The many roles of cholesterol in the hedgehog signaling pathway
- Cell-cell communication is a fundamental biological process critical for animal development and tissue homeostasis. The Hedgehog (HH) pathway is one of these essential communication systems; improper HH signaling results in birth defects and cancer. Signaling is initiated when secreted HH ligands bind to their receptor Patched-1 (PTCH1) on target cells. This blocks PTCH1 activity, freeing the G protein-coupled receptor Smoothened (SMO) to signal across the membrane to intracellular proteins. How PTCH1 inhibits SMO has remained a central mystery in developmental signaling for 25 years. By integrating data from structural studies, CRISPR genetic screens, and novel lipid imaging tools, my thesis research provides a solution to this longstanding question: the second messenger that communicates the signal between PTCH1 and SMO is the accessibility of cholesterol in a membrane compartment. I demonstrate how PTCH1 depletes the membrane of cholesterol and how SMO responds to changes in cholesterol accessibility. Beyond HH signaling, my work reveals how cholesterol can function as an instructive signal to regulate protein activity, a discovery with implications for diverse membrane-related events ranging from signaling to pathogen entry and trafficking. The overall experimental strategy and specific methods I developed can be readily applied to probe the function of cholesterol accessibility in any cellular process of interest. Prior work implicated cholesterol as an endogenous lipid regulator of SMO. However, a major conundrum is presented by the abundance of cholesterol: how can a lipid that makes up 30%-50% of the plasma membrane be used to regulate a signaling pathway? I took a unique approach to resolve this paradox. Using a custom CRISPR library focused on genes involved in lipid regulation, I asked the more general question of which lipids regulate HH signaling in target cells. The outcome of this screen led to the unexpected discovery that sphingomyelin levels negatively regulate HH signaling. Sphingomyelin affects the chemical activity (or "accessibility") of cholesterol by sequestering it into complexes. This highlights a key concept in membrane biology: only the accessible pool of cholesterol is available to interact with proteins and engage in signaling reactions. Consequently, I proposed that accessible cholesterol (rather than total cholesterol) is the endogenous second messenger regulating SMO activity in cells. To understand how PTCH1 uses accessible cholesterol to regulate SMO, I generated tools to measure and visualize accessible and sequestered cholesterol in intact cells. I adapted toxin-based sensors to measure these pools of cholesterol with subcellular resolution and found that HH ligands change cholesterol accessibility selectively in the membrane of the primary cilium. Primary cilia are antenna-like organelles required for HH signaling in all vertebrates. PTCH1 was known to inhibit SMO at cilia, but the reason for this had remained a mystery in the field. I proposed that by compartmentalizing HH signaling in cilia, cells can use accessible cholesterol to communicate between PTCH1 and SMO without interfering with overall cellular cholesterol homeostasis. I also demonstrated that the ciliary membrane is enriched in sphingomyelin, creating a low accessible cholesterol environment compared to the plasma membrane. The unique lipid composition of this organelle likely has implications for other signaling receptors that localize there. Multiple lines of evidence have suggested that PTCH1 acts as a transporter to deplete the membrane of cholesterol. However, the transport directionality and transport mechanism of PTCH1 have remained a mystery. To understand this, I established an assay using Total Internal Reflection Fluorescence Microscopy (TIRFM) to directly measure cholesterol accessibility in the outer leaflet of the membrane in live cells. I find that PTCH1 depletes outer leaflet accessible cholesterol in a manner regulated by HH ligands and the transmembrane potassium gradient. I propose that PTCH1 moves cholesterol from the outer to the inner leaflet in exchange for potassium ion export. This assay can be readily adapted to study the effect of any protein on outer leaflet accessible cholesterol levels. Interestingly, cholesterol is also important for HH ligand biogenesis. HH is the only known protein in the human proteome covalently modified by cholesterol, yet the function of this evolutionarily conserved modification is unknown. In collaboration with Christian Siebold's lab at Oxford, I showed that the cholesterol moiety dramatically increases the potency of HH ligands. This work raises the possibility that the cholesterol moiety of HH ligands evolved to act as a substrate mimetic, inhibiting PTCH1 by clogging a cholesterol transport domain. My work nominated accessible cholesterol as the second messenger between PTCH1 and SMO and provided a mechanism for how PTCH1 can control the accessible cholesterol content of the membrane. A final mystery is how SMO is activated by cholesterol. Crystal structures have identified two cholesterol binding sites on SMO: one in the extracellular cysteine rich domain (CRD) and a second in the center of the 7-transmembrane domain (TMD). I asked which of these sites is regulated by PTCH1 and tested the role of these sites in SMO activation. I show that mutations in the CRD (but not the TMD) reduce the fold-increase in SMO activity triggered by HH ligands, suggesting that this is the site regulated by PTCH1. In contrast, sterol binding to the TMD site boosts SMO activity in the presence and absence of HH ligands. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 Angstroms apart. This work demonstrates that cholesterol functions as both a HH-regulated orthosteric ligand at the CRD and an allosteric ligand at the TMD to regulate SMO activity and HH signaling. In summary, my research has led to a new unified model for HH signaling that reconciles many previously disconnected observations in the literature. It started by testing the model that cholesterol is the mysterious second messenger between PTCH1 and SMO, revealing that only a subset of the total cholesterol molecules are sensed by the HH pathway. Following this work, I took a mechanistic look at how cholesterol is transported by PTCH1 and sensed by SMO. This work has highlighted how little we know about how accessible cholesterol is sensed and regulated in localized compartments and organelles in cells. The methods I have developed to manipulate and measure accessible cholesterol are poised to answer questions which will have broad implications for our understanding of membrane biology and pathology.
|Type of resource
|electronic resource; remote; computer; online resource
|1 online resource.
|Degree committee member
|Degree committee member
|Degree committee member
|Stanford University, Department of Biochemistry
|Statement of responsibility
|Submitted to the Department of Biochemistry.
|Thesis Ph.D. Stanford University 2021.
- © 2021 by Maia Kinnebrew
- This work is licensed under a Creative Commons Attribution Non Commercial 3.0 Unported license (CC BY-NC).
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