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Background: Orthokeratinized odontogenic cyst (OOC) is a rare developmental odontogenic cyst of the jaw. It was originally believed to be a variant of odontogenic keratocyst (OKC) but is now considered to be a distinct entity. OOC usually presents as a single lesion and recurs infrequently. On the other hand, OKC often presents with multiple lesions and displays locally aggressive behavior and a high recurrence rate associated with the protein patched homolog 1 ( PTCH1 ) gene mutation. Multiple OOC cases are extremely rare and seem to be aggressive, but their pathogenesis is not fully understood. This study aimed to determine the clinical, pathological, and genetic characteristics of multiple OCC. Methods: Three cases of multiple OOC were evaluated for clinical and histological findings, and immunohistochemical expression of Ki-67 and Bcl-2. Furthermore, PTCH1 mutations were analyzed by next-generation sequencing using a custom panel to cover the entire exon of PTCH1 . Results: The three cases of multiple OOC included two men and one woman with a mean age of 25.3 years old (range, 18–38 years old). Each case had two or three OOCs (total of seven OOCs), all of which were simultaneously detected. Of the seven OOCs that manifested as multiple jaw cysts, seven (100%) occurred in the posterior regions, four (57.1%) occurred in the mandible, and four (57.1%) were associated with an impacted tooth. Histological examination revealed cysts lined by orthokeratinized stratified squamous epithelium. Immunohistochemistry showed a low Ki-67 labeling index and no Bcl-2 expression in the seven OOCs. No pathogenic PTCH1 mutations were detected in any of the seven OOCs. None of the patients had any other symptoms or signs of recurrence at the last follow-up (6–60 months). Conclusion: Multiple OOCs appeared to occur more often in younger patients than solitary OOC. Both multiple and solitary OOCs may be related diseases within the entity of odontogenic cysts. Multiple OOCs are clinicopathologically and genetically distinct from OKC.

The oncoprotein Smoothened (SMO), a G-protein-coupled receptor (GPCR) of the Frizzled-class (class-F), transduces the Hedgehog signal from the tumour suppressor Patched-1 (PTCH1) to the glioma-associated-oncogene (GLI) transcription factors, which activates the Hedgehog signalling pathway 1 , 2 . It has remained unknown how PTCH1 modulates SMO, how SMO is stimulated to form a complex with heterotrimeric G proteins and whether G-protein coupling contributes to the activation of GLI proteins 3 . Here we show that 24,25-epoxycholesterol, which we identify as an endogenous ligand of PTCH1, can stimulate Hedgehog signalling in cells and can trigger G-protein signalling via human SMO in vitro. We present a cryo-electron microscopy structure of human SMO bound to 24( S ),25-epoxycholesterol and coupled to a heterotrimeric G i protein. The structure reveals a ligand-binding site for 24( S ),25-epoxycholesterol in the 7-transmembrane region, as well as a G i -coupled activation mechanism of human SMO. Notably, the G i protein presents a different arrangement from that of class-A GPCR–G i complexes. Our work provides molecular insights into Hedgehog signal transduction and the activation of a class-F GPCR. Cryo-electron microscopy structure of the human Smoothened protein bound to 24( S ),25-epoxycholesterol and a heterotrimeric G i protein provides insights into the activation of a Frizzled-class G-protein-coupled receptor and Hedgehog signal transduction.

The Hedgehog (Hh) signaling pathway coordinates cell–cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO). PTCH1, a transporter-like tumor-suppressor protein, binds to Hh ligands, but SMO, a G-protein-coupled-receptor family oncoprotein, transmits the Hh signal across the membrane. Recent structural, biochemical and cell-biological studies have converged at the surprising model that a specific pool of plasma membrane cholesterol, termed accessible cholesterol, functions as a second messenger that conveys the signal between PTCH1 and SMO. Beyond solving a central puzzle in Hh signaling, these studies are revealing new principles in membrane biology: how proteins respond to and remodel cholesterol accessibility in membranes and how the cholesterol composition of organelle membranes is used to regulate protein function. The Hedgehog (Hh) receptor PTCH1 uses its transporter-like function to inhibit the GPCR SMO by limiting the pool of accessible membrane cholesterol. Cholesterol acts as a ligand for SMO to activate downstream signaling.

The Hedgehog (Hh) signaling pathway is important in embryogenesis; overactivation is associated with cancer. Central to the pathway is the membrane receptor Patched 1 (Ptch1), which indirectly inhibits a G protein–coupled receptor called Smoothened. This inhibition is relieved when Ptch1 binds the secreted protein Hh. Gong et al. report the cryo–electron microscopy structures of human Ptch1 alone and in complex with its Hh ligand at 3.9 and 3.6 Å, respectively. Both structures include two steroid-shaped densities, and mutational analysis indicates that the interaction between Ptch1 and Hh is steroid-dependent. Science , this issue p. eaas8935 The cryo–electron microscopy structure of the human receptor for the regulator of development and regeneration shows steroid dependency. The Hedgehog (Hh) pathway involved in development and regeneration is activated by the extracellular binding of Hh to the membrane receptor Patched (Ptch). We report the structures of human Ptch1 alone and in complex with the N-terminal domain of human Sonic hedgehog (ShhN) at resolutions of 3.9 and 3.6 angstroms, respectively, as determined by cryo–electron microscopy. Ptch1 comprises two interacting extracellular domains, ECD1 and ECD2, and 12 transmembrane segments (TMs), with TMs 2 to 6 constituting the sterol-sensing domain (SSD). Two steroid-shaped densities are resolved in both structures, one enclosed by ECD1/2 and the other in the membrane-facing cavity of the SSD. Structure-guided mutational analysis shows that interaction between ShhN and Ptch1 is steroid-dependent. The structure of a steroid binding–deficient Ptch1 mutant displays pronounced conformational rearrangements.

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration 1 . Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma 2 . Hedgehog proteins bind to and inhibit the transmembrane cholesterol transporter Patched-1 (PTCH1), which permits activation of the seven-transmembrane transducer Smoothened (SMO) via a mechanism that is poorly understood. Here we report the crystal structure of active mouse SMO bound to both the agonist SAG21k and to an intracellular binding nanobody that stabilizes a physiologically relevant active state. Analogous to other G protein-coupled receptors, the activation of SMO is associated with subtle motions in the extracellular domain, and larger intracellular changes. In contrast to recent models 3 – 5 , a cholesterol molecule that is critical for SMO activation is bound deep within the seven-transmembrane pocket. We propose that the inactivation of PTCH1 by Hedgehog allows a transmembrane sterol to access this seven-transmembrane site (potentially through a hydrophobic tunnel), which drives the activation of SMO. These results—combined with signalling studies and molecular dynamics simulations—delineate the structural basis for PTCH1–SMO regulation, and suggest a strategy for overcoming clinical resistance to SMO inhibitors. The crystal structure of active mouse SMO in complex with the SAG21k agonist and a stabilizing intracellular binding nanobody reveals the structural basis of SMO regulation by PTCH1.

Hedgehog (HH) signalling governs embryogenesis and adult tissue homeostasis in mammals and other multicellular organisms 1 – 3 . Whereas deficient HH signalling leads to birth defects, unrestrained HH signalling is implicated in human cancers 2 , 4 – 6 . N-terminally palmitoylated HH releases the repression of Patched to the oncoprotein smoothened (SMO); however, the mechanism by which HH recognizes Patched is unclear. Here we report cryo-electron microscopy structures of human patched 1 (PTCH1) alone and in complex with the N-terminal domain of ‘native’ sonic hedgehog (native SHH-N has both a C-terminal cholesterol and an N-terminal fatty-acid modification), at resolutions of 3.5 Å and 3.8 Å, respectively. The structure of PTCH1 has internal two-fold pseudosymmetry in the transmembrane core, which features a sterol-sensing domain and two homologous extracellular domains, resembling the architecture of Niemann–Pick C1 (NPC1) protein 7 . The palmitoylated N terminus of SHH-N inserts into a cavity between the extracellular domains of PTCH1 and dominates the PTCH1–SHH-N interface, which is distinct from that reported for SHH-N co-receptors 8 . Our biochemical assays show that SHH-N may use another interface, one that is required for its co-receptor binding, to recruit PTCH1 in the absence of a covalently attached palmitate. Our work provides atomic insights into the recognition of the N-terminal domain of HH (HH-N) by PTCH1, offers a structural basis for cooperative binding of HH-N to various receptors and serves as a molecular framework for HH signalling and its malfunction in disease. High-resolution structures of the human plasma membrane protein patched 1 alone and in complex with the native form of the ligand sonic hedgehog are determined.

The Hedgehog (HH) signaling pathway is important in development, and excessive HH signaling is associated with cancer. Signaling occurs through the G protein–coupled receptor Smoothened. The pathway is repressed by the membrane receptor Patched-1 (PTCH1), and this inhibition is relieved when PTCH1 binds the secreted protein HH. Two recent papers have described structures of HH bound to PTCH1, but surprisingly, each described a different binding epitope on HH. Qi et al. present a cryo–electron microscopy structure that explains this apparent contradiction by showing that a single HH protein uses both of these interfaces to engage two PTCH1 receptors (see the Perspective by Sommer and Lemmon). Functional assays suggest that both interfaces must be bound for efficient signaling. Science , this issue p. eaas8843 ; see also p. 26 The cryo–electron microscopy structure of a key complex involved in regulating a pathway important in development and cancer is elucidated. Aberrant Hedgehog (HH) signaling leads to various types of cancer and birth defects. N-terminally palmitoylated HH initiates signaling by binding its receptor Patched-1 (PTCH1). A recent 1:1 PTCH1-HH complex structure visualized a palmitate-mediated binding site on HH, which was inconsistent with previous studies that implied a distinct, calcium-mediated binding site for PTCH1 and HH co-receptors. Our 3.5-angstrom resolution cryo–electron microscopy structure of native Sonic Hedgehog (SHH-N) in complex with PTCH1 at a physiological calcium concentration reconciles these disparate findings and demonstrates that one SHH-N molecule engages both epitopes to bind two PTCH1 receptors in an asymmetric manner. Functional assays using PTCH1 or SHH-N mutants that disrupt the individual interfaces illustrate that simultaneous engagement of both interfaces is required for efficient signaling in cells.

To translate insights in developmental biology into medical applications, techniques are needed to ensure correct cell localization. Morphogen gradients allow precise and highly reproducible pattern formation during development. Through in vitro experiments and modeling, Li et al. tested the effects of unusual properties of Hedgehog (HH) signaling. The HH morphogen's receptor, Patched (PTCH), sends an inhibitory signal when no ligand is bound, which is relieved by ligand binding. PTCH also regulates spatial distribution of the signal by sequestering the HH ligand. Furthermore, signaling through the receptor promotes synthesis of more inhibitory receptor. These characteristics help speed gradient formation and explain the robustness of the system to changes in the rate of morphogen production. Science , this issue p. 543 Insights from building a morphogen gradient in cell culture are discussed. In developing tissues, cells estimate their spatial position by sensing graded concentrations of diffusible signaling proteins called morphogens. Morphogen-sensing pathways exhibit diverse molecular architectures, whose roles in controlling patterning dynamics and precision have been unclear. In this work, combining cell-based in vitro gradient reconstitution, genetic rewiring, and mathematical modeling, we systematically analyzed the distinctive architectural features of the Sonic Hedgehog pathway. We found that the combination of double-negative regulatory logic and negative feedback through the PTCH receptor accelerates gradient formation and improves robustness to variation in the morphogen production rate compared with alternative designs. The ability to isolate morphogen patterning from concurrent developmental processes and to compare the patterning behaviors of alternative, rewired pathway architectures offers a powerful way to understand and engineer multicellular patterning.

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.