cbEGF containing proteins in health and disease
Co-workers: Dr Sacha Jensen, Dr Pat Whiteman, Sarah Iqbal, Philip Weisshuhn, Ralf Schneider, Joyce Tay Zi
Rotation students: Ellen Kempston (Wellcome Trust), Ian Robertson (BBSRC)
The calcium binding epidermal growth factor-like (cbEGF) domain is a widely distributed structural motif in extracellular proteins. Missense mutations which alter residues within this domain are associated with a diverse collection of human diseases highlighting the key role of the cbEGF domain in many biological processes including connective tissue function, determination of cell fate, cholesterol metabolism, blood clotting and the visual transduction pathway.
I currently have two main research programmes focussed on different cbEGF-containing proteins. The first involves molecular and cellular methods to study the structure and assembly of the extracellular matrix glycoprotein fibrillin-1 into 10-12nm microfibrils and the mechanisms by which mutations affecting fibrillin-1 cause Marfan syndrome (Fig. 1). Our recent studies have identified the molecular architecture of the main module types within fibrillin-1, from which we have proposed a calcium-stabilised extended structure for fibrillin within the microfibril ( Fig. 2, 3). We are currently studying fibrillin-1 intermolecular interactions in order to gain insight into the process of microfibril assembly. In our second programme we aim to understand the molecular basis of Notch receptor activation by its ligands. The Notch pathway is a universally conserved signal transduction system in metazoan organisms.
Not only does it play a key role in development but the pathway also regulates cell proliferation, apoptosis and angiogenesis. By understanding the molecular mechanisms involved in Notch ligand recognition and activation, it is hoped that novel therapies will be developed to modulate the Notch signal in different biological processes. Our recent studies have identified the molecular architecture of fragments of Notch and its ligand Jagged-1 which interact in a calcium-dependent manner. Furthermore, we have identified a conserved face of the ligand involved in Notch trans-activation and cis-inhibition (Fig.4). Currently we are working towards a greater understanding of Notch receptor organisation at the cell surface and are using site-directed mutagenesis to identify regions of the Notch receptor which are involved in ligand recognition. The Notch programme involves collaborative studies with Prof. S. Lea (X-ray crystallography), Dr C. Redfield (NMR), Dr M. Baron, University of Manchester (Drosophila genetics) and Prof A. Harris/Dr Banham/Dr Saison (antibodies targeting the tumour vasculature).
Other interests within my laboratory include the study of fibulin-5 and CRB1. Mutations affecting these cbEGF proteins result in defects in the visual transduction pathway.
- Whiteman, P., Willis, A.C., Warner, A., Brown, J., Redfield, C. and Handford, P.A. Cellular and Molecular studies of Marfan syndrome mutations identify co-operative protein folding in the cbEGF12-13 region of fibrillin-1. (2007) Hum. Mol. Genet. 16, 907-18
- Davis, J.A., Handford, P.A. & Redfield, C. The N1317H substitution associated with Leber congenital amaurosis results in impaired interdomain packing in human CRB1 EGF domains. (2007) J.Biol.Chem 282, 28807-28814
- Cordle, J., Redfield, C., Stacey, M., van der Merwe, P.A., Willis, A.C., Champion, B.R., Hambleton, S. and Handford, P.A. Localisation of the Delta-Like-1 binding site in human Notch-1 and its modulation by calcium affinity. (2008) J.Biol.Chem, 283, 11785-11793
- Cordle, J.*, Johnson, S.* Tay, J.Z.Y. Roversi, P., Wilkin, M., Hernandez, B., Shimizu, H., Jensen, S., Whiteman, P., Jin, B., Redfield, C. Baron, M., Lea, S.M. & Handford, P.A. A conserved face of the Jagged/Serrate DSL Domain is involved in Notch Trans-Activation and Cis-Inhibition. (2008) Nat.Str.Mol.Biol. ,15:849-57
Jovanovic, J., Iqbal, S., Jensen, S., Mardon, H. and Handford, P.A. Fibrillin-integrin interactions in health and disease. (2008) Biochem.Soc.Trans. 36, 257-262
* Joint first authors
Figure 1: Neonatal region of human fibrillin-1 showing the clustering of missense mutations which cause severe disease
Figure 2: Fibrillin-1 structure and microfibril organisation. High resolution structure of fibrillin-1 hybrid and cbEGF domains (A) supports an extended rather than folded back structure for fibrillin-1 within the microfibril (B). Rotary-shadowed electron microscopy image of 10-12nm microfibril (C)
Figure 3: Cartoon of conserved residues within a cbEGF domain and SDS-PAGE analysis showing proteolytic susceptibility of cbEGF domains in the absence of Ca 2+
Figure 4: Studies of the DSL domain from Jagged/Serrate ligand family identify a Notch binding site. (A) DSL domain structure (B) Functional analysis of DSL mutants in D. melanogaster wing disc