Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened. Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol. Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.

Further insight into the cosubstrate-induced structural change of the melibiose permease (MelB) of Escherichia coli has been sought by investigating the binding and spectroscopic properties of the fluorescent sugar 2′-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl 1-thio-β-d-galactopyranoside (Dns²-S-Gal) and related analogs (Dns³-S-Gal or Dns⁶-S-Gal with a propyl or hexyl instead of an ethyl linker, respectively) interacting with MelB in membrane vesicles or in proteoliposomes. The three analogs efficiently inhibit melibiose transport and bind to MelB in a sodium-dependent fashion. Their dissociation constants (K _d) are in the micromolar range in the presence of NaCl and an order of magnitude higher in its absence. In the presence of NaCl and Dns²-S-Gal, sample excitation at 335 or 297 nm gives rise to a fluorescent signal at around 465 nm, whereas Dns³-S-Gal or Dns⁶-S-Gal emits a fluorescence light at 490 or 506 nm, respectively. Detailed study of the Dns²-S-Gal signal elicited by a 297 nm illumination indicates that a tryptophan-mediated fluorescence resonance energy transfer phenomenon is involved in the response. All fluorescence signals below 500 nm are prevented by addition of melibiose in excess, and the kinetic constants describing their dependence on the probe or NaCl concentrations closely correlate with the probe binding constants. Finally, the Dns²-S-Gal signal recorded in sodium-free medium is red shifted by up to 25 nm from that recorded in the presence of NaCl. Taken together, these results suggest (i) that the fluorescence signals below 500 nm arise from Dns-S-Gal molecules bound to MelB, (ii) the presence of a highly hydrophobic environment close to or at the sugar-binding site, the polarity of which increases on moving away from the sugar-binding site, and (iii) that the interaction of sodium ions with MelB enhances the hydrophobicity of this environment. These results are consistent with the induction of a cooperative change of the structure of the sugar-binding site or of its immediate vicinity by the ions.

Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties. Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside (‘C12-b-M’) as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors and the human Patched protein receptor by approximately 10°C compared to C12-b-M. Another surfactant yielded the highest stabilization of the human Patched protein receptor compared to C12-b-M (13°C) but was inferior for the G protein-coupled receptors. In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b₆f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M.

In the accompanying paper, we demonstrated the presence of a fluorescence resonance energy transfer (FRET) between the tryptophans of the melibiose permease (MelB) of Escherichia coli and a fluorescent sugar, 2′-(N-5-dimethylaminonaphthalene-1-sulfonyl)aminoethyl-1-thio-β-d-galactopyranoside (Dns²-S-Gal) bound at the sugar-binding site (Maehrel, C., Cordat, E., Mus-Veteau, I., and Leblanc, G. (1998) J. Biol. Chem. 273, 33192–33197). To identify the tryptophans that transfer their energy to the fluorescent sugar, we analyzed the FRET properties of MelB mutants carrying the replacement of each of the eight MelB tryptophans by a phenylalanine. The data indicate that Trp⁶⁴, localized in loop 2–3 from the N-terminal domain, and Trp²⁹⁹, localized in helix IX in the C-terminal domain, are responsible for up to 80% of the FRET signal. Moreover, by assuming that only Trp²⁹⁹ transfers energy to Dns²-S-Gal in mutant W64F, whereas only Trp⁶⁴transfers energy to Dns²-S-Gal in mutant W299F, we calculated that Trp²⁹⁹ and Trp⁶⁴ are about 14 and 20 Å away from the probe, respectively. In addition, we observed that mutating Trp³⁴², localized in helix X of the C-terminal domain, produces a significant increase of the polarity of the fluorescent sugar environment, suggesting its proximity to the sugar-binding site. Taken together, these data provide additional support for the suggestion that (i) the sugar-binding site is localized in the C-terminal part of the transporter, probably close to membrane segments IX and X, and (ii) the N-terminal domain, and particularly cytoplasmic loop 2–3, is also close to the sugar-binding site.

Most anticancer drugs fail to eradicate tumors, leading to the development of drug resistance and disease recurrence. The Hedgehog signaling plays a crucial role during embryonic development, but is also involved in cancer development, progression, and metastasis. The Hedgehog receptor Patched (Ptc) is a Hedgehog signaling target gene that is overexpressed in many cancer cells. Here, we show a link between Ptc and resistance to chemotherapy, and provide new insight into Ptc function. Ptc is cleared from the plasma membrane upon interaction with its ligand Hedgehog, or upon treatment of cells with the Hedgehog signaling antagonist cyclopamine. In both cases, after incubation of cells with doxorubicin, a chemotherapeutic agent that is used for the clinical management of recurrent cancers, we observed an inhibition of the efflux of doxorubicin from Hedgehog-responding fibroblasts, and an increase of doxorubicin accumulation in two different cancer cell lines that are known to express aberrant levels of Hedgehog signaling components. Using heterologous expression system, we stringently showed that the expression of human Ptc conferred resistance to growth inhibition by several drugs from which chemotherapeutic agents such as doxorubicin, methotrexate, temozolomide, and 5-fluorouracil. Resistance to doxorubicin correlated with Ptc function, as shown using mutations from Gorlin's syndrome patients in which the Ptc-mediated effect on Hedgehog signaling is lost. Our results show that Ptc is involved in drug efflux and multidrug resistance, and suggest that Ptc contributes to chemotherapy resistance of cancer cells. Mol Cancer Res; 10(11); 1496–508. ©2012 AACR.