A microfibril assembly assay identifies different mechanisms of dominance underlying Marfan syndrome, stiff skin syndrome and acromelic dysplasias

Fibrillin-1 is the major component of the 10–12 nm diameter extracellular matrix microfibrils. The majority of mutations affecting the human fibrillin-1 gene, FBN1, result in Marfan syndrome (MFS), a common connective tissue disorder characterised by tall stature, ocular and cardiovascular defects. Recently, stiff skin syndrome (SSS) and a group of syndromes known collectively as the acromelic dysplasias, which typically result in short stature, skin thickening and joint stiffness, have been linked to FBN1 mutations that affect specific domains of the fibrillin-1 protein. Despite their apparent phenotypic differences, dysregulation of transforming growth factor β (TGFβ) is a common factor in all of these disorders. Using a newly developed assay to track the secretion and incorporation of full-length, GFP-tagged fibrillin-1 into the extracellular matrix, we investigated whether or not there were differences in the secretion and microfibril assembly profiles of fibrillin-1 variants containing substitutions associated with MFS, SSS or the acromelic dysplasias. We show that substitutions in fibrillin-1 domains TB4 and TB5 that cause SSS and the acromelic dysplasias do not prevent fibrillin-1 from being secreted or assembled into microfibrils, whereas MFS-associated substitutions in these domains result in a loss of recombinant protein in the culture medium and no association with microfibrils. These results suggest fundamental differences in the dominant pathogenic mechanisms underlying MFS, SSS and the acromelic dysplasias, which give rise to TGFβ dysregulation associated with these diseases

The conserved C2 phospholipid‐binding domain in Delta contributes to robust Notch signalling

Abstract: Accurate Notch signalling is critical for development and homeostasis. Fine‐tuning of Notch–ligand interactions has substantial impact on signalling outputs. Recent structural studies have identified a conserved N‐terminal C2 domain in human Notch ligands which confers phospholipid binding in vitro. Here, we show that Drosophila ligands Delta and Serrate adopt the same C2 domain structure with analogous variations in the loop regions, including the so‐called β1‐2 loop that is involved in phospholipid binding. Mutations in the β1‐2 loop of the Delta C2 domain retain Notch binding but have impaired ability to interact with phospholipids in vitro. To investigate its role in vivo, we deleted five residues within the β1‐2 loop of endogenous Delta. Strikingly, this change compromises ligand function. The modified Delta enhances phenotypes produced by Delta loss‐of‐function alleles and suppresses that of Notch alleles. As the modified protein is present on the cell surface in normal amounts, these results argue that C2 domain phospholipid binding is necessary for robust signalling in vivo fine‐tuning the balance of trans and cis ligand–receptor interactions

Somatic mutant clones colonize the human esophagus with age.

The extent to which cells in normal tissues accumulate mutations throughout life is poorly understood. Some mutant cells expand into clones that can be detected by genome sequencing. We mapped mutant clones in normal esophageal epithelium from nine donors (age range, 20 to 75 years). Somatic mutations accumulated with age and were caused mainly by intrinsic mutational processes. We found strong positive selection of clones carrying mutations in 14 cancer genes, with tens to hundreds of clones per square centimeter. In middle-aged and elderly donors, clones with cancer-associated mutations covered much of the epithelium, with NOTCH1 and TP53 mutations affecting 12 to 80% and 2 to 37% of cells, respectively. Unexpectedly, the prevalence of NOTCH1 mutations in normal esophagus was several times higher than in esophageal cancers. These findings have implications for our understanding of cancer and aging.Wellcome Trust Cancer Research U

New insights into the structure, assembly and biological roles of 10–12 nm connective tissue microfibrils from fibrillin-1 studies

The 10–12 nm diameter microfibrils of the extracellular matrix (ECM) impart both structural and regulatory properties to load-bearing connective tissues. The main protein component is the calcium-dependent glycoprotein fibrillin, which assembles into microfibrils at the cell surface in a highly regulated process involving specific proteolysis, multimerization and glycosaminoglycan interactions. In higher metazoans, microfibrils act as a framework for elastin deposition and modification, resulting in the formation of elastic fibres, but they can also occur in elastin-free tissues where they perform structural roles. Fibrillin microfibrils are further engaged in a number of cell matrix interactions such as with integrins, bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β (TGFβ). Fibrillin-1 (FBN1) mutations are associated with a range of heritable connective disorders, including Marfan syndrome (MFS) and the acromelic dysplasias, suggesting that the roles of 10–12 nm diameter microfibrils are pleiotropic. In recent years the use of molecular, cellular and whole-organism studies has revealed that the microfibril is not just a structural component of the ECM, but through its network of cell and matrix interactions it can exert profound regulatory effects on cell function. In this review we assess what is known about the molecular properties of fibrillin that enable it to assemble into the 10–12 nm diameter microfibril and perform such diverse role

An investigation into the molecular mechanism of the fibrillin1 - LTBP1 interaction

Many studies have demonstrated a connection between the fibrillin matrix and TGFβ signalling, but at present the mechanistic basis for this link is unclear. An interaction between the C-terminus of Latent TGFβ Binding Protein 1 (LTBP1) and the N-terminus of fibrillin1 has previously been identified, and may have the potential to directly link the fibrillin matrix to TGFβ signalling. To investigate the structural basis for this interaction, several multi-domain fragments of fibrillin1 and LTBP1 were expressed prokaryotically and refolded in vitro. After initial characterisation to confirm folding, the structure, dynamics, and interdomain interactions of these fragments were investigated in more detail using NMR techniques. Domains in both LTBP1 and fibrillin1 appear to demonstrate folds consistent with homologous structures, and while the LTBP1 C-terminal cbEGF14-TB3-EGF3-cbEGF15 region contains many flexible linkers and few interdomain interactions, the fibrillin1 EGF2-EGF3-hyb1-cbEGF1 region appears rigid, with interfaces forming between all domains present. SPR studies were used to demonstrate binding between distinct LTBP1 and fibrillin fragments, suggesting interactions between multiple domains are involved in the LTBP1-fibrillin1 interaction. The binding sites involved were then mapped to specific residues using HSQC titration studies, and structural models for the LTBP1-fibrillin1 interaction were generated based on these data. Predictions from these models were used to target residues for site-directed mutagenesis, based on their potential involvement in salt bridges, and when certain residues were replaced with those of opposite charge, reductions in binding could be seen in the SPR assay. These key residues were consistent with a particular model of the LTBP1-fibrillin1 interaction, as derived from the HSQC titration data. The conservation of potential binding site residues through deuterostome evolution also supports an important biological role for the LTBP-fibrillin interaction.This thesis is not currently available in OR

C-terminal propeptide is required for fibrillin-1 secretion and blocks premature assembly through linkage to domains cbEGF41-43

Fibrillin microfibrils are 10–12 nm diameter, extracellular matrix assemblies that provide dynamic tissues of metazoan species with many of their biomechanical properties as well as sequestering growth factors and cytokines. Assembly of fibrillin monomers into microfibrils is thought to occur at the cell surface, with initial steps including proprotein processing, multimerization driven by the C terminus, and the head-to-tail alignment of adjacent molecules. At present the mechanisms that regulate microfibril assembly are still to be elucidated. We have used structure-informed protein engineering to create a recombinant, GFP-tagged version of fibrillin-1 (GFP-Fbn) to study this process. Using HEK293T cells transiently transfected with GFP-Fbn constructs, we show that (i) the C-terminal propeptide is an essential requirement for the secretion of full-length fibrillin-1 from cells; (ii) failure to cleave off the C-terminal propeptide blocks the assembly of fibrillin-1 into microfibrils produced by dermal fibroblasts; and (iii) the requirement of the propeptide for secretion is linked to the presence of domains cbEGF41-43, because either deletion or exchange of domains in this region leads to cellular retention. Collectively, these data suggest a mechanism in which the propeptide blocks a key site at the C terminus to prevent premature microfibril assembly

Assembly assay identifies a critical region of human fibrillin-1 required for 10-12 nm diameter microfibril biogenesis.

The human FBN1 gene encodes fibrillin-1 (FBN1); the main component of the 10-12 nm diameter extracellular matrix microfibrils. Marfan syndrome (MFS) is a common inherited connective tissue disorder, caused by FBN1 mutations. It features a wide spectrum of disease severity, from mild cases to the lethal neonatal form (nMFS), that is yet to be explained at the molecular level. Mutations associated with nMFS generally affect a region of FBN1 between domains TB3-cbEGF18-the "neonatal region". To gain insight into the process of fibril assembly and increase our understanding of the mechanisms determining disease severity in MFS, we compared the secretion and assembly properties of FBN1 variants containing nMFS-associated substitutions with variants associated with milder, classical MFS (cMFS). In the majority of cases, both nMFS- and cMFS-associated neonatal region variants were secreted at levels comparable to wild type. Microfibril incorporation by the nMFS variants was greatly reduced or absent compared to the cMFS forms, however, suggesting that nMFS substitutions disrupt a previously undefined site of microfibril assembly. Additional analysis of a domain deletion variant caused by exon skipping also indicates that register in the neonatal region is likely to be critical for assembly. These data demonstrate for the first time new requirements for microfibril biogenesis and identify at least two distinct molecular mechanisms associated with disease substitutions in the TB3-cbEGF18 region; incorporation of mutant FBN1 into microfibrils changing their integral properties (cMFS) or the blocking of wild type FBN1 assembly by mutant molecules that prevents late-stage lateral assembly (nMFS)