cbEGF containing proteins in health and disease
Co-workers: Dr Sacha Jensen, Dr Pat Whiteman, Dr Bogusia Korona and Dr Ian Robertson
DPhil Students: Laurie Holt
Rotation students: Johan Hill (Wellcome Trust), Thomas Rowntree (BBSRC)
The calcium binding epidermal growth factor-like (cbEGF) domain is a widely distributed structural motif in extracellular proteins. Missense mutations that 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, assembly and interactions of the extracellular matrix glycoprotein fibrillin-1, and the mechanisms by which disease-causing mutations result in the connective tissue disorders Marfan syndrome and the acromelic dysplasias. Our recent structural studies have enabled us to create a GFP-labelled form of fibrillin-1. This has allowed us to track the fate of normal and mutant fibrillin-1 forms in the process of microfibril assembly using a co-culture system (Fig.1). We are also currently studying fibrillin-1 intermolecular interactions with the LTBPs in order to gain insight into the role of the fibrillin microfibril matrix in regulating the process of TGFβ activation (Fig. 2, 3).
In our second programme we aim to understand the cell surface organisation of the Notch receptor, its interaction with ligands, and its regulation by O-glycosylation using a range of molecular, cellular and whole organism methods. The Notch pathway is a universally conserved signal transduction system in metazoan organisms (Fig.4) 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, including a novel phospholipid-binding domain at the N-terminus of Jagged-1 (Fig.5). Currently we are working towards a greater understanding of the Notch receptor/ligand complex at the cell surface. The Notch programme involves collaborative studies with Prof. S. Lea (X-ray crystallography), Prof. C. Redfield (NMR), Prof. Haltiwanger (O-glycosylation) Prof. A. Woollard (C. elegans).
- 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: GFP-tagged fibrillin-1 and microfibril incorporation assay.
A) Using information gained from the structure of the fibrillin-1 N-terminal domains (FUN-EGF3), we created a GFP-tagged fibrillin-1 construct (GFP-Fbn) in which GFP is inserted into the unstructured N-terminus.
B) HEK293T cells transfected with constructs expressing GFP-tagged fibrillin variants are co-cultured with skin fibroblasts to examine the effects of mutations on fibrillin microfibril assembly. Recombinant fibrillin-1 in the extracellular matrix is detected with an anti-GFP antibody (white arrows). Replacing the arginines at the furin cleavage site with alanines (RRAA mutant) blocks furin processing and inhibits microfibril formation, showing that the C-terminal propeptide regulates microfibril assembly (see Jensen et al . 2014.
Figure 2: Predicted stages in the evolution of fibrillin/LTBP family of proteins and their functional roles (see Robertson et al., 2011).
Figure 3: Structural organisation of the fibrillin-1 binding region of LTBP1 (see Robertson et al, 2014)
Figure 4: The architecture of Notch receptors and their ligands. Notch receptors are single pass transmembrane proteins containing multiple extracellular EGF-like repeats, which are also present in Notch ligands, along with a DSL and MNNL domain. C. elegans also have soluble DSL ligands, not present in humans or Drosophila.
Figure 5: X-ray structure of the C2 domain at the N-terminus of human Jagged-1 and human Notch-1 major ligand-binding domain. Above; Structure of the J1NE3 fragment resolved to 2.5Å as reported by Chillakuri et al. (2013). PDB accession code 4CC1. Below, X-ray structure of the N-1 EGF11-13 fragment of human Notch-1 at 2.5Å, as reported by Cordle et al. (2008). PDB accession code 2VJ3.