(1237-B) Bioluminescent cell-based reporter identifies atypical activator of human Sonic Hedgehog Autoprocessing
Monday, February 5, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: Sonic Hedgehog (SHh) protein signaling maintains a critical role in human development and adult health. SHh signaling is initiated by a self-cleaving precursor protein through a cholesterol-driven mechanism known as autoprocessing. Dysregulation of SHh autoprocessing is linked to developmental disease and cancer. Our recently developed cellular high-throughput luminescent assay for screening human SHh autoprocessing modulators has led to the identification of an atypical, non-steroidal autoprocessing activator called PAPP. Unlike the native substrate cholesterol, which utilizes a hydroxyl group to cleave precursor SHh, PAPP seemingly uses an amine functional group for covalent attack. While promiscuity in Hh autoprocessing has been well studied using Drosophila Hh protein, little evidence has been found of the same substrate tolerance in human SHh protein. Competition binding studies between sterol and PAPP are consistent with ternary complex formation (SHh/sterol/PAPP), potentially indicating an unidentified alternative substrate binding pocket within the SHh autoprocessing domain. Through a panel of Western blotting, mass spectrometry, and protein 19F NMR, our data suggests that PAPP acts as an unusual nucleophilic substrate for precursor SHh autoprocessing. We propose the use of PAPP, along-side our previously published synthetic sterol 2-ACC, as chemical switches for controlled protein of interest (POI) activation using a POI-SHhC(D46A) fusion construct. This technology, which we call Sonic Hedgehog Exported POI for Retention or Dispersal (SHEPRD), allows for selective transport of POI to the cellular membrane or to be secreted from the cell depending on the chemical activator used. Preliminary FACS and microscopy data using a model HA-mScarlet-I3-SHEPRD construct demonstrate HA-mS-I3 membrane retention in cells treated with 2-ACC, and secretion of HA-mS3 into media of cells treated with PAPP.