Biology subjects

Baldock, C.

Publications and source records attributed to Baldock, C..

9 recordsLinked to original sources

bioRxiv2025

Human pluripotent stem cell model of multiple epiphyseal dysplasia with MATN3 mutation identifies altered Matrix organisation and upregulation of the cholesterol biosynthesis pathway

Multiple epiphyseal dysplasia (MED), caused by mutations in MATN3, is a chondrodysplasia affecting the cartilage growth plate and is characterised by delayed epiphyseal ossification, short stature, and early onset osteoarthritis. Here we generated an in vitro human pluripotent stem cell (hPSC) model of cartilage growth-plate development to identify pathogenic mechanisms underlying MED. hPSCs were differentiated to chondrocytes via a mesenchymal intermediate, followed by TGF{beta}3+BMP2 induced chondrogenic pellet culture. MATN3-mutant hPSCs were generated by reprogramming MED patient PBMCs or by CRISPR-Cas9 gene editing to introduce a MATN3 mutation in a hESC line. RNAseq was used to assess chondrogenesis and identify MED pathogenic mechanisms. Transmission electron microscopy (TEM) was used to assess extracellular matrix assembly. The resultant hPSC-derived cartilage pellets displayed a typical cartilage morphology and strongly expressed cartilage matrix markers, e.g., collagen II and matrilin-3. Matrilin-3 protein was detected within both the matrix and cells of heterozygous mutant hPSC-cartilage pellets. RNAseq of mutant hPSC-cartilage pellets revealed significant enrichment for ECM organisation and cholesterol biosynthesis pathway genes as well as sightly increased expression of some unfolded protein response (UPR) marker genes. MATN3 mutant hPSC-derived cartilage pellets displayed abnormal matrix assembly, distended ER, accumulation of lipid droplets, and increased cholesterol content. Our model revealed mutant matrilin-3 induces cholesterol biosynthesis pathway upregulation and abnormal matrix assembly during MED pathogenesis. This study provides new insights into the molecular mechanisms underlying MED and highlights potential therapeutic targets.

cell biology↗

bioRxiv2024

The structural organisation of pentraxin-3 and its interactions with heavy chains of inter-alpha-inhibitor regulate crosslinking of the hyaluronan matrix

Pentraxin-3 (PTX3) is an octameric protein, comprised of eight identical protomers, that has diverse functions in reproductive biology, innate immunity and cancer. PTX3 interacts with the large polysaccharide hyaluronan (HA) to which heavy chains (HCs) of the inter--inhibitor (II) family of proteoglycans are covalently attached, playing a key role in the (non-covalent) crosslinking of HC*HA complexes. These interactions stabilise the cumulus matrix, essential for ovulation and fertilisation in mammals, and are also implicated in the formation of pathogenic matrices in the context of viral lung infections. To better understand the physiological and pathological roles of PTX3 we have analysed how its quaternary structure underpins HA crosslinking via its interactions with HCs. A combination of X-ray crystallography, cryo-electron microscopy (cryo-EM) and AlphaFold predictive modelling revealed that the C-terminal pentraxin domains of the PTX3 octamer are arranged in a central cube, with two long extensions on either side, each formed from four protomers assembled into tetrameric coiled-coil regions, essentially as described by (Noone et al., 2022; doi:10.1073/pnas.2208144119). From crystallography and cryo-EM data, we identified a network of inter-protomer salt bridges that facilitate the assembly of the octamer. Small angle X-ray scattering (SAXS) validated our model for the octameric protein, including the analysis of two PTX3 constructs: a tetrameric Half-PTX3 and a construct missing the 24 N-terminal residues ({Delta}1-24-PTX3). SAXS determined a length of [~]520 [A] for PTX3 and, combined with 3D variability analysis of cryo-EM data, defined the flexibility of the N-terminal extensions. Biophysical analyses revealed that the prototypical heavy chain HC1 does not interact with PTX3 at pH 7.4, consistent with our previous studies showing that, at this pH, PTX3 only associates with HC*HA complexes if they are formed in its presence. However, PTX3 binds to HC1 at acidic pH, and can also be incorporated into pre-formed HC*HA complexes under these conditions. This provides a novel mechanism for the regulation of PTX3-mediated HA crosslinking (e.g., during inflammation), likely mediated by a pH-dependent conformational change in HC1. The PTX3 octamer was found to associate simultaneously with up to eight HC1 molecules and, thus, has the potential to form a major crosslinking node within HC*HA matrices, i.e., where the physical and biochemical properties of resulting matrices could be tuned by the HC/PTX3 composition.

biochemistry↗

bioRxiv2024

An avidity-driven mechanism of extracellular BMP regulation by Twisted gastrulation

During dorsoventral patterning of bilaterian embryos, the conserved regulator Twisted gastrulation (Tsg) modulates BMP signalling by binding Chordin/Short gastrulation (Sog). Here we elucidate the mechanism by which Tsg interacts with Sog/Chordin to promote formation of the inhibitory Tsg-Sog/Chordin-BMP complex and regulate BMP signalling extracellularly. We identify and validate in vitro a hydrophobic interface in the Tsg C-terminal domain that binds Chordin. Mutation of this epitope in Drosophila Tsg (TsgL100A) results in an unexpectedly mild perturbation to embryonic BMP gradient formation. We show that a protosome-specific Tsg C-terminal extension also binds Sog, and the presence of this second binding site allows partial rescue of Sog interaction with TsgL100A in the presence of BMP. Consistent with this, a truncated Tsg protein lacking both Sog binding regions is unable to support BMP gradient formation in vivo. As our data show that disruption of either Sog binding site in Tsg, but not both, can be overcome by Tsg-BMP and Sog-BMP interactions, we present a new avidity-driven mechanism of BMP gradient formation that will be relevant to a broad range of developmental contexts. Summary statementIdentification and mutational analysis of the binding epitopes that mediate Twisted gastrulation interaction with Short gastrulation/Chordin reveals an avidity-based model of embryonic BMP gradient formation.

developmental biology↗

bioRxiv2022

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating Wnt signaling

The formation of multinucleated contractile myofibers from muscle stem cells during myogenesis is indispensable for skeletal muscle formation. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD. However, very few MYOD- regulated effector proteins were shown to be sufficient to promote myogenesis. Here, we identified an unexpected role for the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as a rheostat for Wnt signaling during myogenesis downstream of MYOD. ADAMTSL2 expression was induced during myoblast differentiation and ADAMTSL2 was required for myoblast differentiation. ADAMTSL2 ablation in myogenic precursor cells resulted in aberrant muscle architecture in vivo. The pro-myogenic ADAMTSL2 function was dependent on WNT ligands. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. Finally, we identified a WNT-binding ADAMTSL2 peptide that was sufficient to promote myogenesis. Since ADAMTSL2 was previously described as a negative regulator of TGF{beta} signaling in fibroblasts, ADAMTSL2 now emerges as a signaling node that could integrate and fine-tune WNT, TGF{beta} and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

cell biology↗

bioRxiv2022

Fibrillin microfibril structure identifies long-range effects of inherited pathogenic mutations affecting a key regulatory TGFβ-binding site

Genetic mutations in fibrillin microfibrils cause a range of serious inherited diseases such as Marfan syndrome (MFS) and Weill-Marchesani syndrome (WMS). These diseases typically show major dysregulation of tissue development and growth, particularly in skeletal long bones, but links between the mutations and the diseases are unknown. In this study we reveal the detailed cryo-EM structure of native fibrillin microfibrils from mammalian tissue. The major bead region showed pseudo 8-fold symmetry and a buried protease resistant N-terminal core. Based on this structure, we show a WMS deletion mutant induces a rearrangement with long-range effects blocking interaction with latent TGF{beta}-binding protein (LTBP)-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. These results establish that in complex extracellular protein assemblies, such as in fibrillin, mutations may have long-range structural consequences to disrupt growth factor signalling and cause disease.

biochemistry↗

bioRxiv2022

Modelling the structure of Short Gastrulation and generation of a toolkit for studying its function in Drosophila

A BMP gradient is essential for patterning the dorsal-ventral axis of invertebrate and vertebrate embryos. The extracellular BMP binding protein Short Gastrulation (Sog) in Drosophila plays a key role in BMP gradient formation. In this study, we combine genome editing, structural and developmental approaches to study Sog function in Drosophila. We generate a sog knockout fly stock, which allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, we test the requirement for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, we show that the SogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo. Additionally, we use experimental negative stain EM imaging and hydrodynamic data to validate the AlphaFold structure prediction for Sog. The model suggests a more compact shape than the vertebrate ortholog Chordin and conformational flexibility between the C-terminal von Willebrand C domains. We discuss how this altered compactness may contribute to mechanistic differences in Sog and Chordin function during BMP gradient formation. Summary statementThe authors model the structure of Sog and establish a new sog knockout fly stock that they validate for the testing of specific sog mutations.

developmental biology↗

bioRxiv2021

Proteolysis of fibrillin-2 microfibrils is essential for normal skeletal development

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet specific transition mechanisms ensuring ECM proteostasis and their regulatory impact are poorly defined. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of mouse Adamts6 and Adamts10 mutant embryos, lacking these homologous secreted metalloproteases individually and in combination, along with in vitro analysis of microfibrils, measurement of ADAMTS6-fibrillin affinities and N-terminomics identification of ADAMTS6-cleaved sites, demonstrates a transcriptionally adapted system for fibrillin-2 proteolysis that contributes to postnatal fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development resulting from a drastic reduction of aggrecan, cartilage link protein and impaired BMP, but not TGF{beta} signaling in cartilage. Although ADAMTS6 cleaves fibrillin-1 and fibrillin-2 as well as fibronectin, which provides the initial scaffold for microfibril assembly, primacy of the protease-substrate relationship between ADAMTS6 and fibrillin-2 was unequivocally established by reversal of these defects in Adamts6-/- embryos by genetic reduction of Fbn2, but not Fbn1.

developmental biology↗

bioRxiv2019

How the dual PDZ domain from Postsynaptic density protein 95 clusters ion channels and receptors.

PSD-95 is a member of Membrane Associated Guanylate Kinase class of proteins which form scaffolding interactions with partner proteins including ion and receptor channels. PSD-95 is directly implicated in modulating the electrical responses of excitable cells. The first two PSD-95/Disks Large/Zona Occludens domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of the dual domain in both in apo and ligand-bound form; thermodynamic analysis of ligand association and Small Angle X-ray Scattering of the dual domain in the absence and presence of ligands. These experiments reveal that the ligated double domain forms a scaffold in the complete sense of the word. The concentration of the components in this study is comparable to those found in compartments of excitable cells such as the postsynaptic density and juxta-paranodes of Ranvier. The properties of the dual domain explain the basis of the scaffolding function of PSD-95, and provide a more detailed understanding of the integration of key components of neuronal specializations involved in nervous signal transmission.

biophysics↗

bioRxiv2019

Heavy chain-1 of inter-α-inhibitor has an integrin-like structure with immune regulatory activities

Inter--inhibitor (II) is a proteoglycan essential for mammalian reproduction that also plays a less well-characterised role in inflammation. II is composed of 2 homologous heavy chains (HC1 and HC2) covalently attached to chondroitin sulphate on the bikunin core protein. Prior to ovulation HCs are transferred onto the polysaccharide hyaluronan (HA), thereby stabilising a matrix that is required for fertilisation. Here we show that human HC1 has a structure similar to integrin {beta}-chains and contains a functional MIDAS (metal ion-dependent adhesion site) motif that can mediate self-association of heavy chains, providing a mechanism for matrix crosslinking. Surprisingly, its interaction with RGD-containing integrin ligands, such as vitronectin and the latency-associated peptides of TGF{beta}, occurs in a MIDAS/cation-independent manner. However, HC1 utilises its MIDAS motif to bind to, and inhibit the cleavage of, complement C3, thus identifying it as a novel regulator of innate immunity through inhibition of the alternative pathway C3 convertase.\n\nAbbreviationsADPs, atomic displacement parameter; AUC, analytical ultracentrifugation; CMG2, capillary morphogenesis protein-2; COC, cumulus-oocyte complex; CS, chondroitin sulphate; FB, complement factor B; FnIII; fibronectin type III; HA, hyaluronan; HC, heavy chain; HC*HA, covalent complex of HC with HA; II, inter--inhibitor; ITGA, integrin -chain; ITGB, integrin {beta}-chain; LAP, latency associated peptide; LLC, large latent complex; LTBP, latent TGF{beta} binding protein; MIDAS, metal ion-dependent adhesion site; PI, pre--inhibitor; PTX3, pentraxin-3; rHC1, recombinant HC1; SAXS, small-angle X-ray scattering; SHAP, serum-derived HA binding protein; SLC, small latent complex; TEM8, tumour endothelial marker-8; TGF{beta}, transforming factor {beta}; TSG-6, tumour necrosis factor-stimulated gene-6; TSG-6*HC, covalent complex of TSG-6 and HC; vWFA domain, von Willebrand Factor A domain.

biochemistry↗