Summary Hedgehog (Hh) signaling during development and in postembryonic tissues requires activation of the 7TM oncoprotein Smoothened (Smo), by mechanisms that may involve endogenous lipidic modulators. Exogenous Smo ligands previously identified include the plant sterol cyclopamine (and its therapeutically useful synthetic mimics) and hydroxylated cholesterol derivatives (oxysterols); Smo is also highly sensitive to cellular sterol levels. The relationships between these effects are unclear because the relevant Smo structural determinants are unknown. We identify the conserved extracellular cysteine rich domain (CRD) as the site of action for oxysterols on Smo, involving residues structurally analogous to those contacting the Wnt lipid adduct in the homologous Frizzled CRD; this modulatory effect is distinct from that of cyclopamine mimics, from Hh-mediated regulation, and from the permissive action of cellular sterol pools. These results imply that Hh pathway activity is sensitive to lipid binding at several Smo sites, suggesting mechanisms for tuning by multiple physiological inputs.

The structural features of sterols required to support mammalian cell growth have not been fully defined. Here, we use mutant CHO cells that synthesize only small amounts of cholesterol to test the capacity of various sterols to support growth. Sterols with minor modifications of the side chain (e.g., campesterol, ␤-sitosterol, and desmosterol) supported long-term growth of mutant cells, but sterols with more complex modifications of the side chain, the sterol nucleus, or the 3-hydroxy group did not. After 60 days in culture, the exogenous sterol comprised >90% of cellular sterols. Inactivation of residual endogenous synthesis with the squalene epoxidase inhibitor NB-598 prevented growth in ␤-sitosterol and greatly reduced growth in campesterol. Growth of cells cultured in ␤-sitosterol and NB-598 was restored by adding small amounts of cholesterol to the medium. Surprisingly, enantiomeric cholesterol also supported cell growth, even in the presence of NB-598. Thus, sterols fulfill two roles in mammalian cells: (i) a bulk membrane requirement in which phytosterols can substitute for cholesterol and (ii) other processes that specifically require small amounts of cholesterol but are not enantioselective.ent-cholesterol ͉ phytosterols ͉ NB-598 S terols are essential components of eukaryote membranes. Their incorporation enhances the packing of the acyl chains of phospholipids in the hydrophobic phase of the bilayer, increases its mechanical strength, and reduces its permeability (1). Despite this crucial role, most animal species (e.g., nematodes and arthropods) cannot make sterols and so must get them from the diet. Vertebrates, by contrast, make sterols de novo from acetyl-coenzyme A and so do not require exogenous sterols. Accordingly, studies to probe sterol requirements of eukaryotes have used in invertebrates (2, 3), protazoans (4, 5), or yeast strains defective in sterol synthesis (6-8), so that the sterol composition of the organism can be controlled exogenously.Although phytosterols can account for a substantial portion of total dietary sterols (approximately one-third in humans), vertebrates systematically exclude them from the body. Cholesterol predominates in the membranes of most animals and is virtually the exclusive sterol of vertebrates. Invertebrates such as insects typically convert phytosterols to cholesterol by dealkylating C24 in the side chain, thereby furnishing cholesterol needed by membranes and preventing accumulation of noncholesterol sterols. Mammals and other vertebrates can either make sterols de novo or get cholesterol from the diet. Accumulation of other dietary sterols in these animals is prevented by the action of two ATP-binding cassette transporters, ABCG5 and ABCG8 (9), which function as a heterodimer to limit intestinal absorption and facilitate biliary excretion of noncholesterol sterols (10, 11). Thus, cholesterol comprises the great majority of vertebrate sterols, even in animals ingesting large quantities of phytosterols.The biological basis for selection of cholesterol as t...

Misregulation of protein translation plays a critical role in human cancer pathogenesis at many levels. Silvestrol, a cyclopenta[b]benzofuran natural product, blocks translation at the initiation step by interfering with assembly of the eIF4F translation complex. Silvestrol has a complex chemical structure whose functional group requirements have not been systematically investigated. Moreover, silvestrol has limited development potential due to poor druglike properties. Herein, we sought to develop a practical synthesis of key intermediates of silvestrol and explore structure-activity relationships around the C6 position. The ability of silvestrol and analogues to selectively inhibit the translation of proteins with high requirement on the translation-initiation machinery (i.e., complex 5'-untranslated region UTR) relative to simple 5'UTR was determined by a cellular reporter assay. Simplified analogues of silvestrol such as compounds 74 and 76 were shown to have similar cytotoxic potency and better ADME characteristics relative to those of silvestrol.

The activity (Po) of large-conductance voltage/Ca2+-gated K+ (BK) channels is blunted by cholesterol levels within the range found in natural membranes. We probed BK channel–forming α (cbv1) subunits in phospholipid bilayers with cholesterol and related monohydroxysterols and performed computational dynamics to pinpoint the structural requirements for monohydroxysterols to reduce BK Po and obtain insights into cholesterol’s mechanism of action. Cholesterol, cholestanol, and coprostanol reduced Po by shortening mean open and lengthening mean closed times, whereas epicholesterol, epicholestanol, epicoprostanol, and cholesterol trisnorcholenic acid were ineffective. Thus, channel inhibition by monohydroxysterols requires the β configuration of the C3 hydroxyl and is favored by the hydrophobic nature of the side chain, while having lax requirements on the sterol A/B ring fusion. Destabilization of BK channel open state(s) has been previously interpreted as reflecting increased bilayer lateral stress by cholesterol. Lateral stress is controlled by the sterol molecular area and lipid monolayer lateral tension, the latter being related to the sterol ability to adopt a planar conformation in lipid media. However, we found that the differential efficacies of monohydroxysterols to reduce Po (cholesterol≥coprostanol≥cholestanol>>>epicholesterol) did not follow molecular area rank (coprostanol>>epicholesterol>cholesterol>cholestanol). In addition, computationally predicted energies for cholesterol (effective BK inhibitor) and epicholesterol (ineffective) to adopt a planar conformation were similar. Finally, cholesterol and coprostanol reduced Po, yet these sterols have opposite effects on tight lipid packing and, likely, on lateral stress. Collectively, these findings suggest that an increase in bilayer lateral stress is unlikely to underlie the differential ability of cholesterol and related steroids to inhibit BK channels. Remarkably, ent-cholesterol (cholesterol mirror image) failed to reduce Po, indicating that cholesterol efficacy requires sterol stereospecific recognition by a protein surface. The BK channel phenotype resembled that of α homotetramers. Thus, we hypothesize that a cholesterol-recognizing protein surface resides at the BK α subunit itself.

ATP-binding cassette transporters G5 and G8 are half-transporters expressed on the apical membranes of enterocytes and hepatocytes that limit intestinal uptake and promote secretion of neutral sterols. Genetic defects that inactivate either halftransporter cause accumulation of cholesterol and plant sterols, resulting in premature coronary atherosclerosis. These observations suggest that G5 and G8 promote the translocation of sterols across membranes, but the primary transport substrate of the G5G8 complex has not been directly determined. Here we report the development of a sterol transfer assay using "insideout" membrane vesicles from Sf9 cells expressing recombinant mouse G5 and G8. Radiolabeled cholesterol or sitosterol was transferred from donor liposomes to G5-and G8-containing membrane vesicles in an ATP-dependent and vanadate-sensitive manner; net transfer of cholesterol was associated with an increase in vesicular cholesterol mass. CTP, GTP, and UTP, as well as ATP, supported transfer but with lesser efficiency (ATP Ͼ Ͼ CTP > GTP > UTP). Transfer was specific for sterols and was stereoselective; minimal ATP-dependent and vanadate-sensitive transfer of cholesteryl oleate, phosphatidylcholine, or enantiomeric cholesterol was observed. These studies indicate that G5 and G8 are sufficient for reconstitution of sterol transfer activity in vitro and provide the first demonstration that sterols are direct transport substrates of the G5 and G8 heterodimer. Members of the superfamily of ATP-binding cassette (ABC)2 transporters actively translocate a wide variety of substances, including anions, lipids, peptides, and other compounds across membranes (1). Two ABC half-transporters, ABCG5 (G5) and ABCG8 (G8), expressed in the absorptive cells of the intestine and in hepatocytes play critical roles in the trafficking of cholesterol and other neutral sterols (2). G5 and G8 form heterodimers in the endoplasmic reticulum and are transported to the apical membranes (3, 4). In enterocytes, the G5G8 complex limits the amount of dietary sterols that are incorporated into lipoproteins and delivered to the liver (3, 4). In hepatocytes, G5 and G8 are required for efficient cholesterol secretion into bile, the major pathway for cholesterol elimination in mammals (5). Mutations inactivating either G5 or G8 cause sitosterolemia, a recessive disorder characterized by hypercholesterolemia and phytosterolemia because of increased fractional absorption and reduced biliary secretion of sterols (2, 6). Detailed metabolic studies in genetically modified mice in which G5 and G8 are overexpressed or inactivated confirm the central role of G5 and G8 in sterol trafficking (5, 7). The fractional absorption of sterols is increased and biliary sterol secretion decreased in the G5G8 Ϫ/Ϫ mice, whereas overexpression of G5 and G8 reduces the fractional absorption of dietary cholesterol and promotes biliary cholesterol secretion (5, 7).All ABC transporters share a common molecular architecture, which includes a pair of nucleotide binding domains ...