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Conserved cholesterol-related activities of Dispatched 1 drive Sonic hedgehog shedding from the cell membrane.

ABSTRACT: The Sonic hedgehog (Shh) pathway controls embryonic development and tissue homeostasis after birth. Long-standing questions about this pathway include how the dual-lipidated, firmly plasma membrane-associated Shh ligand is released from producing cells to signal to distant target cells and how the resistance-nodulation-division transporter Dispatched 1 (Disp, also known as Disp1) regulates this process. Here, we show that inactivation of Disp in Shh-expressing human cells impairs proteolytic Shh release from its lipidated terminal peptides, a process called ectodomain shedding. We also show that cholesterol export from Disp-deficient cells is reduced, that these cells contain increased cholesterol amounts in the plasma membrane, and that Shh shedding from Disp-deficient cells is restored by pharmacological membrane cholesterol extraction and by overexpression of transgenic Disp or the structurally related protein Patched 1 (Ptc, also known as Ptch1; a putative cholesterol transporter). These data suggest that Disp can regulate Shh function via controlled cell surface shedding and that membrane cholesterol-related molecular mechanisms shared by Disp and Ptc exercise such sheddase control.

SUBMITTER: Ehring K

PROVIDER: S-EPMC8403983 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Cleavage activates dispatched for Sonic Hedgehog ligand release.

Project description:Hedgehog ligands activate an evolutionarily conserved signaling pathway that provides instructional cues during tissue morphogenesis, and when corrupted, contributes to developmental disorders and cancer. The transmembrane protein Dispatched is an essential component of the machinery that deploys Hedgehog family ligands from producing cells, and is absolutely required for signaling to long-range targets. Despite this crucial role, regulatory mechanisms controlling Dispatched activity remain largely undefined. Herein, we reveal vertebrate Dispatched is activated by proprotein convertase-mediated cleavage at a conserved processing site in its first extracellular loop. Dispatched processing occurs at the cell surface to instruct its membrane re-localization in polarized epithelial cells. Cleavage site mutation alters Dispatched membrane trafficking and reduces ligand release, leading to compromised pathway activity in vivo. As such, convertase-mediated cleavage is required for Dispatched maturation and functional competency in Hedgehog ligand-producing cells.

| S-EPMC5811216 | biostudies-literature

Dispatching plasma membrane cholesterol and Sonic Hedgehog dispatch: two sides of the same coin?

Project description:Vertebrate and invertebrate Hedgehog (Hh) morphogens signal over short and long distances to direct cell fate decisions during development and to maintain tissue homeostasis after birth. One of the most important questions in Hh biology is how such Hh signaling to distant target cells is achieved, because all Hh proteins are secreted as dually lipidated proteins that firmly tether to the outer plasma membrane leaflet of their producing cells. There, Hhs multimerize into light microscopically visible storage platforms that recruit factors required for their regulated release. One such recruited release factor is the soluble glycoprotein Scube2 (Signal sequence, cubulin domain, epidermal-growth-factor-like protein 2), and maximal Scube2 function requires concomitant activity of the resistance-nodulation-division (RND) transporter Dispatched (Disp) at the plasma membrane of Hh-producing cells. Although recently published cryo-electron microscopy-derived structures suggest possible direct modes of Scube2/Disp-regulated Hh release, the mechanism of Disp-mediated Hh deployment is still not fully understood. In this review, we discuss suggested direct modes of Disp-dependent Hh deployment and relate them to the structural similarities between Disp and the related RND transporters Patched (Ptc) and Niemann-Pick type C protein 1. We then discuss open questions and perspectives that derive from these structural similarities, with particular focus on new findings that suggest shared small molecule transporter functions of Disp to deplete the plasma membrane of cholesterol and to modulate Hh release in an indirect manner.

| S-EPMC8589413 | biostudies-literature

Dispatched and scube mediate the efficient secretion of the cholesterol-modified hedgehog ligand.

Project description:The Hedgehog (Hh) signaling pathway plays critical roles in metazoan development and in cancer. How the Hh ligand is secreted and spreads to distant cells is unclear, given its covalent modification with a hydrophobic cholesterol molecule, which makes it stick to membranes. We demonstrate that Hh ligand secretion from vertebrate cells is accomplished via two distinct and synergistic cholesterol-dependent binding events, mediated by two proteins that are essential for vertebrate Hh signaling: the membrane protein Dispatched (Disp) and a member of the Scube family of secreted proteins. Cholesterol modification is sufficient for a heterologous protein to interact with Scube and to be secreted in a Scube-dependent manner. Disp and Scube recognize different structural aspects of cholesterol similarly to how Niemann-Pick disease proteins 1 and 2 interact with cholesterol, suggesting a hand-off mechanism for transferring Hh from Disp to Scube. Thus, Disp and Scube cooperate to dramatically enhance the secretion and solubility of the cholesterol-modified Hh ligand.

| S-EPMC3682496 | biostudies-literature

Cholesterol and CDON Regulate Sonic Hedgehog Release from Pancreatic Cancer Cells.

Project description:Background: Sonic Hedgehog (Shh) is a tightly regulated membrane-associated morphogen and a known driver of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). After processing, Shh remains at the plasma membrane of Shh producing cells, thereby limiting its distribution and signal strength. In PDAC, the release of Shh from tumor cells is necessary to promote a tumor-permissive microenvironment. Mechanisms regulating Shh sequestration and/or release from tumor cells to signal distant stromal cells are not well known. Previously, our laboratory demonstrated that the Drosophila transmembrane protein Boi, sequesters Hh at the membrane of Hh-producing cells. In response to dietary cholesterol or in the absence of boi, Hh is constitutively released to promote proliferation in distant cells. In this study, we investigated the conservation of this mechanism in mammals by exploring the role of the human boi homolog, CDON, in PDAC. Methods: Using PDAC cell-lines BxPC-3, Capan-2, and MIA PaCa-2, along with normal pancreatic epithelial cells (PDEC), we investigated Shh expression via Immunoblot and real-time, quantitative polymerase chain reaction in addition to Shh release via enzyme-linked immunoassay following cholesterol treatment and/or transfection with either RNA interference to reduce CDON expression or with human CDON to increase expression. Results: Consistent with our Boi model, CDON suppresses Shh release, which is alleviated in response to dietary cholesterol. However, over-expressing CDON suppresses cholesterol-mediated Shh release in some PDAC contexts, which may be relative to the mutational burden of the cells. Conclusion: Identifying mechanisms that either sequester or stimulate Shh release from the tumor cell membrane may provide new avenues to reduce signaling between the tumor and its surrounding environment, which may restrain tumor development.

| S-EPMC8252898 | biostudies-literature

Cytoneme delivery of Sonic Hedgehog from ligand-producing cells requires Myosin 10 and a Dispatched-BOC/CDON co-receptor complex.

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| S-EPMC7968926 | biostudies-literature

An emerging role of Sonic hedgehog shedding as a modulator of heparan sulfate interactions.

Project description:Major developmental morphogens of the Hedgehog (Hh) family act at short range and long range to direct cell fate decisions in vertebrate and invertebrate tissues. To this end, Hhs are released from local sources and act at a distance on target cells that express the Hh receptor Patched. However, morphogen secretion and spreading are not passive processes because all Hhs are synthesized as dually (N- and C-terminal) lipidated proteins that firmly tether to the surface of producing cells. On the cell surface, Hhs associate with each other and with heparan sulfate (HS) proteoglycans. This raises the question of how Hh solubilization and spreading is achieved. We recently discovered that Sonic hedgehog (Shh) is solubilized by proteolytic processing (shedding) of lipidated peptide termini in vitro. Because unprocessed N termini block Patched receptor binding sites in the cluster, we further suggested that their proteolytic removal is required for simultaneous Shh activation. In this work we confirm inactivity of unprocessed protein clusters and demonstrate restored biological Shh function upon distortion or removal of N-terminal amino acids and peptides. We further show that N-terminal Shh processing targets and inactivates the HS binding Cardin-Weintraub (CW) motif, resulting in soluble Shh clusters with their HS binding capacities strongly reduced. This may explain the ability of Shh to diffuse through the HS-containing extracellular matrix, whereas other HS-binding proteins are quickly immobilized. Our in vitro findings are supported by the presence of CW-processed Shh in murine brain samples, providing the first in vivo evidence for Shh shedding and subsequent solubilization of N-terminal-truncated proteins.

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PTCHD1 Binds Cholesterol but Not Sonic Hedgehog, Suggesting a Distinct Cellular Function.

Project description:Deleterious mutations in the X-linked Patched domain-containing 1 (PTCHD1) gene may account for up to 1% of autism cases. Despite this, the PTCHD1 protein remains poorly understood. Structural similarities to Patched family proteins point to a role in sterol transport, but this hypothesis has not been verified experimentally. Additionally, PTCHD1 has been suggested to be involved in Hedgehog signalling, but thus far, the experimental results have been conflicting. To enable a variety of biochemical and structural experiments, we developed a method for expressing PTCHD1 in Spodoptera frugiperda cells, solubilising it in glycol-diosgenin, and purifying it to homogeneity. In vitro and in silico experiments show that PTCHD1 function is not interchangeable with Patched 1 (PTCH1) in canonical Hedgehog signalling, since it does not repress Smoothened in Ptch1-/- mouse embryonic fibroblasts and does not bind Sonic Hedgehog. However, we found that PTCHD1 binds cholesterol similarly to PTCH1. Furthermore, we identified 13 PTCHD1-specific protein interactors through co-immunoprecipitation and demonstrated a link to cell stress responses and RNA stress granule formation. Thus, our results support the notion that despite structural similarities to other Patched family proteins, PTCHD1 may have a distinct cellular function.

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Quiescent sox2(+) cells drive hierarchical growth and relapse in sonic hedgehog subgroup medulloblastoma.

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Cryo-EM structure of the Hedgehog release protein Dispatched.

Project description:The Hedgehog (Hh) signaling pathway controls embryonic development and adult tissue homeostasis in multicellular organisms. In Drosophila melanogaster, the pathway is primed by secretion of a dually lipid-modified morphogen, Hh, a process dependent on a membrane-integral protein Dispatched. Although Dispatched is a critical component of the pathway, the structural basis of its activity has, so far, not been described. Here, we describe a cryo-electron microscopy structure of the D. melanogaster Dispatched at 3.2-Å resolution. The ectodomains of Dispatched adopt an open conformation suggestive of a receptor-chaperone role. A three-dimensional reconstruction of Dispatched bound to Hh confirms the ability of Dispatched to bind Hh but using a unique mode distinct from those previously observed in structures of Hh complexes. The structure may represent the state of the complex that precedes shedding of Hh from the surface of the morphogen-releasing cell.

| S-EPMC7159904 | biostudies-literature

Architecture of Dispatched, a Transmembrane Protein Responsible for Hedgehog Release.

Project description:The evolutionarily conserved Hedgehog (Hh) signaling pathway is crucial for programmed cell differentiation and proliferation. Dispatched (Disp) is a 12-transmembrane protein that plays a critical role in the Hedgehog (Hh) signaling pathway by releasing the dually lipidated ligand HhN from the membrane, a prerequisite step to the downstream signaling cascade. In this study, we focus on the Disp from water bear, a primitive animal known as the most indestructible on Earth. Using a zebrafish model, we show that the water bear homolog possesses the function of Disp. We have solved its structure to a 6.5-Å resolution using single-particle cryogenic electron microscopy. Consistent with the evolutional conservation of the pathway, the water bear Disp structure is overall similar to the previously reported structures of the fruit fly and human homologs. Although not revealing much detail at this resolution, the water bear Disp shows a different conformation compared to published structures, suggesting that they represent different functional snapshots.

| S-EPMC8453165 | biostudies-literature

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