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Conflict of interest statement
Introduction The Hedgehog (Hh) family of secreted protein signals patterns many tissues and structures during embryogenesis (Chiang et al., 1996, Dessaud et al., 2008, Ingham, 1993) and, post-embryonically, governs tissue homeostasis and regeneration by regulating stem cell activity (Goodrich et al., 1997, Shin et al., 2011, Teglund and Toftgård, 2010). Impaired Hedgehog signaling is associated with birth defects, including holoprosencephaly (HPE) (Chiang et al., 1996, Roessler et al., 1996), whereas aberrant pathway activation leads to formation of ectodermally derived cancers such as basal cell carcinoma (BCC) and medulloblastoma (Teglund and Toftgård, 2010). Recent work also reveals that pathway activity in stromal Fmoc-Tyr(tBu)-OH actually restrains cancer growth and progression in certain cancers of endodermal origin (Gerling et al., 2016, Lee et al., 2014, Lee et al., 2016, Rhim et al., 2014, Shin et al., 2014). The mammalian Hedgehog family comprises Sonic hedgehog (SHH), Desert hedgehog (DHH), and Indian hedgehog (IHH), which diverge in their expression patterns, but all utilize a common transduction machinery. The major receptor for mammalian Hedgehog signals is Patched1 (PTCH1) (Chen and Struhl, 1996, Fuse et al., 1999, Ingham et al., 1991, Stone et al., 1996), a 12-pass transmembrane protein. PTCH1 suppresses activity of the 7-pass transmembrane protein Smoothened (SMO), maintaining Hedgehog pathway quiescence. When PTCH1 is bound by Hedgehog, PTCH1 suppression of SMO is lifted, permitting SMO-mediated pathway activation (Goodrich et al., 1997, Ingham and McMahon, 2001, Ingham et al., 1991; Figure S1A). PTCH1 binding also serves to sequester the Hedgehog protein, shaping graded tissue responses to Hedgehog signals. Both SMO regulation and Hedgehog sequestration are conserved in metazoans ranging from insects to mammals (Chen and Struhl, 1996, Ingham and McMahon, 2001) and are needed to prevent inappropriate pathway activity. Loss-of-function PTCH1 mutations account for about 85% of BCC (Johnson et al., 1996), a cancer with over 1 million patients treated annually in the United States alone (Rogers et al., 2015), making PTCH1 perhaps the most commonly mutated tumor suppressor. Little is known regarding the biochemical function of PTCH1. Several observations, including homology of PTCH1 to the resistance-nodulation-division (RND) family of bacterial transporters, led to a model suggesting that PTCH1 may act as a transporter that controls access of certain modulatory lipids to SMO, with the binding of Hedgehog acting to inhibit this transport activity (Taipale et al., 2002). A large body of work encompassing genetic, cell biological, biochemical, and structural approaches has shown that cholesterol is required for SMO activation (Byrne et al., 2016, Cooper et al., 2003, Huang et al., 2016, Huang et al., 2018, Luchetti et al., 2016, Myers et al., 2013, Myers et al., 2017, Xiao et al., 2017). Whether PTCH1 regulates SMO by acting on cellular cholesterol has not been addressed.
Results
Discussion The structure of PTCH1 is consistent with the proposed hypothesis that PTCH1 functions as an RND-like transporter to control access of modulatory ligands to SMO (Taipale et al., 2002). The striking similarity in the transmembrane domain between PTCH1 and bacterial RND homologs and the requirement of a charged triad for PTCH1 activity together suggest that PTCH1 may be capable of harnessing energy from ions flowing down an electrochemical gradient to drive conformational switching. Although the PTCH1 extracellular domain differs significantly from that of the well-characterized bacterial transporter AcrB, the cavity positioned between sandwich domains in PTCH1, HpnN, and NPC1 is similar, suggesting a potentially similar transport mechanism. The possibility that HpnN may transport hopanoids (Doughty et al., 2011), bacterial lipids with sterol-like rigid rings, along with its sequence similarity to PTCH1 led to the proposal that PTCH1 might originate from HpnN in evolution (Hausmann et al., 2009). In addition, NPC1 is proposed to be a cholesterol transporter and is required for efflux of lysosomal cholesterol. The conduit in NPC1 thus may also be utilized for cholesterol transport, but apparently in the opposite direction, in which cholesterol moves from the lumenal domain into the membrane.