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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CilDyn (Molecular analysis of the Hedgehog signal transduction complex in the primary cilium)

Teaser

Summary

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 Âµm 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 down to the ultimate 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 spatial and temporal 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 in a variety of contexts. Hh dysfunction leads to cancer, severe developmental defects and neurodegenerative diseases. Loss-of-function mutations in Hh signalling cause developmental defects, whilst gain-of-function mutations, especially in the Hh signal transducer Smoothened and the intracellular Hh signal protein SUFU are oncogenic. Abnormal Hh pathway activation is associated with the development of various different cancers (e.g. skin, brain, breast and pancreas). Recent results underline the clinical potential of modulating the Hh pathway. 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 against advanced BCC. However, SMO mutations have been observed in MB and BCC patients that confer drug resistance. In order to overcome resistance, our molecular analyses on 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?
Secreted signalling molecules, prominent amongst which are the Hedgehog (Hh) proteins, orchestrate the development of multicellular organisms, the allocation of cells to specific lineages and the growth and morphogenesis of different organs. I aim 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 intracellular 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. Hh reporter assays) methods.

OBJECTIVE 2: To define the dynamic distribution of the intracellular 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 live-cell fluorescence microscopy.

OBJECTIVE 3: To test the functional consequences of the cilium-specific EVC complex on the Hh signal transduction machinery. We will identify and structurally characterise potential interaction determinants and relate these to the dynamics o

Work performed

Selected highlights:

1. Structural characterization of the Hh signal transducer and G protein coupled receptor Smoothened:
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. Sterols, such as 20(S)-OHC activate signalling via the CRD-binding site. We showed that cholesterol occupies this 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 vismodegib-binding and resistance observed in the clinic. Whereas 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. This work already resulted in two high-profile publications (Byrne et al Nature 2016, Juchetti et al Elife 2016).

2. Development of an effective and innovative mammalian expression system for structural studies:
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. We are now able to produce milligram-scale quantities of the full-length membrane protein SMO from litre-scale suspension cultures and also similar yields for 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. A manuscript detailing the development and step-by-step application of this technique is currently in revision with the journal Nature Protocols.

Final results

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 intracellular Hh signal components and multi-subunit complexes. Ultimately, we aim to obtain and integrated picture of the Hh intracellular machinery and how this is dynamically and spatially regulated at the primary cilium.