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bioRxiv · 10.1101/2022.06.27.497760

Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput.

Ciulla, D.·Dranchak, P.·Pezzullo, J.·Mancusi, R.·Psaras, A.·Rai, G.·Giner, J.-L.·Inglese, J.·Callahan, B.
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Abstract

The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursors enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2- carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/497760v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@63c641org.highwire.dtl.DTLVardef@6cee38org.highwire.dtl.DTLVardef@18bd310org.highwire.dtl.DTLVardef@cdfdc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Ciulla, D., Dranchak, P., Pezzullo, J., Mancusi, R., Psaras, A., Rai, G., Giner, J.-L., Inglese, J., Callahan, B.. 2022-06-27. Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput.. https://doi.org/10.1101/2022.06.27.497760

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