SummaryRecent studies suggest that the sterol metabolic network participates in the interferon (IFN) antiviral response. However, the molecular mechanisms linking IFN with the sterol network and the identity of sterol mediators remain unknown. Here we report a cellular antiviral role for macrophage production of 25-hydroxycholesterol (cholest-5-en-3β,25-diol, 25HC) as a component of the sterol metabolic network linked to the IFN response via Stat1. By utilizing quantitative metabolome profiling of all naturally occurring oxysterols upon infection or IFN-stimulation, we reveal 25HC as the only macrophage-synthesized and -secreted oxysterol. We show that 25HC can act at multiple levels as a potent paracrine inhibitor of viral infection for a broad range of viruses. We also demonstrate, using transcriptional regulatory-network analyses, genetic interventions and chromatin immunoprecipitation experiments that Stat1 directly coupled Ch25h regulation to IFN in macrophages. Our studies describe a physiological role for 25HC as a sterol-lipid effector of an innate immune pathway.

Oxysterols are a class of endogenous signaling molecules that can activate the Hedgehog pathway, which plays critical roles in development, regeneration and cancer. However, it has been unclear how oxysterols influence Hedgehog signaling, including whether their effects are mediated through a protein target or indirectly through effects on membrane properties. To answer this question, we synthesized the enantiomer and an epimer of the most potent oxysterol, 20(S)-hydroxycholesterol. Using these molecules, we show that the effects of oxysterols on Hedgehog signaling are exquisitely stereoselective, consistent with their function through a specific protein target. We present several lines of evidence that this protein target is the 7-pass transmembrane protein Smoothened, a major drug target in oncology. Our work suggests that these enigmatic sterols, which have multiple effects on cell physiology, may act as ligands for signaling receptors and provides a generally applicable framework for probing their mechanism of action.

The Hedgehog (Hh) signal is transduced across the membrane by the heptahelical protein Smoothened (Smo), a developmental regulator, oncoprotein and drug target in oncology. We present the 2.3 Å crystal structure of the extracellular cysteine rich domain (CRD) of vertebrate Smo and show that it binds to oxysterols, endogenous lipids that activate Hh signaling. The oxysterol-binding groove in the Smo CRD is analogous to that used by Frizzled 8 to bind to the palmitoleyl group of Wnt ligands and to similar pockets used by other Frizzled-like CRDs to bind hydrophobic ligands. The CRD is required for signaling in response to native Hh ligands, showing that it is an important regulatory module for Smo activation. Indeed, targeting of the Smo CRD by oxysterol-inspired small molecules can block signaling by all known classes of Hh activators and by clinically relevant Smo mutants.DOI: http://dx.doi.org/10.7554/eLife.01340.001

Neurosteroids are potent blockers of neuronal low-voltage activated (T-type) Ca(2+) channels and potentiators of GABA(A) ligand-gated channels, but their effects in peripheral pain pathways have not been studied previously. To investigate potential analgesic effects and the ion channels involved, we tested the ability of locally injected 5alpha-reduced neurosteroids to modulate peripheral thermal nociception to radiant heat in adult rats in vivo and to modulate GABA(A) and T-type Ca(2+) channels in vitro. The steroid anesthetic alphaxalone (ALPX), the endogenous neurosteroid allopregnanolone (3alpha5alphaP), and a related compound ((3alpha,5alpha,17beta)-3-hydroxyandrostane-17-carbonitrile, (ACN)), induced potent, dose-dependent, enantioselective anti-nociception in vivo and modulation of both T-type Ca(2+) currents and GABA(A)-mediated currents in vitro. Analgesic effects of ALPX were incompletely antagonized by co-injections of the GABA(A) receptor antagonist bicuculline. The neurosteroid analogue ((3alpha,5alpha)-3-hydroxy-13,24-cyclo-18,21-dinorchol-22-en-24-ol (CDNC24), a compound with GABAergic but not T-type activity, was not analgesic. However, (3beta,5alpha,17beta)-17-hydroxyestrane-3-carbonitrile (ECN)), which has effects on T-type channels but not on GABA(A) receptors, also induced potent enantioselective peripheral anti-nociception. ECN increased pain thresholds less than ALPX, 3alpha5alphaP and ACN. However, when an ineffective dose of CDNC24 was combined with ECN, anti-nociceptive activity was greatly enhanced, and this effect was bicuculline-sensitive. These results strongly suggest that GABA(A) channels do not contribute to baseline pain transmission, but they can enhance anti-nociception mediated by blockade of T-type Ca(2+) channels. In conclusion, we demonstrate that potent peripheral analgesia induced by 5alpha-reduced neurosteroid is mediated in part by effects on T-type Ca(2+) channels. Our results also reveal a role of GABA-gated ion channels in peripheral nociceptive signaling.

Neurosteroids are endogenous modulators of neuronal excitability and nervous system development and are being developed as anesthetic agents and treatments for psychiatric diseases. While gamma amino-butyric acid Type A (GABAA) receptors are the primary molecular targets of neurosteroid action, the structural details of neurosteroid binding to these proteins remain ill defined. We synthesized neurosteroid analogue photolabeling reagents in which the photolabeling groups were placed at three positions around the neurosteroid ring structure, enabling identification of binding sites and mapping of neurosteroid orientation within these sites. Using middle-down mass spectrometry (MS), we identified three clusters of photolabeled residues representing three distinct neurosteroid binding sites in the human α1β3 GABAA receptor. Novel intrasubunit binding sites were identified within the transmembrane helical bundles of both the α1 (labeled residues α1-N408, Y415) and β3 (labeled residue β3-Y442) subunits, adjacent to the extracellular domains (ECDs). An intersubunit site (labeled residues β3-L294 and G308) in the interface between the β3(+) and α1(−) subunits of the GABAA receptor pentamer was also identified. Computational docking studies of neurosteroid to the three sites predicted critical residues contributing to neurosteroid interaction with the GABAA receptors. Electrophysiological studies of receptors with mutations based on these predictions (α1-V227W, N408A/Y411F, and Q242L) indicate that both the α1 intrasubunit and β3-α1 intersubunit sites are critical for neurosteroid action.