New insights into the extracellular regulation of Notch and TGF-beta signaling pathways
Two new pieces of MRC-funded work from the Handford lab on Notch signalling regulation by membrane lipids and TGF beta sequestration by fibrillin-1/LTBP1 have recently been published.
New mode of Notch activity regulation by membrane lipids
Fig 1: Cartoon to show Notch ligands interact with membrane as well as with the Notch receptor via their C2 domain (shown in pale green)
The Notch signalling pathway regulates many developmental processes and its misregulation underlies a wide range of human disorders. The presence of multiple Notch receptors/ligands, and post-translational modification of Notch by the Fringe O-glycosyltransferases are two known ways in which the Notch pathway signal is modulated in specific biological contexts. Research from the Handford and Lea groups (R. Suckling & B. Korona et al., published in EMBO J with associated commentary and front cover), has now highlighted a third way in which the Notch signal may be modulated - by membrane-lipid interactions with Notch ligands. Previous work from these groups identified a known phospholipid-binding module, at the N-terminus of the Notch ligand Jagged-1. In this new study, structural work confirmed the presence of this module in other Notch ligands: Jagged-2 and Delta-like-4. Liposome-binding assays highlighted quantitative differences in the way each ligand bound. Further insight was provided by inclusion of the ligand-binding portion of Notch in these assays, which was found to enhance liposome binding to ligand. This suggests that coupling of the receptor / ligand-binding event with the membrane composition in which Notch sits provides an additional way by which to tune the Notch signal in specific physiological contexts (Figure 1).
Structural data allowed a subset of Jagged-1 mutations associated with extra-hepatic biliary atresia (EHBA), a rare disorder of bile duct formation, to be mapped to regions close to the membrane-binding region, but distinct from the Notch-binding region. Recombinant proteins containing these disease-causing substitutions were found to be defective in liposome binding but not Notch binding, and importantly were defective in Notch activation. These data provide the first evidence of the physiological importance of membrane lipid binding for Notch activity, and pave the way for further model organism studies.
New insights into how elastic fibres sequester the growth factor TGF-β.
All the cells in our body are surrounded by a complex web of proteins and sugars known as the extracellular matrix (ECM). This network gives our tissues strength and resilience, but also confers dynamic properties such as elasticity. The elastic fibres that allow our skin, lung and arteries to stretch and recoil were once thought of purely as a structural component; however, in the last few decades, studies of inherited disorders of elastic fibres have demonstrated that they also play important regulatory roles in cell signalling and controlling the bioavailability of important growth factors such as TGF-β and bone morphogenic proteins (BMPs).
Fibrillin forms large macromolecular assemblies called 10-12nm microfibrils that are a vital component of the elastic fibre architecture and are essential for the assembly of the elastic fibre itself. Marfan syndrome, a disease which results in eye, heart and skeletal defects, is caused when patients have one defective fibrillin-1 (FBN1) gene out of two, resulting in a deficiency of functional microfibrils. A breakthrough in understanding Marfan syndrome came when looking at mouse models of fibrillin deficiency. Many of the tissue defects observed could be reversed by reducing TGF-β signalling through antibody infusion or drug treatment. However, the exact mechanisms by which fibrillin and elastic fibres regulate TGF-β have proved to be difficult to pin down.
In work from the Handford and Redfield groups, recently published in ‘Structure’, Robertson et al. have used nuclear magnetic resonance spectroscopy and a variety of other techniques, to define, at the residue-specific level, the binding sites which anchor TGF-β to fibrillin microfibrils via its partner protein Latent TGF-β binding protein 1 (LTBP1). Their studies showed that LTBP1 binds at two discrete sites on opposite faces of the fibrillin molecule. A flexible linker identified in previous work allows the two fibrillin-binding domains of LTBP1 to wrap around fibrillin and act as a grappling hook (Figure 2). Mutagenesis studies confirmed that this hook is secured by specific salt bridges between fibrillin and LTBP-1.
Fig 2: LTBP binds to fibrillin in the extracellular matrix at a site adjacent to the TGFbeta/LAP complex
The ‘bipartite’ mode of interaction is particularly interesting as a nearby site on LTBP1 covalently binds to the pro-peptide ‘straight-jacket’ of TGF-β. To bind to its receptors TGF-β must first be released from this straight-jacket or ‘activated’, and in vivo this process appears to rely on integrin proteins on the surface of cells, which bind to the straight jacket and can physically pull it apart. In addition to integrins providing the pulling force, they need something to pull against. The LTBP/fibrillin interaction may act as an anchorage point for this process in vivo. Revealing these molecular details will help inform further physiological studies to dissect accurately the molecular contributions of LTBP and fibrillin to the complex biology of TGF-β signalling.
Ultimately TGF-β is a growth factor of profound importance in our development, but it can also be a destructive factor in many acquired diseases, such as fibrosis, autoimmunity, and cancer. Understanding the details of this growth factor’s regulation in the ECM may reveal new drug targets that can precisely modulate its release in specific tissue and disease contexts.