Oxysterols are a class of endogenous cellular lipids derived from cholesterol that have been implicated in the pathophysiology of atherosclerosis, inborn errors of metabolism, inflammation and cancer. In many cases, the proteins and molecular pathways through which these enigmatic molecules exert their powerful biological effects remain unknown. We established collaboration between a synthetic organic chemist and a cell biologist to understand how oxysterols activate the Hedgehog (Hh) signaling system, a pathway that plays important roles in development, regeneration and cancer. We discovered that a specific oxysterol, 20(S)-OHC, is an allosteric activator of the 7-pass transmembrane protein Smoothened (Smo), a human oncoprotein and key drug target in oncology. This finding significantly expands the regulatory scope of oxysterols as signaling molecules, demonstrating their capacity to function as direct agonists for both a human on co-protein and a signaling receptor. Based on preliminary work, we hypothesize that endogenous 20(S)-OHC functions as a second-messenger in Hh signaling. Using a combination of mutagenesis, photo affinity labeling, and mass spectrometry, we will map the region of Smo that interacts with 20(S)-OHC to provide a biochemical portrait of this novel class of receptor-ligand interaction (Aim 1). Using quantitative mass spectrometry and a click chemistry-based imaging assay, we will ask if Hedgehog signaling can alter cellular levels or distribution of 20(S)-OHC (Aim 2). Finally, we will develop and characterize novel Hh pathway inhibitors that are inspired by oxysterol scaffolds (Aim 3). We expect three major outcomes to emerge from the successful completion of this project: (1) An answer to the question of how Smo is regulated in cells, perhaps the longest-standing mystery in the Hh pathway, (2) a biochemical understanding of how oxysterols engage and regulate 7-pass signaling receptors, and (3) the development of an integrative toolkit that can be deployed to dissect any other oxysterol- regulated cellular process. To accomplish these goals, we have recruited a team of investigators with complementary expertise in cell biology, protein biochemistry, synthetic chemistry, and mass spectrometry.

Public Health Relevance

Cholesterol-derived molecules are known to play important roles in a variety of diseases, ranging from cancer to cardiovascular disease. Using the power of chemical biology to study how these enigmatic molecules influence the Hedgehog pathway, a signaling cascade crucial for human development and cancer, we strive to develop general strategies to unlock their function and therapeutic potential.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM106078-03
Application #
9059731
Study Section
Intercellular Interactions Study Section (ICI)
Program Officer
Dunsmore, Sarah
Project Start
2014-09-05
Project End
2018-04-30
Budget Start
2016-05-01
Budget End
2017-04-30
Support Year
3
Fiscal Year
2016
Total Cost
Indirect Cost
Name
Stanford University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
009214214
City
Stanford
State
CA
Country
United States
Zip Code
94304
Byrne, Eamon Fx; Luchetti, Giovanni; Rohatgi, Rajat et al. (2018) Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates. Curr Opin Cell Biol 51:81-88
Byrne, Eamon F X; Sircar, Ria; Miller, Paul S et al. (2016) Structural basis of Smoothened regulation by its extracellular domains. Nature 535:517-522
Luchetti, Giovanni; Sircar, Ria; Kong, Jennifer H et al. (2016) Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling. Elife 5:
Marada, Suresh; Navarro, Gemma; Truong, Ashley et al. (2015) Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling. PLoS Genet 11:e1005473
Montgomery, Scott R; Nargizyan, Taya; Meliton, Vicente et al. (2014) A novel osteogenic oxysterol compound for therapeutic development to promote bone growth: activation of hedgehog signaling and osteogenesis through smoothened binding. J Bone Miner Res 29:1872-85
Peyrot, Sara M; Nachtergaele, Sigrid; Luchetti, Giovanni et al. (2014) Tracking the subcellular fate of 20(s)-hydroxycholesterol with click chemistry reveals a transport pathway to the Golgi. J Biol Chem 289:11095-110