 483, 618–622 (2012)

    CAS  ADS  Article  Google Scholar 

  25. 25

    Wang, T. et al. Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1. J. Mol. Biol. 386, 1011–1023 (2009)

    CAS  Article  Google Scholar 

  26. 26

    Matsumoto, M. L. et al. Engineering and structural characterization of a linear polyubiquitin-specific antibody. J. Mol. Biol. 418, 134–144 (2012)

    CAS  Article  Google Scholar 

  27. 27

    Rual, J. F. et al. Towards a proteome-scale map of the human protein–protein interaction network. Nature 437, 1173–1178 (2005)

    CAS  ADS  Article  Google Scholar 

  28. 28

    Maretto, S. et al. Mapping Wnt/β-catenin signaling during mouse development and in colorectal tumors. Proc. Natl Acad. Sci. USA 100, 3299–3304 (2003)

    CAS  ADS  Article  Google Scholar 

  29. 29

    HogenEsch, H. et al. A spontaneous mutation characterized by chronic proliferative dermatitis in C57BL mice. Am. J. Pathol. 143, 972–982 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30

    Seymour, R. E. et al. Spontaneous mutations in the mouse Sharpin gene result in multiorgan inflammation, immune system dysregulation and dermatitis. Genes Immun. 8, 416–421 (2007)

    CAS  Article  Google Scholar 

  31. 31

    Rahighi, S. et al. Specific recognition of linear ubiquitin chains by NEMO is important for NF-κB activation. Cell 136, 1098–1109 (2009)

    CAS  Article  Google Scholar 

  32. 32

    Mainardi, P. C. et al. The natural history of Cri du Chat Syndrome. A report from the Italian Register. Eur. J. Med. Genet. 49, 363–383 (2006)

    Article  Google Scholar 

  33. 33

    Mainardi, P. C. et al. Clinical and molecular characterisation of 80 patients with 5p deletion: genotype-phenotype correlation. J. Med. Genet. 38, 151–158 (2001)

    CAS  Article  Google Scholar 

  34. 34

    Zhang, X. et al. High-resolution mapping of genotype-phenotype relationships in cri du chat syndrome using array comparative genomic hybridization. Am. J. Hum. Genet. 76, 312–326 (2005)

    CAS  Article  Google Scholar 

  35. 35

    Ye, S., Dhillon, S., Ke, X., Collins, A. R. & Day, I. N. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res. 29, e88 (2001)

    CAS  Article  Google Scholar 

  36. 36

    Adams, D. J. et al. A genome-wide, end-sequenced 129Sv BAC library resource for targeting vector construction. Genomics 86, 753–758 (2005)

    CAS  Article  Google Scholar 

  37. 37

    DasGupta, R. & Fuchs, E. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557–4568 (1999)

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Ambulos, N. P., Jr, Duvall, E. J. & Lovett, P. S. Method for blot-hybridization analysis of mRNA molecules from Bacillus subtilis. Gene 51, 281–286 (1987)

    CAS  Article  Google Scholar 

  39. 39

    Kim, F. A. et al. The vHNF1 homeodomain protein establishes early rhombomere identity by direct regulation of Kreisler expression. Mech. Dev. 122, 1300–1309 (2005)

    CAS  Article  Google Scholar 

  40. 40

    Vecchi, A. et al. Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. Eur. J. Cell Biol. 63, 247–254 (1994)

    CAS  PubMed  Google Scholar 

  41. 41

    Abramoff, M. D., Magalhaes, P. J. & Ram, S. J. Image Processing with ImageJ. Biophotonics Intl 11, 36–42 (2004)

    Google Scholar 

  42. 42

    Torban, E., Wang, H. J., Groulx, N. & Gros, P. Independent mutations in mouse Vangl2 that cause neural tube defects in looptail mice impair interaction with members of the Dishevelled family. J. Biol. Chem. 279, 52703–52713 (2004)

    CAS  Article  Google Scholar 

  43. 43

    Veeman, M. T., Axelrod, J. D. & Moon, R. T. A second canon. Functions and mechanisms of β-catenin-independent Wnt signaling. Dev. Cell 5, 367–377 (2003)

    CAS  Article  Google Scholar 

  44. 44

    Ernst, A. et al. A strategy for modulation of enzymes in the ubiquitin system. Science 339, 590–595 (2013)

    CAS  ADS  Article  Google Scholar 

  45. 45

    Kean, M. J., Couzens, A. L. & Gingras, A. C. Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins. Methods 57, 400–408 (2012)

    CAS  Article  Google Scholar 

  46. 46

    Dunham, W. H. et al. A cost–benefit analysis of multidimensional fractionation of affinity purification-mass spectrometry samples. Proteomics 11, 2603–2612 (2011)

    CAS  Article  Google Scholar 

  47. 47

    Liu, G. et al. ProHits: integrated software for mass spectrometry-based interaction proteomics. Nature Biotechnol. 28, 1015–1017 (2010)

    CAS  Article  Google Scholar 

  48. 48

    Choi, H. et al. Analyzing protein-protein interactions from affinity purification-mass spectrometry data with SAINT. Curr. Protoc. Bioinform. Ch. 8, Unit 8.15. (2012)

  49. 49

    Choi, H. et al. SAINT: probabilistic scoring of affinity purification-mass spectrometry data. Nature Methods 8, 70–73 (2011)

    CAS  Article  Google Scholar 

Download references

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).

Author information

Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Frank Sicheri or Sabine P. Cordes.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.