Structure and function of the hedgehog receptor patched
Abstract/Contents
- Abstract
- The Hedgehog (Hh) family of protein signals specifies a host of cell types during embryogenesis in the manner of a classic morphogen, and post- embryonically orchestrates stem cell self-renewal and differentiation to regulate tissue regeneration and homeostasis. Impaired Hedgehog signaling during embryonic development leads to birth defects including holoprosencephaly. Abnormality in the Hedgehog pathway activity is also frequent in cancers. Aberrant Hedgehog pathway activation results in basal cell carcinoma and medulloblastoma, whereas progression of several other cancers, including bladder and colon cancer, is marked by loss of the pathway activity. The primary receptor for the Hedgehog signal in mammals is a twelve- transmembrane protein Patched (PTCH1). PTCH1 constitutively suppresses a downstream 7-TM protein Smoothened (SMO) and maintains the quiescence of the pathway. When Patched binds to Hedgehog, the inhibition of SMO is lifted, allowing SMO to activate downstream signaling events. The mechanism by which PTCH1 regulates SMO has been a key question in the field. PTCH1 does not form a stable complex with SMO and suppresses it catalytically. Based on the observation that PTCH1 is homologous to the Resistance, Nodulation and Division (RND) family of transporters in bacteria, which transport hydrophobic substrates, and that SMO is modulated by a series of lipid-like ligands, it has been proposed that PTCH1 may function as a RND-like transporter and modulate SMO by redistribution of its ligands. In this thesis, I determined the structure of PTCH1 with cryo-EM and compared its structure to that of bacterial RND transporters. PTCH1 transmembrane domain bears strong resemblance to RND transporters, suggesting a conserved role in transport. The extracellular domains are distinct from those in the well-characterized RND protein AcrB, but are similar to the extracellular domains of HpnN, a bacterial RND transporter specialized in hopanoid lipid transport. A cavity in PTCH1 extracellular domain is structurally homologous to the transport conduit in HpnN. Cell-based assays further demonstrated that PTCH1 reduces accessible cholesterol in the inner leaflet of the plasma membrane and that this PTCH1 effect is reversed by ShhN treatment and is abrogated by mutations in the transport conduit. In addition, PTCH1 activity to suppress SMO correlates with its effect on cholesterol, as mutations that abolish one effect also affect the other. I further investigated the conformational changes by selection for nanobodies specific for a PTCH1 conformation. After in vitro evolution for higher affinity, one of the conformation-specific nanobodies exhibited activity in vivo. In mice that received Adeno-associated virus (AAV) encoding the evolved nanobody, cells showing Hedgehog pathway activity expanded to larger regions in the lingual epithelium as compared to only within the taste buds in control animals. Taken together, the evidence shows that PTCH1 is an RND-like transporter and suppresses SMO via its transport activity. Stabilizing a particular conformation of PTCH1 causes its inactivation and novel PTCH1 inhibitors or Hedgehog pathway agonists may be developed following this mechanism.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2019; ©2019 |
| Publication date | 2019; 2019 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Zhang, Yunxiao |
|---|---|
| Degree supervisor | Beachy, Philip Arden |
| Thesis advisor | Beachy, Philip Arden |
| Thesis advisor | Jackson, Peter K. (Peter Kent) |
| Thesis advisor | Weis, William I |
| Thesis advisor | Wysocka, Joanna, Ph. D |
| Degree committee member | Jackson, Peter K. (Peter Kent) |
| Degree committee member | Weis, William I |
| Degree committee member | Wysocka, Joanna, Ph. D |
| Associated with | Stanford University, Institute for Stem Cell Biology and Regenerative Medicine. |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Yunxiao Zhang. |
|---|---|
| Note | Submitted to the Institute of Stem Cell Biology and Regenerative Medicine. |
| Thesis | Thesis Ph.D. Stanford University 2019. |
| Location | electronic resource |
Access conditions
- Copyright
- © 2019 by Yunxiao Zhang
- License
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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