Dishevelled localization and function are differentially regulated by structurally distinct sterols.

Aberrant cholesterol synthesis, characterized by depletion of cellular cholesterol and accumulation of sterol precursors, is directly linked to a group of rare human diseases exhibiting broad clinical phenotypes and developmental malformations. Despite decreased cholesterol content being shared across these diseases, patients exhibit distinct clinical manifestations associated with specific tissues or systems with varying degrees of phenotypic overlap. This tissue specificity may result from disease-specific disruption of important critical signaling pathways due to the inability of sterol precursors to structurally compensate for cholesterol. We previously determined that the cholesterol precursor 7-dehydrocholesterol (7DHC), which accumulates in Smith-Lemli-Opitz syndrome, perturbs Dishevelled 2 (DVL2) protein function due to reduced binding affinity of 7DHC to DVL2's PDZ domain (Postsynaptic density 95, Disc large 1, and Zonular occludens 1 domain) in comparison to cholesterol, resulting in loss of canonical Wnt/β-catenin signaling (1). We hypothesized that structural differences between cholesterol and alternative sterols will inhibit binding with DVL2 and other PDZ-domain containing proteins, producing dire cellular consequences and directly affecting Wnt signaling (2). Protein homology modeling and ligand-protein docking analysis suggested a differential binding affinity and specificity of DVL2 for cholesterol versus alternative sterols. Using PM-mimetic vesicles incorporating cholesterol versus alternative sterols in liposome/DVL2 pulldowns, we experimentally observed a reduced binding affinity between DVL2 and alternate sterols. Surface plasmon resonance analysis to define the kinetics of protein-sterol interaction corroborates in silicomodeling and pulldowns. Our data suggests orientation of the beta- and alpha-sterol face within the DVL2-PDZ domain binding pocket is predictive of DVL2-sterol interaction. At the cellular level, we have also observed disruption of DVL2-sterol binding shifts the subcellular localization of DVL2 from the plasma membrane to the nucleus. Increased nuclear DVL2 promotes non-canonical WNT signaling and DVL2 protein-protein binding with nuclear factors. Our work suggests that disrupted sterol homeostasis broadly impacts cellular signaling in a sterol-specific and pathway-specific manner. Defining the structural and biochemical requirements for sterol interactions with DVL2 and other cholesterol-binding proteins will help define the critical role sterol homeostasis plays in human health. (1)Francis KR et al, Nature medicine. 2016;22(4):388-96. (2) Sheng R et al, Nature communications. 2012;3:1249.