Regulation of hedgehog signaling by cholesterol

Abstract/Contents

Abstract

The Hedgehog (Hh) pathway is a highly conserved signaling program required for proper embryonic development and adult tissue maintenance. Core Hh pathway components were discovered by genetic screening in Drosophila nearly 40 years ago, but some of the molecular events that transduce the signal between components remain mysterious. A long-standing mystery in the field is how secreted Hh ligands transmit signals across the membrane bi-layer of recipient cells? Signaling at this step is mediated by two integral membrane proteins, the Hh ligand receptor Patched (PTCH) and the G-protein coupled receptor (GPCR) Smoothened (SMO). In resting cells, PTCH represses SMO without a direct physical interaction. Hh ligands bind to and inactivate PTCH allowing SMO to adopt an active conformation. A widely accepted model posits that PTCH regulates SMO indirectly by controlling its access to a small molecule second messenger. The component of the Hh pathway that transduces the signal across the membrane is SMO, a Frizzled-class GPCR. Like other GPCRs, SMO activity is modulated by numerous small molecules including the anticancer drug vismodegib. Small molecules interact with SMO via at least two distinct binding-sites, one located in its heptahelical transmembrane domain (7TMD) and the second in its extracellular cysteine-rich domain (CRD). How SMO is regulated and whether PTCH inhibits its activation using either of these binding-sites remains an open question. The SMO CRD harbors a highly conserved sterol binding-site, select mutations in which diminish SMO signaling by native Hh ligands. From these observations, I hypothesized that endogenous molecules able to bind this site would inform the physiological mechanism of SMO regulation. Using the isolated CRD and an affinity reagent incorporating the CRD agonist 20(S)-hydroxycholesterol (20(S)-OHC), I tested lipids extracted and purified from animal tissue that could compete for this interaction. From this approach, numerous oxidized cholesterol metabolites emerged whose bioavailability in cellular assays depended on their delivery as soluble methyl-β-cyclodextrin (MβCD) inclusion complexes. By delivering cholesterol as a soluble MβCD complex, I made the serendipitous discovery that cholesterol is sufficient to activate the Hh pathway. Using genetic epistasis and pharmacological approaches, I determined cholesterol acts on SMO to trigger Hh signaling. My discovery complimented recent crystallographic data describing the first full-length crystal structure of SMO. Unexpectedly, the structure was solved together with a ligand bound to the extracellular CRD, the ligand turned out to be cholesterol. Using the structure to inform SMO mutagenesis, I concluded that the activating effect of cholesterol on SMO is mediated through the same CRD binding-site observed in the crystal structure. Interestingly, SMO also depended on its cholesterol binding-site for responding to native Hh ligands. Specifically, mutations that impaired cholesterol binding also disrupted endogenous signaling. From these data, I hypothesized how signaling between PTCH and SMO might be regulated by nominating cholesterol as a second messenger for Hh signaling. This model of Hh regulation requires PTCH to directly or indirectly influence cholesterol availability for binding to SMO. Coincidentally, PTCH is homologous to the mammalian lysosomal protein Niemann Pick C1 (NPC1). Assuming PTCH functions like NPC1 as a cholesterol transporter, PTCH might inhibit SMO by sequestering or shuttling cholesterol away to prevent its access to SMO. Hh ligand binding to PTCH would inhibit cholesterol transport, cholesterol would bind to the CRD driving SMO activation and downstream signaling. Although my data are consistent with this model, I have not proven PTCH functions to influence cholesterol levels or availability to SMO. Cholesterol shares a long history with the Hh field as a necessary component for supporting proper SMO activity. My discovery is distinct from early studies showing cholesterol acts solely as a necessary component for Hh signaling. Contrasting this, I showed that cholesterol is also sufficient to activate Hh signaling. Importantly, these instructive effects coincide with a region of SMO unique from that required to mediate cholesterol's necessary effects. Specifically, cholesterol is required by the SMO 7TMD while the extracellular CRD mediates its instructive effects. Finally, I must also consider separate and competing models to explain my data. For example, is cholesterol only an allosteric regulator of SMO signaling? In fact, allosteric modulators influence GPCR activity directly to promote receptor activation alone, or indirectly to modify the pharmacological properties of a separate ligand binding-site. In the latter case, cholesterol may influence binding of a separate ligand to the 7TMD or other binding-sites awaiting future discovery. Also, consistent with an allosteric role, cholesterol may act as a SMO cofactor or prosthetic group required for supporting high-level responses driven by Hh ligands. Finally, by uncovering cholesterol and its unique ability to activate SMO, I have advanced the Hh field by providing new hypotheses and clear future directions.

Description

Type of resource	text
Form	electronic; electronic resource; remote
Extent	1 online resource.
Publication date	2017
Issuance	monographic
Language	English

Creators/Contributors

Associated with	Luchetti, Giovanni
Associated with	Stanford University, Department of Biochemistry.
Primary advisor	Rohatgi, Rajat
Thesis advisor	Rohatgi, Rajat
Thesis advisor	Artandi, Steven E
Thesis advisor	Harbury, Pehr
Thesis advisor	Yeh, Ellen
Advisor	Artandi, Steven E
Advisor	Harbury, Pehr
Advisor	Yeh, Ellen

Subjects

Genre	Theses

Bibliographic information

Statement of responsibility	Giovanni Luchetti.
Note	Submitted to the Department of Biochemistry.
Thesis	Thesis (Ph.D.)--Stanford University, 2017.
Location	electronic resource

Access conditions

Copyright

© 2017 by Giovanni Luchetti

License

This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).

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