Molecular insights into the Patched1 drug efflux inhibitory activity of panicein A hydroquinone: a computational study.

Human Hedgehog receptor Patched1 (PTCH1) is able to efflux chemotherapeutics of different chemical structure out of cancer cells thus contributing to multidrug resistance phenomena in tumor treatment. A screening of natural compounds purified from marine sponges led to the identification of the first PTCH1 efflux inhibitor, panicein A hydroquinone (PAH), demonstrated to increase doxorubicin toxicity in vitro and vemurafenib toxicity in vitro and in vivo. In this work we combined different computational techniques to gain molecular insights of the inhibitory activity of PAH and some of its active and inactive analogues. We first performed a thorough characterization and druggability analysis of the main putative substrate binding pockets known from available cryo-electron microscopy structures. Further, dynamical descriptors of the active and inactive PAH analogues were extracted from microsecond-long all-atom molecular dynamics simulations in water solution. Finally, a blind ensemble docking methodology coupled with the conformational analysis of compounds enabled rationalization of the interaction between PTCH1 and PAH and derivatives in terms of their intrinsic physico-chemical properties. Our results suggest that the Neck pocket is the preferential binding site for PAH analogues on PTCH1, and that compounds assuming an open cylindric-like shape in solution are most likely to be good binders for PTCH1.

Cholesterol access in cellular membranes controls Hedgehog signaling.

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.

Patched 1-interacting Peptide Represses Fibrosis in Pancreatic Cancer to Augment the Effectiveness of Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is resistant to immunotherapy. As a factor of resistance, the dense fibrosis of this cancer acts as a barrier to inhibit immune cell infiltration into a tumor. We examined the influence of a Hedgehog signal inhibitor, Patched 1-interacting peptide, on fibrosis, infiltration of immune cells, and immunotherapeutic effects on PDAC. We found that this peptide inhibited proliferation and migration of cancer-associated fibroblasts and cancer cells. Furthermore, this peptide reduced the production of extracellular matrix and transforming growth factor ß1 in cancer-associated fibroblasts and induced expression of HLA-ABC in PDAC cells and interferon-γ in lymphocytes. In vivo, the peptide suppressed fibrosis of PDAC and increased immune cell infiltration into tumors. The combination of this peptide and an anti-programmed death-1 antibody augmented the antitumor effect, and this combination showed the same effect in experiments using cancer cells and autologous lymphocytes. These results indicate that, in addition to the direct effect of tumor suppression, the Patched 1-interacting peptide increases the infiltration of immune cells by reducing fibrosis of PDAC and consequently enhances the effect of immunotherapy. Therefore, treatment with this peptide may be a novel therapy with 2 different mechanisms: direct tumor suppression and enhancing the immune response against PDAC.

The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.

Structural basis of sterol recognition by human hedgehog receptor PTCH1.

Hedgehog signaling is central in embryonic development and tissue regeneration. Disruption of the pathway is linked to genetic diseases and cancer. Binding of the secreted ligand, Sonic hedgehog (ShhN) to its receptor Patched (PTCH1) activates the signaling pathway. Here, we describe a 3.4-Å cryo-EM structure of the human PTCH1 bound to ShhNC24II, a modified hedgehog ligand mimicking its palmitoylated form. The membrane-embedded part of PTCH1 is surrounded by 10 sterol molecules at the inner and outer lipid bilayer portion of the protein. The annular sterols interact at multiple sites with both the sterol-sensing domain (SSD) and the SSD-like domain (SSDL), which are located on opposite sides of PTCH1. The structure reveals a possible route for sterol translocation across the lipid bilayer by PTCH1 and homologous transporters.

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.

The protein-specific activities of the transmembrane modules of Ptch1 and Ptch2 are determined by their adjacent protein domains.

Signaling through the Hedgehog (Hh) pathway is mediated by the Patched (Ptch) family of proteins. Although the vertebrate Ptch proteins Ptch1 and Ptch2 harbor two closely related transmembrane modules related to sterol-sensing domains (SSDs), the role of these closely related receptors in the Hh pathway are not equivalent. Ptch1 is essential for development and appears to be the principal receptor mediating responses to Hh ligands, whereas Ptch2 is nonessential, and its role in Hh-signaling remains ambiguous. We hypothesized that the SSDs of the Ptch proteins function as generic modules whose protein-specific activities are determined by the adjacent cytoplasmic and luminal domains. We first showed that individual N-terminal and C-terminal halves of Ptch1 associated noncovalently to mediate ligand-dependent regulation of Hh signaling. The analogous regions of Ptch2 also interacted noncovalently but did not repress the Hh pathway. However, the SSD of Ptch2 were capable of repressing Hh signaling, as determined using chimeric proteins where the SSDs of Ptch1 were replaced by those from Ptch2. Replacement of the SSDs of Ptch1 with the analogous regions from the cholesterol transporter NPC1 failed to produce a chimeric protein capable of Hh repression. Further refinement of the specific regions in Ptch1 and Ptch2 revealed that specific cytoplasmic domains of Ptch1 were necessary but not sufficient for repression of Hh signaling and that the two principal luminal domains of Ptch1 and Ptch2 were interchangeable. These data support a model where the SSDs of the Ptch family proteins exhibit generic activities and that the adjacent cytoplasmic and luminal domains determine their protein-specific activities.

Two Patched molecules engage distinct sites on Hedgehog yielding a signaling-competent complex.

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.

Structures of human Patched and its complex with native palmitoylated sonic hedgehog.

Hedgehog (HH) signalling governs embryogenesis and adult tissue homeostasis in mammals and other multicellular organisms1-3. Whereas deficient HH signalling leads to birth defects, unrestrained HH signalling is implicated in human cancers2,4-6. 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) protein7. 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-receptors8. 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.

Structural basis for the recognition of Sonic Hedgehog by human Patched1.

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.

Measuring quantitative effects of methylation on transcription factor-DNA binding affinity.

Methylation of CpG (cytosine-phosphate-guanine) dinucleotides is a common epigenetic mark that influences gene expression. The effects of methylation on transcription factor (TF) binding are unknown for most TFs and, even when known, such knowledge is often only qualitative. In reality, methylation sensitivity is a quantitative effect, just as changes to the DNA sequence have quantitative effects on TF binding affinity. We describe Methyl-Spec-seq, an easy-to-use method that measures the effects of CpG methylation (mCPG) on binding affinity for hundreds to thousands of variants in parallel, allowing one to quantitatively assess the effects at every position in a binding site. We demonstrate its use on several important DNA binding proteins. We calibrate the accuracy of Methyl-Spec-seq using a novel two-color competitive fluorescence anisotropy method that can accurately determine the relative affinities of two sequences in solution. We also present software that extends standard methods for representing, visualizing, and searching for matches to binding site motifs to include the effects of methylation. These tools facilitate the study of the consequences for gene regulation of epigenetic marks on DNA.

Detergent-solubilized Patched purified from Sf9 cells fails to interact strongly with cognate Hedgehog or Ihog homologs.

Patched (Ptc) is a twelve-pass transmembrane protein that functions as a receptor for the Hedgehog (Hh) family of morphogens. In addition to Ptc, several accessory proteins including the CDO/Ihog family of co-receptors are necessary for proper Hh signaling. Structures of Hh proteins bound to members of the CDO/Ihog family are known, but the nature of the full Hh receptor complex is not well understood. We have expressed the Drosophila Patched and Mouse Patched-1 proteins in Sf9 cells and find that Sonic Hedgehog will bind to Mouse Patched-1 in isolated Sf9 cell membranes but that purified, detergent-solubilized Ptc proteins do not interact strongly with cognate Hh and CDO/Ihog homologs. These results may reflect a nonnative conformation of detergent-solubilized Ptc or that an additional factor or factors lost during purification are required for high-affinity Ptc binding to Hh.