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Molecular analysis of the Hedgehog signal transduction complex in the primary cilium

Periodic Reporting for period 3 - CilDyn (Molecular analysis of the Hedgehog signal transduction complex in the primary cilium)

Reporting period: 2018-08-01 to 2020-01-31

What is the problem/issue being addressed?
The unexpected connection between the primary cilium and cell-to-cell signalling is one of the most exciting discoveries in cell and developmental biology in the last decade. In particular, the Hedgehog (Hh) pathway relies on the primary cilium to fulfil its fundamental functions in orchestrating vertebrate development. This microtubule-based antenna, up to 5 µm long, protrudes from the plasma membrane of almost every human cell and is the essential compartment for the entire Hh signalling cascade. All its molecular components, from the most upstream transmembrane Hh receptor PTCH1 down to the transcription factors, are dynamically localised and enriched in the primary cilium. The aim of this proposal, which combines structural biology and live cell imaging, is to understand the function and signalling consequences of the multivalent interactions between Hh signal transducer proteins as well as their regulation in the primary cilium.

Why is it important for society?
The Hh pathway orchestrates the development of multicellular organisms, the allocation of cells to specific lineages and proliferation and survival. Hh dysfunction leads to cancer, developmental and neurodegenerative diseases. Abnormal Hh pathway activation is associated with development of various cancers (e.g. skin, brain and pancreas). Recent results underline the clinical potential of modulating the Hh pathway, since SMO inhibition led to dramatic tumour shrinkage in meduloblastoma (MB) and basal cell carcinoma (BCC) patients, and there are now two SMO inhibitors in the clinic. However, SMO mutations have been observed in BCC and MB patients that confer resistance to drug treatment. Our molecular analyses on SMO and downstream Hh signal components has the potential to identify novel druggable targets and therapeutic strategies to circumvent the shortcomings of existing therapies.

What are the overall objectives?
The overall objective is to determine the detailed architecture of multi-protein Hh signalling complexes and investigate the consequences of structure-guided disruptions of various protein-protein interfaces. This information will be integrated into analyses of spatial and temporal processes in the primary cilium, the centre of Hh signal transduction, where the whole Hh machinery is dynamically regulated. Specifically, our objectives are:

OBJECTIVE 1: To unravel the molecular architecture of the Hh signal transduction machinery. We will identify interaction domains, purify stable binary and higher order complexes and solve their structures using X-ray crystallography and cryo electron microscopy. This analysis will be combined with biophysical (e.g. protein-protein interaction measurements) and cellular (e.g. reporter assays) methods.

OBJECTIVE 2: To define the dynamic distribution of the Hh signal transduction machinery in the primary cilium. We will study the spatial and temporal movements in primary cilia upon Hh activation, processes that were never studied before in live cells, and elucidate the functional consequences of trafficking using state-of-the-art fluorescence microscopy.
Selected highlights:

1. Structural characterization of the Hh signal transducer, oncoprotein and GPCR Smoothened (SMO) ((Byrne Nature 2016, Luchetti Elife 2016):
SMO belongs to the Frizzled-class GPCRs composed of an extracellular (CRD) and transmembrane (TMD) domain. We determined the SMO crystal structure, the first structure of any GPCR with an ectodomain, and identified two separable ligand-binding sites, one in the TMD and one in the CRD. The TMD-binding site binds to synthetic ligands like the anticancer drug vismodegib. We showed that cholesterol occupies the CRD-binding site and activates Hh signalling. We also determined the structure of SMO with vismodegib. Vismodegib-binding transmits a conformational change to the CRD resulting in loss of cholesterol-binding, thus revealing how GPCRs are controlled by ligand-regulated interactions between their extracellular and transmembrane domains. Several SMO residues mutated in vismodegib-resistant cancers are located close to the vismodegib-binding site, explaining loss of binding and resistance observed in the clinic. Whereas the CRD-binding site is essential for signalling, mutations in the TMD site blocking vismodegib-binding display normal Hh activity. Our identification of a ligand-binding site in the CRD essential for SMO activation opens new therapeutic avenues especially for cancers resistant to conventional therapy.

2. Development of an effective and innovative mammalian expression system for structural studies ((Elegheert Nature Protocols 2019):
For efficient production of our target proteins and complexes, we improved our transient transfection protocols that have been traditionally used in our laboratory. We designed and implemented a lentiviral plasmid suite for constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Inducible protein expression also allows full control over expression parameters and leads to milligram-scale quantities of target proteins and complexes from litre-scale suspension cultures ( e.g. for the GPCR SMO, the 12 TM helix-containing HH receptor Patched-1 (PTCH1) and some of our binary and ternary Hh signal transduction complexes). A feature of our vector suite is the bicistronic expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting. This strategy conveniently allows the rapid production of HEK293 cell lines that stably co-express multiple proteins of interest, and as such is suitable for protein complexes.

3. Structural studies on Hh receptor-ligand complexes (Rudolf Nature Chem Biol 2019):
HH ligands are covalently coupled to two lipids- a palmitoyl group at the N-terminus and a cholesteryl group at the C-terminus. While the palmitoyl group plays a role in inactivating PTCH1, the main HH receptor, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with the re-assessment of prior cryo-EM structures, we find that the HH-attached cholesterol binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signalling. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, SHH inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core. Since PTCH1 is thought to function as a cholesterol transporter, we suggest that HH ligands evolved the ability to inhibit PTCH1 by linking its substrate cholesterol to a protein chain that arrests the transport cycle.
We aim to determine the architecture of multi-protein Hh signalling complexes and investigate the consequences of structure-guided disruptions of various protein-protein interfaces. Our work already resulted in a detailed molecular snapshots of the Hh signal transducer SMO in different signaling states and we are now extending our analyses towards Hh multi-subunit complexes. Ultimately, we aim to obtain and integrated picture of the Hh machinery and how this is dynamically and spatially regulated at the primary cilium.
Molecular mechansims of HH-receptor interactions (A) and HH signal transduction (B).