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Improved Sonic Hedgehog Protein Autoprocessing Assay in Cells Using Luciferase Reporter System
Author(s) -
Ciulla Daniel,
Mancusi Rebecca,
Psaras Alexandra,
Ghotra Sahib,
Callahan Brian
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.01785
Subject(s) - sonic hedgehog , luciferase , hek 293 cells , transfection , hedgehog signaling pathway , mutant , cell culture , chemistry , microbiology and biotechnology , vismodegib , biology , signal transduction , biochemistry , genetics , gene
Dysregulated cell signaling by Sonic hedgehog (Hh) proteins is implicated in developmental malformations and sporadic tumor growth. Biosynthesis of Sonic Hh requires cholesterolysis, a specialized autoprocessing event involving peptide‐bond cleavage and subsequent cholesterol modification. Cholesterolysis provides a potential site for targeted therapy. We are pursuing an in‐cell assay to monitor cholesterolysis activation and inhibition in multi‐well plates to improve on conventional techniques such as the Western Blot. Our chimeric reporter construct that fuses luciferase to human Sonic hedgehog cholesterolysis‐active domain (nLuc‐SHhC) appears to provide sensitive and quantitative measurement of cholesterolysis in HEK293 cells. Construct function was initially assessed by transient transfection. We are now preparing stable cell lines using CRISPR‐Cas9 system as a means for targeted gene insertion. Transient transfections demonstrate 5–10 fold difference in luciferase luminescence between catalytically active Sonic Hh and cholesterolysis‐defective mutant constructs. Wildtype construct yields a stronger luminescence in the supernatant, while the mutant Hh constructs show signal predominately in cell lysate. Additionally, stable cell lines show an even greater fold difference based on preliminary results. We intend to apply the reporter system introduced here for structure/function studies of cholesterolysis and for assessment of potential inhibitors targeting cholesterolysis. Support or Funding Information Funding for this research has been graciously provided by National Cancer Institute (Grant R01 CA206592) and Department of Defense (Grant W81XWH‐14‐1‐0155).