Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast |
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Authors: | Olivier Joubert Damien Fleury Michel Bidet Martial Ruat Patrick Mollat Isabelle Mus-Veteau |
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Institution: | a Laboratoire de Biologie et de Physiopathologie des Systèmes Intégrés, CNRS FRE 3094, Faculté des Sciences, (Université de Nice - Sophia Antipolis), Parc Valrose, F-06108 Nice Cedex 2, France b Galapagos SASU, 102 avenue Gaston Roussel, F-93230 Romainville, France c Laboratoire de Chimie Bioorganique et des Systèmes Moléculaires Vectoriels, Université d'Avignon et des Pays du Vaucluse, F-84000 Avignon, France d Signal Transduction and Developmental Neuropharmacology, CNRS UPR 9040, 1 Avenue de la Terrasse F-91198 Gif-Yvette, France |
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Abstract: | The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction. |
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Keywords: | Hh Hedgehog Shh Sonic Hedgehog hSmo human Smoothened hPtc human Patched AOX1 alcohol oxidase 1 PMSF phenylmethylsulfonyl fluoride CBD calmodulin binding domain HA hemagglutinin A PCR polymerase chain reaction SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis DTT dithiothreitol DDM d-maltoside" target="_blank">n-dodecyl-β-d-maltoside Ni-NTA Ni2+-nitrilotriacetate FSs fluorinated surfactants F6-TAC C6F13C2H4-S-poly-Tris-(hydroxymethyl)aminomethane F8-TAC C8F17C2H4-S-poly-Tris-(hydroxymethyl)aminomethane SPR surface plasmon resonance |
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