Hedgehog pathway activation through nanobody-mediated conformational blockade of the Patched sterol conduit.

Activation of the Hedgehog pathway may have therapeutic value for improved bone healing, taste receptor cell regeneration, and alleviation of colitis or other conditions. Systemic pathway activation, however, may be detrimental, and agents amenable to tissue targeting for therapeutic application have been lacking. We have developed an agonist, a conformation-specific nanobody against the Hedgehog receptor Patched1 (PTCH1). This nanobody potently activates the Hedgehog pathway in vitro and in vivo by stabilizing an alternative conformation of a Patched1 "switch helix," as revealed by our cryogenic electron microscopy structure. Nanobody-binding likely traps Patched in one stage of its transport cycle, thus preventing substrate movement through the Patched1 sterol conduit. Unlike the native Hedgehog ligand, this nanobody does not require lipid modifications for its activity, facilitating mechanistic studies of Hedgehog pathway activation and the engineering of pathway activating agents for therapeutic use. Our conformation-selective nanobody approach may be generally applicable to the study of other PTCH1 homologs.

Smoothened stimulation by membrane sterols drives Hedgehog pathway activity.

Hedgehog signalling is fundamental to embryonic development and postnatal tissue regeneration1. Aberrant postnatal Hedgehog signalling leads to several malignancies, including basal cell carcinoma and paediatric medulloblastoma2. 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 models3-5, 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.

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Rapid, direct activity assays for Smoothened reveal Hedgehog pathway regulation by membrane cholesterol and extracellular sodium.

Hedgehog signaling specifies tissue patterning and renewal, and pathway components are commonly mutated in certain malignancies. Although central to ensuring appropriate pathway activity in all Hedgehog-responsive cells, how the transporter-like receptor Patched1 regulates the seven-transmembrane protein Smoothened remains mysterious, partially due to limitations in existing tools and experimental systems. Here we employ direct, real-time, biochemical and physiology-based approaches to monitor Smoothened activity in cellular and in vitro contexts. Patched1-Smoothened coupling is rapid, dynamic, and can be recapitulated without cilium-specific proteins or lipids. By reconstituting purified Smoothened in vitro, we show that cholesterol within the bilayer is sufficient for constitutive Smoothened activation. Cholesterol effects occur independently of the lipid-binding Smoothened extracellular domain, a region that is dispensable for Patched1-Smoothened coupling. Finally, we show that Patched1 specifically requires extracellular Na+ to regulate Smoothened in our assays, raising the possibility that a Na+ gradient provides the energy source for Patched1 catalytic activity. Our work suggests a hypothesis wherein Patched1, chemiosmotically driven by the transmembrane Na+ gradient common to metazoans, regulates Smoothened by shielding its heptahelical domain from cholesterol, or by providing an inhibitor that overrides this cholesterol activation.

The Stromal Niche for Epithelial Stem Cells: A Template for Regeneration and a Brake on Malignancy.

Stromal restraint of cancer growth and progression-emerging as a widespread phenomenon in epithelial cancers such as bladder, pancreas, colon, and prostate-appears rooted in stromal cell niche activity. During normal tissue repair, stromal niche signals, often Hedgehog-induced, promote epithelial stem cell differentiation as well as self-renewal, thus specifying a regenerating epithelial pattern. In the case of cancerous tissue, stromal cell-derived differentiation signals in particular may provide a brake on malignant growth. Understanding and therapeutic harnessing of the role of stroma in cancer restraint may hinge on our knowledge of the signaling programs elaborated by the stromal niche.

Production of recombineering substrates with standard-size PCR primers.

Recombineering is a powerful method for DNA manipulation. It has advantages over restriction endonuclease-based methods and is usually rapid. Typically, recombineering uses long PCR primers (c. 65 bases), each of which contains a small region of target homology (c. 45 bases). We have developed a simple, albeit somewhat less rapid, strategy to create recombineering substrates that can use primers of ≤ 35 bases for all steps. The regions of homology can be several hundred base pairs in length to (1) increase the chance of obtaining the desired clone and/or (2) allow coliphage-based recombineering in some non-Escherichia coli bacteria. The method uses cloning techniques to construct a template for the generation of the recombineering substrate. Because the template is made from cloned DNA segments, the segments (including those for the homology regions) can be readily changed. During construction of the template plasmid, potential background transformants arising from the vector without insert are significantly reduced by cloning each segment with two restriction endonucleases that produce noncompatible ends. We have used this method to change the bla gene of pACYC177 to aadA, to add the MCS-lacZα region from pBBR1MCS to IncQ plasmid vectors, and to make an oriT(IncP) -aacC1 cassette and add it to a plasmid.