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Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Cherry AL, Finta C, Karlström M, Jin Q, Schwend T, Astorga-Wells J, Zubarev RA, Del Campo M, Criswell AR, de Sanctis D, Jovine L, Toftgård R - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction.It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation.These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition and Center for Biosciences, Karolinska Institutet, Novum, Hälsovägen 7, SE-141 83 Huddinge, Sweden.

ABSTRACT
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

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The IDR regulates SUFU activity. (a) SUFU-FL (FL), SUFU-Δ (Δ) and SUFU-SH (SH) all bind GLI1 when co-expressed in Cos-7 cells, as shown by Co-IP. (b, c) SUFU-FL and SUFU-Δ repress GLI1-induced reporter gene activity in HEK 293 cells (b) and suppress constitutive pathway activity in Sufu−/− cells (c) with similar efficiency. (d) SUFU-Δ, SUFU-SH and SUFU-IDRfly (IDRfly) expressed in Sufu−/− cells are unable to mediate Hh pathway reactivation induced by the SMO agonist SAG. Error bars indicate the range of data in three parallel samples. (e) Schematic model of the regulatory role of SUFU with and without an intact IDR. Green and red colours indicate activated and repressed states of proteins, respectively.
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fig8: The IDR regulates SUFU activity. (a) SUFU-FL (FL), SUFU-Δ (Δ) and SUFU-SH (SH) all bind GLI1 when co-expressed in Cos-7 cells, as shown by Co-IP. (b, c) SUFU-FL and SUFU-Δ repress GLI1-induced reporter gene activity in HEK 293 cells (b) and suppress constitutive pathway activity in Sufu−/− cells (c) with similar efficiency. (d) SUFU-Δ, SUFU-SH and SUFU-IDRfly (IDRfly) expressed in Sufu−/− cells are unable to mediate Hh pathway reactivation induced by the SMO agonist SAG. Error bars indicate the range of data in three parallel samples. (e) Schematic model of the regulatory role of SUFU with and without an intact IDR. Green and red colours indicate activated and repressed states of proteins, respectively.

Mentions: The observed physical differences between MBP-SUFU constructs with and without the IDR suggest a functional role of this domain. In agreement with the thermophoresis data, there was no remarkable difference in GLI1 binding observed in co-immunoprecipitation (Co-IP) experiments with SUFU-FL, SUFU-SH and SUFU-Δ (Fig. 8 ▶a). Similarly, transcriptional activity induced by both GLI1 and GLI2 was efficiently repressed by SUFU-FL, SUFU-Δ and SUFU-SH in transient transfection assay experiments (Fig. 8 ▶b and Supplementary Figs. S10a and S10b), and deletion of the IDR had no considerable effect on repression of the constitutive Hh pathway activity in Sufu−/− MEFs (Fig. 8 ▶c). Collectively, these results imply that the IDR in SUFU is dispensable for GLI binding and repression activity in cells without upstream pathway activation.


Structural basis of SUFU-GLI interaction in human Hedgehog signalling regulation.

Cherry AL, Finta C, Karlström M, Jin Q, Schwend T, Astorga-Wells J, Zubarev RA, Del Campo M, Criswell AR, de Sanctis D, Jovine L, Toftgård R - Acta Crystallogr. D Biol. Crystallogr. (2013)

The IDR regulates SUFU activity. (a) SUFU-FL (FL), SUFU-Δ (Δ) and SUFU-SH (SH) all bind GLI1 when co-expressed in Cos-7 cells, as shown by Co-IP. (b, c) SUFU-FL and SUFU-Δ repress GLI1-induced reporter gene activity in HEK 293 cells (b) and suppress constitutive pathway activity in Sufu−/− cells (c) with similar efficiency. (d) SUFU-Δ, SUFU-SH and SUFU-IDRfly (IDRfly) expressed in Sufu−/− cells are unable to mediate Hh pathway reactivation induced by the SMO agonist SAG. Error bars indicate the range of data in three parallel samples. (e) Schematic model of the regulatory role of SUFU with and without an intact IDR. Green and red colours indicate activated and repressed states of proteins, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852661&req=5

fig8: The IDR regulates SUFU activity. (a) SUFU-FL (FL), SUFU-Δ (Δ) and SUFU-SH (SH) all bind GLI1 when co-expressed in Cos-7 cells, as shown by Co-IP. (b, c) SUFU-FL and SUFU-Δ repress GLI1-induced reporter gene activity in HEK 293 cells (b) and suppress constitutive pathway activity in Sufu−/− cells (c) with similar efficiency. (d) SUFU-Δ, SUFU-SH and SUFU-IDRfly (IDRfly) expressed in Sufu−/− cells are unable to mediate Hh pathway reactivation induced by the SMO agonist SAG. Error bars indicate the range of data in three parallel samples. (e) Schematic model of the regulatory role of SUFU with and without an intact IDR. Green and red colours indicate activated and repressed states of proteins, respectively.
Mentions: The observed physical differences between MBP-SUFU constructs with and without the IDR suggest a functional role of this domain. In agreement with the thermophoresis data, there was no remarkable difference in GLI1 binding observed in co-immunoprecipitation (Co-IP) experiments with SUFU-FL, SUFU-SH and SUFU-Δ (Fig. 8 ▶a). Similarly, transcriptional activity induced by both GLI1 and GLI2 was efficiently repressed by SUFU-FL, SUFU-Δ and SUFU-SH in transient transfection assay experiments (Fig. 8 ▶b and Supplementary Figs. S10a and S10b), and deletion of the IDR had no considerable effect on repression of the constitutive Hh pathway activity in Sufu−/− MEFs (Fig. 8 ▶c). Collectively, these results imply that the IDR in SUFU is dispensable for GLI binding and repression activity in cells without upstream pathway activation.

Bottom Line: Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction.It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation.These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biosciences and Nutrition and Center for Biosciences, Karolinska Institutet, Novum, Hälsovägen 7, SE-141 83 Huddinge, Sweden.

ABSTRACT
Hedgehog signalling plays a fundamental role in the control of metazoan development, cell proliferation and differentiation, as highlighted by the fact that its deregulation is associated with the development of many human tumours. SUFU is an essential intracellular negative regulator of mammalian Hedgehog signalling and acts by binding and modulating the activity of GLI transcription factors. Despite its central importance, little is known about SUFU regulation and the nature of SUFU-GLI interaction. Here, the crystal and small-angle X-ray scattering structures of full-length human SUFU and its complex with the key SYGHL motif conserved in all GLIs are reported. It is demonstrated that GLI binding is associated with major conformational changes in SUFU, including an intrinsically disordered loop that is also crucial for pathway activation. These findings reveal the structure of the SUFU-GLI interface and suggest a mechanism for an essential regulatory step in Hedgehog signalling, offering possibilities for the development of novel pathway modulators and therapeutics.

Show MeSH
Related in: MedlinePlus