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 protostome-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.

Chordin is a cysteine-rich protein which acts as a regulator of bone morphogenetic protein (BMP) signaling in the extracellular matrix. Acting in concert with twisted gastrulation (TWSG1), chordin works as an antagonist of BMP signaling by binding tightly to the growth factor and is a vital component of the network of interactions that establish developmental signaling gradients. Chordin is known to interact with BMP ligands via its four von-Willebrand factor type C domains, but the function of the large central four CHRD domains were previously unknown. Here we show that these domains interact strongly with sulfated glycosaminoglycans (GAGs) and provide evidence for the location of the binding site using X-ray crystallographic analysis combined with mutagenesis and biophysical techniques. Additionally, we report the first recombinant expression and purification of the complete functional chordin, TWSG1, BMP2, BMP7 complex which was used to demonstrate that the four CHRD domains are largely redundant with respect to the role of chordin as an inhibitor of BMP ligands. We therefore propose that the four CHRD domains of chordin have relevance in the diffusion and localization of chordin-TWSG1-BMP complexes at the tissue and organismal level, mediated by their interaction with GAGs or proteoglycans.

Abstract Transforming growth factor-β (TGFβ) is a potent cytokine that controls all aspects of cellular behavior. TGFβ is secreted in complex with its prodomain and latent TGFβ-binding protein-1 (LTBP1), forming the large latent complex (LLC), which through interaction with the extracellular matrix enables integrin-mediated activation. Although TGFβ structures are known, the influence of LTBP1 on the structure and activity of TGFβ is unknown. Here, we report the LLC cryo-EM structure comprising the LTBP1 eight-cysteine domain covalently bound to TGFβ, revealing a hydrophobic interface between TGFβ and LTBP1. Structure-guided mutagenesis shows that the interface is important for complex formation and TGFβ activity. Our structure supports a contralateral domain swapped architecture in the LLC, and simulations show that this architecture requires increased force to overcome barriers for integrin-mediated activation, while the covalent attachment of TGFβ to LTBP1 redistributes force to reduce unfolding barriers. These insights will be important for therapeutic strategies targeting TGFβ.

OBJECTIVE: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. DESIGN:hPSCs were differentiated to chondrocytes via a mesenchymal intermediate, followed by TGFβ3+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. RESULTS: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. CONCLUSION: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.

Understanding osteoporosis requires moving beyond bone mass alone and considering the multiscale determinants of bone quality, from extracellular matrix composition to tissue architecture and mechanical competence. Physical activity is one of the most effective non-pharmacological strategies for preserving skeletal health, suggesting that muscle-derived signals may contribute to the maintenance of bone quality during aging. Among these signals, irisin, an exercise-induced myokine, has emerged as a regulator of bone metabolism with potential benefits for skeletal structure and mechanical performance. However, whether irisin influences bone through direct interactions with matrix components or primarily through cellular and tissue-level responses remains unclear. In this study, we combine in vitro binding assays, in silico modeling, and in vivo preclinical analyses to investigate the effects of irisin on bone across multiple length scales. We examine potential interactions with type I collagen, hydroxyapatite, and heparan sulfate, and evaluate molecular, structural, and mechanical changes in aged bone. Our findings indicate that irisin enhances cortical bone mineral density, improves cortical geometry, and modulates osteogenic and mitochondrial gene expression, whereas direct binding to matrix components appears limited. These results provide new insight into how exercise-mimetic molecules may influence bone quality and support further investigation of irisin-based strategies for age-related osteoporosis.

BACKGROUND:Spontaneous spinal CSF leaks are associated with connective tissue diseases including Marfan syndrome and Loeys-Dietz syndrome, which are caused by mutations in genes that influence the content and integrity of the extracellular matrix. Patients with spontaneous spinal CSF leaks without a defined connective tissue disease diagnosis can show subtle or non-specific connective tissue disease manifestations, suggesting that mutations in extracellular matrix proteins might contribute to more common presentations of this condition. By doing a whole-exome sequencing study, we aimed to elucidate the genetic basis of spontaneous spinal CSF leaks. METHODS:Through retrospective medical record review at Cedars-Sinai Hospital (Los Angeles, USA), we identified 42 individuals who had lateral spontaneous (ie, type 1b) spinal CSF leaks. We did a whole-exome sequencing study in these individuals and compared these data with the results of whole-exome sequencing for three independent control cohorts (2244 unrelated and unaffected adults recruited from various sites in the USA and 714 and 913 individuals recruited to separate sequencing initiatives at the University of Antwerp, Belgium). We used an in-silico prediction tool to establish the location of variants in the tertiary structure of the protein encoded by the top candidate gene. We also tested wild-type and mutant protein fragments for integrin-mediated binding to human dural fibroblasts in vitro. With CRISPR-Cas9 gene editing, we generated three mouse models harbouring different variants in the top candidate gene equivalent to variants found in individuals with type 1b spontaneous spinal CSF leaks and compared them with an established mouse model of Marfan syndrome. Intrathecal infusion testing was used to establish dural integrity and CSF leak properties in these mice. FINDINGS:Whole exome sequencing on 42 unrelated individuals with type 1b spontaneous spinal CSF leaks who had been seen between Jan 1, 2006 and Dec 31, 2019 (35 [83%] were women, seven [17%] were men, 38 [90%] were White, two [5%] were Black, and two [5%] were Hispanic) identified potential causative genes. Nine (21%) of 42 individuals with type 1b spontaneous spinal CSF leak had rare functional variants in FBN2. Significant enrichment in rare functional FBN2 variants was observed on comparison of the patient cohort with the Mendel discovery cohort (177 [8%] of 2244, p=0·041, odds ratio (OR) 3·18 [95% CI 1·50-6·76]) and the Belgian whole-exome (51 [7%] of 714, p=0·004, OR 3·55 [1·61-7·81]) and Belgian thoracic aortic aneurysm and dissection (45 [5%] of 913, p=0·0003, OR 5·26 [2·38-11·66]) validation cohorts. FBN2 variants in individuals with type 1b spontaneous spinal CSF leaks showed a non-random domain distribution in fibrillin-2, with enrichment in TGF-β binding protein-like (TB) domains. Two out of three tested variants reduced fibrillin-2 fragment adhesion to human dural fibroblast in vitro. Mice carrying variants equivalent to the three FBN2 variants in TB domains found in individuals with type 1b spontaneous spinal CSF leak (Fbn2A1052T/+, Fbn2D1581V/+, and Fbn2M2387T/+) showed a predisposition for dural rupture on controlled leak induction. Marfan syndrome mice (Fbn1C1039G/+) had increased meningeal compliance. INTERPRETATION:Rare deleterious variants in FBN2 might cause type 1b spontaneous spinal CSF leak, supporting the integration of FBN2 genetic testing into clinical practice. As in other connective tissue diseases, the disruption of cell adhesion to extracellular matrix proteins might participate in the pathophysiology of spontaneous spinal CSF leaks. The generation of mouse models of spontaneous spinal CSF leaks will help the development of pharmacological therapeutic strategies. FUNDING:The Howard Hughes Medical Institute, the Marfan Foundation, the Pease-Scheeler Fund, and the Biotechnology and Biological Sciences Research Council.

The separation of individual digits is dependent on establishment of digit-interdigit periodicity, remodeling of the interdigital mesenchyme, and invagination of interdigital epithelial tongues. In Protein O-glucosyltransferase 2 and 3 double knockout (Poglut2/3 DKO) mice, digits 2 and 3 are fused, suggesting a defect in one or more processes. POGLUT2/3 add O-linked glucose to epidermal growth factor-like (EGF) repeats. Syndactyly is also observed when genes encoding the POGLUT2/3 substrates fibrillin 2 (FBN2) or both Nidogen 1 and 2 (NID1/2) are knocked out, suggesting that O-glucosylation is important for their function or localization. In this study, we evaluated the distribution of these substrates during digit separation and the effects of the Poglut2/3 DKO on their localization and cell behavior. During digit separation, the FBNs underwent a dramatic reorganization. Aberrant levels and distribution of the FBNs were observed in the Poglut2/3 DKO and microfibrils isolated from Poglut2/3 DKO skin showed altered periodicity in Fibrillin microfibrils. In contrast, the Poglut2/3 DKO had no effect on the levels or localization of NID1. In Poglut2/3 DKOs, bone morphogenetic protein (BMP) signaling was reduced during digit development, especially in the anterior autopod. Early anterior reduction of BMP signaling could potentially affect spacing of digits 2 & 3. While later reduction of BMP signaling in the Poglut2/3 DKO in the digit 2-3 region was likely responsible for defects in clearance of interdigital mesenchyme and interdigital tongue morphogenesis. These results highlight the importance of POGLUT2/3 mediated O-glucosylation for FBN microfibril organization and raise the possibility that O-glucose modulates the biological or physical properties of the FBN microfibril network.

To ensure safety, pharmaceuticals are rigorously tested for lipopolysaccharide (LPS) contamination, as this can trigger severe immune reactions in patients. Low Endotoxin Recovery (LER), describing the masking of spiked LPS controls in Limulus Amebocyte Lysate (LAL) assays, has been associated with the presence of chelating agents and surfactants in pharmaceutical formulations. The addition of excipients, such as Mg2+, have shown the ability to mitigate the effects of LER, however, inconsistencies in various studies regarding the influence of the excipients on LPS aggregate characteristics and LER occurrence hinder a clear understanding of the mechanisms underlying LER. In this study, dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS) were employed to systematically assess the impact of chelating agents, surfactants, and divalent cations on the size and shape of LPS aggregates across various formulations. Our results indicate that surfactant-only formulations generally reduce LPS aggregate size, whereas chelating agent-only formulations do not. Notably, the smallest aggregates were observed when both chelating agents and surfactants were present, with the extent of size reduction being specific to the particular excipients used. Additionally, Mg2+ generally inhibited the excipients' capacity to decrease aggregate size, most effectively in phosphate-containing samples. Despite these variations in size, the overall aggregate shape remained largely unchanged in all formulations. These findings suggest that LPS aggregate size or shape does not distinguish formulations causing LER; instead, factors such as the characteristics of the LPS aggregate surface in different formulations should be explored in the future.

Collagen, a protein known for its long lifespan, is susceptible to accumulation of advanced glycation end products (AGEs) with age. These AGEs are considered markers that indicate the aging severity and influence the mechanics of tissues, leading to fragile bones and hardened skin. While many cross-linking AGEs have been widely studied for their ability to reduce the elasticity of biological tissues, contributing to skin hardening and fragile bones, through strong covalent bonds, non-cross-linking AGEs, or AGE adducts, are typically investigated as indicators of aging or as signaling factors in pathological conditions. However, recent experimental findings have revealed that the number of AGE adducts in aged bone is comparable to enzymatic cross-links, which are significantly more abundant than cross-linking AGEs. Based on these observations, we consider one of the most abundant AGE adducts - carboxymethyllysine (CML) - and employ molecular dynamics simulations to explore its direct impact on the mechanical and conformational properties of single tropocollagen molecules. Our models demonstrate that tropocollagen peptides, constructed based on sequences experimentally identified with sites of CML modifications in type I collagen derived from human cortical bone, exhibit heterogeneous behaviors under tensile testing. Still, most of these modified peptides display compromised structural stability, reduction in structural strength, and diminished energy dissipation ability when tension is applied. This study highlights the potential impact of non-cross-linking AGEs on collagen behavior at molecular scale and provides insights into the mechanisms underlying these modifications. Gaining a deeper understanding of the role of AGE adducts and their contribution to the aging process may pave the way for future solutions in antiaging research.

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-α-inhibitor (IαI) 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 (Δ1-24-PTX3). SAXS determined a length of ∼520 Å 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.

Bone morphogenetic protein 10 (BMP-10) is crucial for endothelial cell signaling via activin receptor-like kinase 1 (ALK1), a pathway central to vascular homeostasis and angiogenesis. Dysregulated BMP-10 signaling contributes to cardiovascular diseases and cancer, highlighting the need to control ALK1-mediated endothelial responses to BMP-10 for therapeutic development. BMP-10 biosynthesis involves processing by proprotein convertases (PPCs) resulting in a non-covalently associated prodomain-growth factor (PD-GF) complex (CPLX), similar to other TGF-β superfamily ligands. However, the molecular requirements for BMP-10 bioactivity remain unclear. We investigated how PPC processing impacts BMP-10 structure, bioactivity, and its interaction with the extracellular matrix (ECM) protein fibrillin-1. Molecular dynamics simulations post-in silico cleavage of the BMP-10 dimer model as well as negative staining and transmission electron microscopy (TEM) revealed that PD processing increases BMP-10 flexibility converting it from a latent wide-angle conformation to a bioactive CPLX which can adopt a V-shape with tighter angle. Only processed BMP-10 demonstrated high potency in HUVEC and C2C12 cells and robust binding to immobilized BMP receptors. Circular dichroism and interaction studies revealed that the N-terminal region of the BMP-10 PD is rich in alpha-helical content, which is essential for efficient complexation with the BMP-10 GF. Binding studies and TEM analyses showed that only the processed BMP-10 CPLX interacts with the N-terminal region of fibrillin-1, causing a conformational change that renders it into a closed ring-shaped conformation. These findings suggest that PD processing induces specific folding events at the PD-GF interface, which is critical for BMP-10 bioactivity and its targeting to the ECM.

Collagen VI links the cell surface to the extracellular matrix to provide mechanical strength to most mammalian tissues, and is linked to human diseases including muscular dystrophy, fibrosis, cardiovascular disease and osteoarthritis. Collagen VI assembles from heterotrimers of three different α-chains into microfibrils, but there are many gaps in our knowledge of the molecular assembly process. Here, we determine the structures of both heterotrimeric mini-collagen VI constructs and collagen VI microfibrils, from mammalian tissue, using cryogenic-electron microscopy. These structures reveal a cysteine-rich coiled coil region involved in trimerisation as well as microfibril assembly. Furthermore, our structures show that pathogenic mutations are located at interaction sites involved in different steps of collagen VI assembly, from the trimeric-coiled coil region that mediates heterotrimerisation, to clusters of mutations in the triple-helical region involved in microfibril formation. Our microfibril structure provides a template for understanding supramolecular assembly, and offers a platform for rationale design of therapeutics for collagen VI pathologies.

Bone Morphogenetic Protein (BMP) signalling is tightly regulated extracellularly by specific protein-protein interactions. During dorsoventral patterning of vertebrate and invertebrate embryos, the conserved regulator Twisted gastrulation (Tsg) precisely modulates BMP signalling by binding Chordin/Short gastrulation (Sog) to promote formation of the inhibitory Tsg-Sog/Chordin-BMP ternary complex. Here we elucidate the mechanism by which Tsg interacts with Sog/Chordin to modulate BMP signalling extracellularly. Using AlphaFold predictions, we identify a Chordin binding epitope in the Tsg C-terminal domain, which we validate using in vitro binding studies with targeted point mutants. Introduction of the equivalent point mutation into Drosophila Tsg, to disrupt Tsg-Sog interaction, results in an unexpectedly mild perturbation to embryonic dorsoventral patterning in vivo , in the form of a shallower BMP gradient. Using binding assays, we provide a molecular explanation for this mild phenotype by showing that the BMP ligand can partially rescue ternary complex formation when the Tsg-Sog interaction is disrupted. Additionally, we show that an evolutionary divergent Tsg C-terminal extension is essential for full Tsg function in Drosophila embryos. Based on these findings we propose that Tsg promotes formation of a Tsg-Sog/Chordin-BMP complex by an avidity-driven mechanism, which will be relevant to a broad range of developmental contexts. ### Competing Interest Statement The authors have declared no competing interest.

Fibrillin-1, an extracellular matrix (ECM) protein encoded by the FBN1 gene, serves as a microfibril scaffold crucial for elastic fiber formation and homeostasis in pliable tissue such as the skin. Aside from causing Marfan syndrome, some mutations in FBN1 result in scleroderma, marked by hardened and thicker skin which limits joint mobility. Here, we describe a tight skin phenotype in the Fbn1G234D/G234D mice carrying a corresponding variant of FBN1 in the hybrid1 domain that was identified in a patient with familial aortic dissection. Unlike scleroderma, skin thickness and collagen fiber abundance do not change in the Fbn1G234D/G234D mutant skin. Instead, increased collagen cross-links were observed. In addition, short elastic fibers were sparsely located underneath the panniculus muscle layer, and an abundance of thin, aberrant elastic fibers was increased within the subcutaneous fascia, which may have tightened skin attachment to the underlying skeletal muscle. Structurally, Fbn1G234D/G234D microfibrils have a disrupted shoulder region that shares similarities with hybrid1 deletion mutant microfibrils. We then demonstrate the consequence of fibrillin-1 G234D mutation on dermal fibroblast functions. Mutant primary fibroblasts produce fewer elastic fibers, exhibit slower migration and increased cell stiffness. Moreover, secretome from mutant fibroblasts are marked by enhanced secretion of ECM, ECM-modifying enzymes, proteoglycans and cytokines, which are pro-tissue repair/fibrogenic. The transcriptome of mutant fibroblasts displays an increased expression of myogenic developmental and immune-related genes. Our study proposes that imbalanced ECM homeostasis due to a fibrillin-1 G234Dmutation impacts fibroblast properties with potential ramifications on skin function.

Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.

Myogenesis is the process that generates multinucleated contractile myofibers from muscle stem cells during skeletal muscle development and regeneration. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD1. Here, we identified the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as part of a Wnt-dependent positive feedback loop, which augmented or sustained MYOD1 expression and thus promoted myoblast differentiation. ADAMTSL2 depletion resulted in severe retardation of myoblast differentiation in vitro and its ablation in myogenic precursor cells resulted in aberrant skeletal muscle architecture. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. We identified the WNT-binding ADAMTSL2 peptide, which was sufficient to promote myogenesis in vitro. Since ADAMTSL2 was previously described as a negative regulator of TGFβ signaling in fibroblasts, ADAMTSL2 now emerges as a signaling hub that could integrate WNT, TGFβ and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.