2. Academic Validation
  3. Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB

Positive selection CRISPR screens reveal a druggable pocket in an oligosaccharyltransferase required for inflammatory signaling to NF-κB

  • Cell. 2024 Apr 25;187(9):2209-2223.e16. doi: 10.1016/j.cell.2024.03.022.
Benjamin L Lampson 1,  Ana S Ramίrez 2,  Marta Baro 3,  Lixia He 1,  Mudra Hegde 4,  Vidyasagar Koduri 5,  Jamie L Pfaff 1,  Ruth E Hanna 4,  Julia Kowal 2,  Nitin H Shirole 1,  Yanfeng He 1,  John G Doench 4,  Joseph N Contessa 3,  Kaspar P Locher 6,  William G Kaelin Jr 7
Affiliations

Affiliations

  • 1 Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA.
  • 2 Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland.
  • 3 Department of Therapeutic Radiology, Yale University School of Medicine, New Haven, CT 06510, USA.
  • 4 Genetic Perturbation Platform, Broad Institute, Cambridge, MA 02142, USA.
  • 5 Division of Hematology, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02215, USA.
  • 6 Institute of Molecular Biology and Biophysics, Eidgenössische Technische Hochschule (ETH), Zürich, Switzerland. Electronic address: locher@mol.biol.ethz.ch.
  • 7 Department of Medical Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, MA 02215, USA. Electronic address: william_kaelin@dfci.harvard.edu.
PMID: 38670073 DOI: 10.1016/j.cell.2024.03.022
Abstract

Nuclear factor κB (NF-κB) plays roles in various diseases. Many inflammatory signals, such as circulating lipopolysaccharides (LPSs), activate NF-κB via specific receptors. Using whole-genome CRISPR-Cas9 screens of LPS-treated cells that express an NF-κB-driven suicide gene, we discovered that the LPS receptor Toll-like Receptor 4 (TLR4) is specifically dependent on the oligosaccharyltransferase complex OST-A for N-glycosylation and cell-surface localization. The tool compound NGI-1 inhibits OST complexes in vivo, but the underlying molecular mechanism remained unknown. We did a CRISPR base-editor screen for NGI-1-resistant variants of STT3A, the catalytic subunit of OST-A. These variants, in conjunction with cryoelectron microscopy studies, revealed that NGI-1 binds the catalytic site of STT3A, where it traps a molecule of the donor substrate dolichyl-PP-GlcNAc2-Man9-Glc3, suggesting an uncompetitive inhibition mechanism. Our results provide a rationale for and an initial step toward the development of STT3A-specific inhibitors and illustrate the power of contemporaneous base-editor and structural studies to define drug mechanism of action.

Keywords

CRISPR screening; N-glycosylation; NF-κB; Toll-like receptors; inhibition mechanism; oligosaccharyltransferase.

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