Notum deacylates Wnt proteins to suppress signalling activity

Abstract

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

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: Notum specifically inhibits Wnt signalling.
Figure 2: Notum requires the GAGs of glypicans to inhibit Wingless signalling.
Figure 3: hNOTUM structure and GAG binding.
Figure 4: Enzymatic activity of hNOTUM.
Figure 5: Wnt-deacylation by Notum.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The crystal structures reported in this paper have been deposited in the Protein Data Bank (PDB) under accession numbers 4WBH, 4UYU, 4UYW, 4UZL, 4UYZ, 4UZ1, 4UZ5, 4UZ6, 4UZ7, 4UZ9, 4UZA, 4UZQ, 4UZJ and 4UZK.

References

  1. 1

    Freeman, M. Feedback control of intercellular signalling in development. Nature 408, 313–319 (2000)

  2. 2

    Takada, R. et al. Monounsaturated fatty acid modification of Wnt protein: its role in Wnt secretion. Dev. Cell 11, 791–801 (2006)

  3. 3

    Janda, C. Y., Waghray, D., Levin, A. M., Thomas, C. & Garcia, K. C. Structural basis of Wnt recognition by Frizzled. Science 337, 59–64 (2012)

  4. 4

    Willert, K. et al. Wnt proteins are lipid-modified and can act as stem cell growth factors. Nature 423, 448–452 (2003)

  5. 5

    Tang, X. et al. Roles of N-glycosylation and lipidation in Wg secretion and signaling. Dev. Biol. 364, 32–41 (2012)

  6. 6

    Clevers, H. & Nusse, R. Wnt/& β-catenin signaling and disease. Cell 149, 1192–1205 (2012)

  7. 7

    Kim, S. E. et al. Wnt Stabilization of β−Catenin Reveals Principles for Morphogen Receptor-Scaffold Assemblies. Science 340, 867–870 (2013)

  8. 8

    Niehrs, C. The complex world of WNT receptor signalling. Nature Rev. Mol. Cell Biol. 13, 767–779 (2012)

  9. 9

    Zhang, X. et al. Tiki1 is required for head formation via Wnt cleavage-oxidation and inactivation. Cell 149, 1565–1577 (2012)

  10. 10

    Giráldez, A. J., Copley, R. R. & Cohen, S. M. HSPG modification by the secreted enzyme Notum shapes the Wingless morphogen gradient. Dev. Cell 2, 667–676 (2002)

  11. 11

    Gerlitz, O. & Basler, K. Wingful, an extracellular feedback inhibitor of Wingless. Genes Dev. 16, 1055–1059 (2002)

  12. 12

    Filmus, J., Capurro, M. & Rast, J. Glypicans. Genome Biol. 9, 224 (2008)

  13. 13

    Yan, D. & Lin, X. Shaping morphogen gradients by proteoglycans. Cold Spring Harb. Perspect. Biol. 1, a002493 (2009)

  14. 14

    Bornemann, D. J., Duncan, J. E., Staatz, W., Selleck, S. & Warrior, R. Abrogation of heparan sulfate synthesis in Drosophila disrupts the Wingless, Hedgehog and Decapentaplegic signaling pathways. Development 131, 1927–1938 (2004)

  15. 15

    Kreuger, J., Perez, L., Giraldez, A. J. & Cohen, S. M. Opposing activities of Dally-like glypican at high and low levels of Wingless morphogen activity. Dev. Cell 7, 503–512 (2004)

  16. 16

    Traister, A., Shi, W. & Filmus, J. Mammalian Notum induces the release of glypicans and other GPI-anchored proteins from the cell surface. Biochem. J. 410, 503–511 (2008)

  17. 17

    Häcker, U., Nybakken, K. & Perrimon, N. Heparan sulphate proteoglycans: the sweet side of development. Nature Rev. Mol. Cell Biol. 6, 530–541 (2005)

  18. 18

    Petersen, C. P. & Reddien, P. W. Polarized notum activation at wounds inhibits Wnt function to promote planarian head regeneration. Science 332, 852–855 (2011)

  19. 19

    Chang, M. V., Chang, J. L., Gangopadhyay, A., Shearer, A. & Cadigan, K. M. Activation of wingless targets requires bipartite recognition of DNA by TCF. Curr. Biol. 18, 1877–1881 (2008)

  20. 20

    Flowers, G. P., Topczewska, J. M. & Topczewski, J. A zebrafish Notum homolog specifically blocks the Wnt/β-catenin signaling pathway. Development 139, 2416–2425 (2012)

  21. 21

    Torisu, Y. et al. Human homolog of NOTUM, overexpressed in hepatocellular carcinoma, is regulated transcriptionally by β-catenin/TCF. Cancer Sci. 99, 1139–1146 (2008)

  22. 22

    Baena-López, L. A., Nojima, H. & Vincent, J.-P. Integration of morphogen signalling within the growth regulatory network. Curr. Opin. Cell Biol. 24, 166–172 (2012)

  23. 23

    Alberts, L. J, et al. Molecular Biology of the Cell Ch. 2 (Garland Science, 2008)

  24. 24

    Basler, K. & Struhl, G. Compartment boundaries and the control of Drosophila limb pattern by hedgehog protein. Nature 368, 208–214 (1994)

  25. 25

    Alexandre, C., Jacinto, A. & Ingham, P. W. Transcriptional activation of hedgehog target genes in Drosophila is mediated directly by the cubitus interruptus protein, a member of the GLI family of zinc finger DNA-binding proteins. Genes Dev. 10, 2003–2013 (1996)

  26. 26

    Teleman, A. A. & Cohen, S. M. Dpp gradient formation in the Drosophila wing imaginal disc. Cell 103, 971–980 (2000)

  27. 27

    Whalen, D. M., Malinauskas, T., Gilbert, R. J. C. & Siebold, C. Structural insights into proteoglycan-shaped Hedgehog signaling. Proc. Natl Acad. Sci. USA 110, 16420–16425 (2013)

  28. 28

    Belenkaya, T. Y. et al. Drosophila Dpp morphogen movement is independent of dynamin-mediated endocytosis but regulated by the glypican members of heparan sulfate proteoglycans. Cell 119, 231–244 (2004)

  29. 29

    Lum, L. et al. Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells. Science 299, 2039–2045 (2003)

  30. 30

    Akiyama, T. et al. Dally regulates Dpp morphogen gradient formation by stabilizing Dpp on the cell surface. Dev. Biol. 313, 408–419 (2008)

  31. 31

    You, J., Belenkaya, T. & Lin, X. Sulfated is a negative feedback regulator of wingless in Drosophila. Dev. Dyn. 240, 640–648 (2011)

  32. 32

    Kirkpatrick, C. A., Dimitroff, B. D., Rawson, J. M. & Selleck, S. B. Spatial regulation of Wingless morphogen distribution and signaling by Dally-like protein. Dev. Cell 7, 513–523 (2004)

  33. 33

    Baeg, G. H. & Perrimon, N. Functional binding of secreted molecules to heparan sulfate proteoglycans in Drosophila. Curr. Opin. Cell Biol. 12, 575–580 (2000)

  34. 34

    Nardini, M. & Dijkstra, B. W. α/β hydrolase fold enzymes: the family keeps growing. Curr. Opin. Struct. Biol. 9, 732–737 (1999)

  35. 35

    Krissinel, E. & Henrick, K. Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr. D 60, 2256–2268 (2004)

  36. 36

    Wu, D. et al. Crystal structure of human esterase D: a potential genetic marker of retinoblastoma. FASEB J. 23, 1441–1446 (2009)

  37. 37

    Duncan, J. A. & Gilman, A. G. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein α subunits and p21(RAS). J. Biol. Chem. 273, 15830–15837 (1998)

  38. 38

    Laskowski, R. A., Watson, J. D. & Thornton, J. M. ProFunc: a server for predicting protein function from 3D structure. Nucleic Acids Res. 33, W89–W93 (2005)

  39. 39

    Orfila, C. et al. Expression of mung bean pectin acetyl esterase in potato tubers: effect on acetylation of cell wall polymers and tuber mechanical properties. Planta 236, 185–196 (2012)

  40. 40

    Xu, Q. et al. Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair. Cell 116, 883–895 (2004)

  41. 41

    Pepinsky, R. B. et al. Identification of a palmitic acid-modified form of human Sonic hedgehog. J. Biol. Chem. 273, 14037–14045 (1998)

  42. 42

    Resh, M. D. Covalent lipid modifications of proteins. Curr. Biol. 23, R431–R435 (2013)

  43. 43

    Rios-Esteves, J. & Resh, M. D. Stearoyl CoA desaturase is required to produce active, lipid-modified Wnt proteins. Cell Reports 4, 1072–1081 (2013)

  44. 44

    Reichsman, F., Smith, L. & Cumberledge, S. Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction. J. Cell Biol. 135, 819–827 (1996)

  45. 45

    Fuerer, C., Habib, S. J. & Nusse, R. A Study on the Interactions Between Heparan Sulfate Proteoglycans and Wnt Proteins. Dev. Dyn. 239, 184–190 (2010)

  46. 46

    Cruciat, C.-M. & Niehrs, C. Secreted and transmembrane wnt inhibitors and activators. Cold Spring Harb. Perspect. Biol. 5, a015081 (2013)

  47. 47

    de Lau, W., Peng, W. C., Gros, P. & Clevers, H. The R-spondin/Lgr5/Rnf43 module: regulator of Wnt signal strength. Genes Dev. 28, 305–316 (2014)

  48. 48

    Zebisch, M. et al. Structural and molecular basis of ZNRF3/RNF43 transmembrane ubiquitin ligase inhibition by the Wnt agonist R-spondin. Nature Commun. 4, 2787 (2013)

  49. 49

    Beckett, K. et al. Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes. Traffic 14, 82–96 (2013)

  50. 50

    Vincent, J.-P., Kolahgar, G., Gagliardi, M. & Piddini, E. Steep differences in wingless signaling trigger myc-independent competitive cell interactions. Dev. Cell 21, 366–374 (2011)

  51. 51

    Baena-Lopez, L. A., Alexandre, C., Mitchell, A., Pasakarnis, L. & Vincent, J. P. Accelerated homologous recombination and subsequent genome modification in Drosophila. Development 140, 4818–4825 (2013)

  52. 52

    Yagi, R., Mayer, F. & Basler, K. Refined LexA transactivators and their use in combination with the Drosophila Gal4 system. Proc. Natl Acad. Sci. USA 107, 16166–16171 (2010)

  53. 53

    Alexandre, C., Baena-Lopez, A. & Vincent, J.-P. Patterning and growth control by membrane-tethered Wingless. Nature 505, 180–185 (2014)

  54. 54

    Marois, E., Mahmoud, A. & Eaton, S. The endocytic pathway and formation of the Wingless morphogen gradient. Development 133, 307–317 (2006)

  55. 55

    Doering, T. L., Englund, P. T. & Hart, G. W. Detection of glycophospholipid anchors on proteins. Curr. Prot. Prot. Sci. Chapter 12, Unit 12.15 (2001)

  56. 56

    Fukui, S., Feizi, T., Galustian, C., Lawson, A. M. & Chai, W. Oligosaccharide microarrays for high-throughput detection and specificity assignments of carbohydrate-protein interactions. Nature Biotechnol. 20, 1011–1017 (2002)

  57. 57

    Palma, A. S., Feizi, T., Childs, R. A., Chai, W. & Liu, Y. The neoglycolipid (NGL)-based oligosaccharide microarray system poised to decipher the meta-glycome. Curr. Opin. Chem. Biol. 18, 87–94 (2014)

  58. 58

    Aricescu, A. R., Lu, W. & Jones, E. Y. A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr. D 62, 1243–1250 (2006)

  59. 59

    Malinauskas, T., Aricescu, A. R., Lu, W., Siebold, C. & Jones, E. Y. Modular mechanism of Wnt signaling inhibition by Wnt inhibitory factor 1. Nature Struct. Mol. Biol. 18, 886–893 (2011)

  60. 60

    Gagliardi, M., Hernandez, A., McGough, I. J. & Vincent, J.-P. Inhibitors of endocytosis prevent Wnt/Wingless signalling by reducing the level of basal β-Catenin/Armadillo. J. Cell Sci. 127, 4918–4926 (2014)

  61. 61

    Glise, B. et al. Shifted, the Drosophila ortholog of Wnt inhibitory factor-1, controls the distribution and movement of Hedgehog. Dev. Cell 8, 255–266 (2005)

Download references

Acknowledgements

We thank K. Dingwell for supplying purified mWnt3A, H. Bellen for anti-Senseless, C. Alexandre for plasmids and advice, W. Chai for glycosaminoglycan probes, T. Holder for suggestions, T. Malinauskas and C. Lorenz for advice and technical support, T. Walter for technical support with crystallization, W. Lu and Y. Zhao for help with tissue culture, and the organisers of the EMBO Wnt meeting 2012 where our collaboration began. We thank staff at Diamond Light Source beamlines (i02, i03, i04, i04-1, i24) for assistance with data collection (proposal mx8423). This work was supported by the MRC (U117584268 to J.-P.V.; G0900084 to E.Y.J.), the UK Research Council Basic Technology Initiative (Glycoarrays Grant GRS/79268 and EPSRC Translational Grant EP/G037604/1), the Wellcome Trust (Biomedical Resource Grants WT093378MA and WT099197MA) to T.F., the European Union (ERC grant WNTEXPORT; 294523 to J.-P.V., a Marie Curie IEF grant to M.Z.), Cancer Research UK (C375/A10976 to E.Y.J.), and the Japan Society for the Promotion of Science (to S.K.). T.-H.C. was funded by a Nuffield Department of Medicine Prize Studentship in conjunction with Clarendon and Somerville College Scholarships. The Wellcome Trust Centre for Human Genetics is supported by Wellcome Trust Centre grant 090532/Z/09/Z.

Author information

Experimental contributions were as follows: Drosophila developmental genetics (P.F.L. and S.K.); Drosophila cell-based assays (S.K.); human cell-based assays (M.Z. and T.-H.C.); mass spectrometry (S.H., S.K. and A.P.S.); glycan arrays (Y.L., S.K. and T.F.); enzymatic assays (M.Z.); structural biology (M.Z.); peptide synthesis (G.B. and N.O’R.). The project was conceived by S.K., P.F.L., M.Z., E.Y.J. and J.-P.V. The first draft of the paper was written by M.Z., E.Y.J. and J.-P.V. with substantial contributions from P.F.L., S.K. and A.P.S. All authors contributed to the design and interpretation of experiments.

Correspondence to Matthias Zebisch or E. Yvonne Jones or Jean-Paul Vincent.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Notum modulates Wingless, but not Dpp or Hedgehog signalling.

a, b, Overexpression of dNotum-V5 with the apterous-Gal4 driver, which is expressed in the dorsal compartment, prevents expression of Senseless (Sens) (b, middle), a Wingless target gene, but has little effect on phospho-Mad (pMad) immunoreactivity (b), an indicator of Dpp signalling. c, Loss of notum activity, achieved by generating large patches of notumKO tissue (see Methods), marked by the loss of GFP, leads to broadening of Senseless expression but does not affect pMad immunoreactivity. dg, Strong, but not complete, reduction of notum activity led to ectopic wing margin bristles (compare insets in d and e) but had no significant effect on wing area, which is sensitive to Dpp signalling (f) (P = 0.26, Student’s t-test), or on the distance between L3 and L4 veins, which is affected by changes in Hedgehog signalling61 (g) (P = 0.41, Student’s t-test). In total, 19 control (notum141/+) and 17 mutant (notum141/KO) wings were analysed. Error bars in f and g are s.d.

Extended Data Figure 2 dNotum does not cleave the GPI anchor of glypicans.

a, b, Ectopic expression of Senseless caused by NRT-wingless, as well as endogenous Senseless, is suppressed by co-expression of dNotum. NRT-wingless and notum are expressed in a vertical band under the control of dpp-Gal4. c, Western blot analysis of phase-separated extracts of S2 cells transfected with a plasmid expressing HA-tagged Dally. In control extracts, Dally is found largely in the detergent (D) phase. Coexpression of dNotum–V5 from a plasmid had no effect, while treatment with PIPLC shifted all detectable Dally to the aqueous (A) phase. d, dNotum-V5 expression as in c was sufficient to suppress Wingless-induced TOPFlash activity. Cells were transfected with a dual luciferase TOPFlash reporter60 along with a mock plasmid (−), tubulin::wingless (Wg), or tubulin::wingless + actin::notum-V5 (Wg + Notum). eh, Extracellular Dlp in control (e, g), PIPLC-treated (f) or apterous-Gal4 UAS-notum-V5 (h) imaginal discs. il, Extracellular anti-GFP staining of imaginal discs from gene trap line expressing Dally–GFP fusion protein. Discs were treated with a mock solution (i) or PIPLC (j) (same discs as in e or f, respectively, but here showing Dally protein). In a separate experiment, dNotum was overexpressed with apterous-Gal4 in the Dally-GFP background (l). No change in the distribution of extracellular GFP could be seen compared to that in control discs (k, no apterous-Gal4).

Extended Data Figure 3 dNotum requires Dally to inhibit Wingless signalling.

a, Wingless and Senseless expression in a dally−/− wing imaginal disc expressing NRT-wingless and notum under the control of dpp-Gal4. Some senseless expression remains, indicating that, in the absence of Dally, dNotum is a poor inhibitor of NRT-Wingless-induced (as well as endogenous) signalling. bd, Anterior margin of wings from control, spalt (sal)-Gal4 UAS-notum-V5, and sal-Gal4 UAS-notum-V5 dally−/− animals. Removal of dally rescues the loss of margin bristles caused by dNotum overexpression.

Extended Data Figure 4 dNotum binds to sulfated glycans.

Binding of dNotum-V5 to a GAG oligosaccharide array, detected by immunofluorescence. CSA/B/C, chondroitin sulfate A/B/C; HA, hyaluronic acid, hep, heparin; HS, heparan sulfate. Details on the array are provided in the Methods.

Extended Data Figure 5 Additional structural information on Notum.

a, Topology plot of hNOTUM. β-strands are shown as numbered triangles and α-helices as circles labelled in alphabetical order from the N to C terminus (NT to CT). Structural elements conserved among most α/β-hydrolases are outlined in grey. b, Comparison of the two most conformationally distinct hNOTUM structures (from crystal forms III and V). Crystal form III is the most structurally different. All other structures superimpose with root mean squared deviation (r.m.s.d.) of <0.7 Å. Circles highlight the most flexible regions. c, Comparison between the structures of hNOTUM (form V) and dNotum (form I). The circle highlights the lack of a cysteine bridge in dNotum.

Extended Data Figure 6 Structural and biophysical analysis of heparin binding.

a, Heparin affinity chromatography of wild-type hNOTUM and selected surface variants. be, Close-up views of additional sulfate binding sites on hNOTUM, crystal form III. Each view is accompanied with SPR heparin affinity data corresponding to each hNOTUM variant.

Extended Data Figure 7 Relation of Notum to other esterases of the α/β hydrolase family.

a, Comparison between hNOTUM and human esterase D (hESTD), showing structural relatedness. hNOTUM is also related to hAPT1, a cytosolic esterase used in this study as a positive control for fatty acid esterase activity. In the views shown here, the hNOTUM structure has been rotated by 90° around the x axis relative to the structure shown in Fig. 3b. b, Rootless phylogenetic tree of animal Notum proteins (red) and plant pectin acetylesterases (PAE, green). Extent of sequence identity to hNOTUM is shown next to species name. Percentages between branches indicate sequence identity between neighbours.

Extended Data Figure 8 Substrates and inhibitors of hNOTUM.

a, Inhibition of hNOTUM activity on pNP-butyrate (pNP4) by PMSF (30 min pre-incubation with 2 mM PMSF) as well as by Triton X-100 and CHAPS (0.5%). Presence of 20 mM SOS and 50 mg l−1 heparin results in a minor increase of esterase activity. The height of each bar represents activity relative to the mean of four control samples lacking the additives. b, Saturable inhibition of hNOTUM by Triton X-100. Triton X-100 inhibits many esterases owing to binding to the acyl binding pocket through its hydrophobic group. c, Lack of inhibition of Norrin-mediated β-catenin stabilization by Notum. Recombinant Norrin was pretreated with hNOTUMcore at a concentration sufficient to suppress Wnt3A-mediated signalling. d, e, Saturation kinetics of the action of hNOTUM on pNP-octanoate (pNP8, d) and pNP-butyrate (pNP4, e). The activity was normalized to the Amax calculated for hNOTUMcore. The activity values for the larger, full length protein were adjusted to compensate for the increased mass. Apparent Km values in d were corrected for the inhibition caused by Triton X-100. f, Saturation inhibition kinetics with myristoleic and palmitoleic acid. pNP8 was used at a concentration of 1 mM and 250 μM, respectively.

Extended Data Figure 9 Additional mass spectrometric analysis of the hNOTUM deacylase activity.

a, Mass spectra of CHGLSGSCEVK from trypsinized Wnt3A protein mock-treated or treated with hNOTUMcore. Left-hand graph is the same as that shown in Fig. 5a, while the right-hand side shows the results of a separate experiment performed with the labels reversed. b, Duplicate LC–MS peak areas with label reversal. Irrespective of the nature of the label (grey indicates light label, black indicates heavy label), hNOTUMcore triggered an increase in peak area of the delipidated Wnt3A tryptic peptide. c, d, Two control Wnt3A cysteine-containing peptides from the same data set were not affected by hNOTUMcore. e, Activity of hNOTUMcore and its Ser232Ala variant on a synthetic disulphide-bonded Wnt3A peptide (CHGLSGSCEVK) palmitoleoylated on the first serine. Both lipidated and unlipidated peptide could be detected by MALDI-TOF. Incubation with hNOTUMcore, but not its Ser232Ala variant, caused significant delipidation (peak corresponding to delipidated peptide is marked by asterisk). Quantification of triplicate experiments is shown in Fig. 5c. f, MALDI-TOF analysis shows that neither hNOTUMcore nor its Ser232Ala variant delipidated a synthetic SHH peptide (CGPGRGFGKRR) palmitoylated on its N-terminal cysteine. Quantification of triplicate experiments is shown in Fig. 5d (peak corresponding to lipidated peptide is marked by black triangle). g, Two-dimensional active site schematic relating to Fig. 5e. Additional hydrogen bonds and electron pair movements thought to occur during hydrolysis by the wild type protein are shown in grey. h, Close-up view on the myristoleate active site complex of hNOTUMcore (crystal form I). The experimental omit electron density is contoured at 2σ.

Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary References and Supplementary Table 1. (PDF 206 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kakugawa, S., Langton, P., Zebisch, M. et al. Notum deacylates Wnt proteins to suppress signalling activity. Nature 519, 187–192 (2015) doi:10.1038/nature14259

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.