The Juxtacrine “Canonical” Notch signaling relies on direct cell-cell contact between the transmembrane ligands expressing “sender” cells and receptors expressing “receiver” cells (Fig.1). Interaction of the Notch receptor with the Notch ligand lead to γ-secretase dependent cleavage of both signaling proteins, followed by respective intracellular domain release (LICDs/NICDs)1–6. NICD directly translocate into the nucleus to drive transcription of Notch target genes through switching the CSL transcriptional repressor conformation into activator7,8. The LICDs were shown to elicit response in the “sender” cells expressing Jag16,9–12, Delta/Dll1/44,5,13–17. Importantly, Jag1ICD destabilizes N1ICD11 by direct interaction with the tumour-suppressor Fxbw7 E3 ligase12,18–20, and its interaction with Afadin protein suggests it could play a role also in signaling cross-talks21,22, however the full extent of its role in vivo is unknown.
Fig.1: Scheme of the “Canonical” Notch and “Bi-directional” Ligand signaling.
Extrahepatic biliary atresia (EHBA) together with Alagille syndrome (ALGS) (manifesting intrahepatic bile duct paucity) are two major pediatric liver pathologies requiring surgical intervention, reviewed in23–25. While disrupted Notch signaling is a well-known driver of ALGS, with mutations in JAGGED1 (JAG1) causing up to 94% ALGS cases26,27, its role in EHBA28 remains severely understudied. EHBA-associated symptoms strongly overlap with processes regulated by Notch. As reviewed in24, 5-15% of EHBA patients show defects in L-R asymmetry, known to be co-regulated by Notch signaling29–31, these also include developmental heart defects, a feature shared with ALGS32,33. Interestingly, expression of bona fide Notch transcription factor RBPJ-κ and its target gene HES1 was suppressed in the postnatal liver of rhesus rotavirus-induced mouse model of EHBA34.
Collectively, this data provides strong support for addressing the potential role of Notch in EHBA in a mouse model that would recapitulate the disease-causing mutation.
The JAG1ICD represents 1/10 of the full-length JAG1 protein, yet, only 1 ALGS-causing mutation was reported in the JAG1ICD region from the almost 400 identified so far (HGMD database)25. This suggests the ICD region is either more tolerant to mutagenesis or so sensitive that its alteration leads to embryonic lethality. The 1 ALGS causing missense mutation targets the putative NLS sequence11,35 of the JAG1ICD (R1097W) with an unknown effect on Notch signaling36. The other functional motif of the ICDs of DLL1/4 and JAG1 is the c-term PDZ domain37, which interacts with the structural protein Afadin (AF6)9,38 that can down-regulate Notch signaling21. Afadin binding affinity was further increased by the JAG1PDZ mutation R1213Q38. Strikingly, this mutation can cause EHBA28 (Fig.2A, B).
Hypothesis: Notch Ligand ICDs act through multiple mechanisms and in biological contexts as “Non-canonical” modulators of Notch signaling by down-regulating the NICD levels in the ligand-expressing cells.
AIM: To reveal the requirements of NLS and PDZ motifs in Notch ligand ICD signaling, we will develop and characterize novel mice strains carrying the ALGS (R1097W) and EHBA (R1213Q) disease-causing mutations in Jag1ICD, and perform a “rescue” experiments using mouse strain with inducible expression of the wild-type JAG1ICD (JAG1ICD GOF) (Fig.2C).
EXPECTED OUTCOMES AND SIGNIFICANCE
This project will expand our knowledge of the indispensable inter-cellular communication, and provide the liver community with a new genetic model of EHBA and a unique, JAG1ICD-focused model of ALGS. Our findings on the roles of LICD NLS and PDZ motifs in bi-directional and “Non-canonical” Notch would open new avenues of research for the Notch field and can lead to development of therapeutics for Notch genetic disorders and cancer.
Fig.2: JAG1 ICD. (A) Scheme of JAG1 proteins. (B) JAG1 NLS sequence is conserved across vertebrates (C) Planned new mouse strains.
Funding and project approval:
This project is currently supported by the PRIMUS Funding PRIMUS/21/SCI/006 (600000Euro/5y).
Required application materials:
How to submit application materials:
Please send email with the required application materials to dr. Jan Mašek: firstname.lastname@example.org, Faculty of Science, Charles University, Prague.
The application deadline is July 23, 2021.
For more information please visit the webpage of the JUNIOR Fund project of the Charles University.
1- Brou, C. et al. Mol. Cell 5, 207–216 (2000).
2. Mumm, J. S. et al. Mol. Cell 5, 197–206 (2000).
3. Mishra-Gorur, K., Rand, M. D., Perez-Villamil, B. & Artavanis-Tsakonas, S. J. Cell Biol. 159, 313–324 (2002).
4. Bland, C. E., Kimberly, P. & Rand, M. D. J. Biol. Chem. 278, 13607–13610 (2003). 5. Ikeuchi, T. & Sisodia, S. S. J. Biol. Chem. 278, 7751–7754 (2003). 6. LaVoie, M. J. & Selkoe, D. J. J. Biol. Chem. 278, 34427–37 (2003). 7. Wilson, J. J. & Kovall, R. A. Cell 124, 985–996 (2006).
8. Nam, Y., Sliz, P., Song, L., Aster, J. C. & Blacklow, S. C. Cell 124, 973–983 (2006). 9. Ascano, J. M., Beverly, L. J. & Capobianco, A. J. J. Biol. Chem. 278, 8771–8779 (2003).
10. Kiyota, T. & Kinoshita, T. Mech. Dev. 121, 573–585 (2004).
11. Metrich, M. et al. Cardiovasc. Res. 108, 74–86 (2015).
12. Kim, M. Y. et al. Exp. Cell Res. 317, 2438–2446 (2011).
13. Kolev, V. et al. FEBS Lett. 579, 5798–5802 (2005).
14. Bordonaro, M., Tewari, S., Atamna, W. & Lazarova, D. L. Exp. Cell Res. 317, 1368– 1381 (2011).
15. Hiratochi, M. et al. Nucleic Acids Res. 35, 912–922 (2007).
16. Jung, J. et al. Mol. Cells 32, 161–165 (2011).
17. Forghany, Z., Robertson, F., Lundby, A., Olsen, J. V. & Baker, D. A. J. Biol. Chem. 293, jbc.M117.819045 (2017).
18. Öberg, C. et al. J. Biol. Chem. 276, 35847–35853 (2001).
19. Tsunematsu, R. et al. J. Biol. Chem. 279, 9417–9423 (2004).
20. Tetzlaff, M. T. et al. Proc. Natl. Acad. Sci. U. S. A. 101, 3338–3345 (2004). 21. Carmena, A., Speicher, S. & Baylies, M. PLoS One 1, (2006). 22. D’Souza, B., Miyamoto, A. & Weinmaster, G. The many facets of Notch ligands. Oncogene vol. 27 5148–5167 (Nature Publishing Group, 2008).
23. Asai, A., Miethke, A. & Bezerra, J. A. Nature Reviews Gastroenterology and Hepatology vol. 12 342–352 (2015).
24. Bezerra, J. A. et al. Hepatology vol. 68 1163–1173 (2018).
25. Mašek, J. & Andersson, E. R. Development 144, 1743–1763 (2017). 26. Alagille, D., Odièvre, M., Gautier, M. & Dommergues, J. P. P. J. Pediatr. 86, 63–71 (1975).
27. Watson, G. H. & Miller, V. Arch. Dis. Child. 48, 459–66 (1973). 28. Kohsaka, T. et al. Hepatology 36, 904–912 (2002).
29. Raya, A. et al. Genes Dev. 17, 1213–1218 (2003).
30. Raya, Á. et al. Nature 427, 121–128 (2004).
31. Krebs, L. T. et al. Genes Dev. 17, 1207–1212 (2003).
32. Scheppke, L. et al. Blood 119, 2149–2158 (2012).
33. C. Glaeser, D. K. A. C. R. K. S. S. U. S. I. H. Hum. Genet. 119, 671 (2006). 34. Rui-Zhong, Z. et al. World J. Gastroenterol. 24, 3260–3272 (2018). 35. Chelsky, D., Ralph, R. & Jonak, G. Mol. Cell. Biol. 9, 2487–2492 (1989). 36. Daniela Marchetti, M. R. I. L. P. Hum. Genet. 126, 329 (2009). 37. Six, E. M. et al. J. Biol. Chem. 279, 55818–55826 (2004).
38. Popovic, M. et al. J. Mol. Recognit. 24, 245–253 (2011).