Schematic overview of Hedgehog (Hh) signal transduction. Reception of the Hh ligand (HhN, red) ...
The morphogen Hedgehog (Hh) is a secreted, extracellular protein which activates an essential cellular pathway in the embryogenesis of various organisms (Beachy et al). Hence, as well as its crucial role in embryonic development, Hh signalling is key in adult stem cell regulation in various tissues and species highlighting its potential importance in the field of regenerative medicine.
The transduction of Hh signalling in cells is mediated by the two multi-transmembrane proteins Patched (Ptc) and Smoothened (Smo). Ptc is the Hh receptor and inhibits the signalling activity of Smo unless bound to Hh. Additional proteins regulate activity, release and transport by direct interaction with Hh ligands. These Hh co-receptors include the interference hedgehog protein (Ihog), the growth-arest specific protein 1 (Gas1), glypican 3 (GPC3) and the hedgehog-interacting protein (Hip) (reviewed in Beachy et al).
The importance of Hh signalling in human disease has become increasingly clear; mutations in Hh pathway components often cause severe developmental defects and are associated with numerous cancers. In vivo evidence suggests that blocking excessive Hh signalling may provide a route to unique mechanism-based therapies against cancer (Rubin & de Sauvage). However, with little structural information on Hh receptors, the molecular mechanisms underlying the biology/pathology of Hh signalling essentially remain a black box. Our aim is to understand how the Hh ligand is sensed by the signal receiving cell and, ultimately, how the signal is transduced through the cell membrane. To archive this, we are using a combination of X-ray crystallography, biochemical, biophysical and cell culture-based methods. The student’s project will synergize with my Wellcome Trust RCDF programme and can be sub-divided into a set of inter-related aims:
Examples of the structural, biophysical and functional techniques we use are available in our recent publications (Aricescu et al, Bishop et al). The above aims are given in increasing order of high risk/high payoff and deliberately contain considerable redundancy in terms of the amount of research/results that could be expected of a doctoral student.
This project provides an opportunity for training in modern techniques in the field of Structural Biology of so-called difficult human proteins. The student will gain experience in molecular biology, cell culture and protein expression, biochemical and biophysical analyses and X-ray crystallography. Our expertise in glycoprotein complexes, established cell culture facilities and highly parallelized approaches will support this project. The work will also benefit from world class in-house X-ray and EM facilities and regular access to the synchrotrons (DIAMOND Light Source and ESRF Grenoble). Structural results from this project will provide atomic level detail which will be crucial in guiding functional studies and the student will have the opportunity to interface with functional collaborators to relate structural and biophysical results to functional studies in vivo.
Protein Science & Structural Biology and Physiology, Cellular & Molecular Biology
Project reference number: 118
| Name | Department | Institution | Country | |
|---|---|---|---|---|
| Dr Christian Siebold | Structural Biology | Oxford University | UK | christian.siebold@strubi.ox.ac.uk |
2010. Interactions between Hedgehog proteins and their binding partners come into view. Genes Dev., 24 (18), pp. 2001-12. Read abstract | Read more
Hedgehog (Hh) proteins are secreted signaling molecules that mediate essential tissue-patterning events during embryonic development and function in tissue homeostasis and regeneration throughout life. Hh signaling is regulated by multiple mechanisms, including covalent lipid modification of the Hh protein and interactions with multiple protein and glycan partners. Unraveling the nature and effects of these interactions has proven challenging, but recent structural and biophysical studies of Hh proteins and active fragments of heparin, Ihog, Cdo, Boc, Hedgehog-interacting protein (Hhip), Patched (Ptc), and the monoclonal antibody 5E1 have added a new level of molecular detail to our understanding of how Hh signal response and distribution are regulated within tissues. We review these results and discuss their implications for understanding Hh signaling in normal and disease states. Hide abstract
2006. Targeting the Hedgehog pathway in cancer. Nat Rev Drug Discov, 5 (12), pp. 1026-33. Read abstract | Read more
Several key signalling pathways, such as Hedgehog, Notch, Wnt and BMP-TGFbeta-Activin (bone morphogenetic protein-transforming growth factor-beta-Activin), are involved in most processes essential to the proper development of an embryo. It is also becoming increasingly clear that these pathways can have a crucial role in tumorigenesis when reactivated in adult tissues through sporadic mutations or other mechanisms. We will focus here on the Hedgehog pathway, which is abnormally activated in most basal cell carcinomas, and discuss potential therapeutic opportunities offered by the progress made in understanding this signalling pathway. Hide abstract
2007. Structure of a tyrosine phosphatase adhesive interaction reveals a spacer-clamp mechanism. Science, 317 (5842), pp. 1217-20. Read abstract | Read more
Cell-cell contacts are fundamental to multicellular organisms and are subject to exquisite levels of control. Human RPTPmu is a type IIB receptor protein tyrosine phosphatase that both forms an adhesive contact itself and is involved in regulating adhesion by dephosphorylating components of cadherin-catenin complexes. Here we describe a 3.1 angstrom crystal structure of the RPTPmu ectodomain that forms a homophilic trans (antiparallel) dimer with an extended and rigid architecture, matching the dimensions of adherens junctions. Cell surface expression of deletion constructs induces intercellular spacings that correlate with the ectodomain length. These data suggest that the RPTPmu ectodomain acts as a distance gauge and plays a key regulatory function, locking the phosphatase to its appropriate functional location. Hide abstract
2009. Structural insights into hedgehog ligand sequestration by the human hedgehog-interacting protein HHIP. Nat. Struct. Mol. Biol., 16 (7), pp. 698-703. Read abstract | Read more
Hedgehog (Hh) morphogens have fundamental roles in development, whereas dysregulation of Hh signaling leads to disease. Multiple cell-surface receptors are responsible for transducing and/or regulating Hh signals. Among these, the Hedgehog-interacting protein (Hhip) is a highly conserved, vertebrate-specific inhibitor of Hh signaling. We have solved a series of crystal structures for the human HHIP ectodomain and Desert hedgehog (DHH) in isolation, as well as HHIP in complex with DHH (HHIP-DHH) and Sonic hedgehog (Shh) (HHIP-Shh), with and without Ca2+. The interaction determinants, confirmed by biophysical studies and mutagenesis, reveal previously uncharacterized and distinct functions for the Hh Zn2+ and Ca2+ binding sites--functions that may be common to all vertebrate Hh proteins. Zn2+ makes a key contribution to the Hh-HHIP interface, whereas Ca2+ is likely to prevent electrostatic repulsion between the two proteins, suggesting an important modulatory role. This interplay of several metal binding sites suggests a tuneable mechanism for regulation of Hh signaling. Hide abstract