Search

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., (© 2025 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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 Fbn1 G234D/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 Fbn1 G234D/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, Fbn1 G234D/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 G234D mutation impacts fibroblast properties with potential ramifications on skin function., Competing Interests: Declaration of competing interest The authors declare no conflict of interest, (Copyright © 2024. Published by Elsevier B.V.)

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., Competing Interests: Declaration of competing interest A.J.D. is a co-founder and employee of Link Biologics Limited, and A.J.D and R.J.D. are both shareholders in the company. Link Biologics is developing a biological drug based on human TSG-6., (Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

Declines in soil multifunctionality (e.gsoil capacity to provide food and energy) are closely related to changes in the soil microbiome (e.g., diversity) Determining ecological drivers promoting such microbiome changes is critical knowledge for protecting soil functions. However, soil-microbe interactions are highly variable within environmental gradients and may not be consistent across studies. Here we propose that analysis of community dissimilarity (β-diversity) is a valuable tool for overviewing soil microbiome spatiotemporal changes. Indeed, β-diversity studies at larger scales (modelling and mapping) simplify complex multivariate interactions and refine our understanding of ecological drivers by also giving the possibility of expanding the environmental scenarios. This study represents the first spatial investigation of β-diversity in the soil microbiome of New South Wales (800,642 km 2 ), Australia. We used metabarcoding soil data (16S rRNA and ITS genes) as exact sequence variants (ASVs) and UMAP (Uniform Manifold Approximation and Projection) as the distance metric. β-Diversity maps (1000-m resolution)-concordance correlations of 0.91-0.96 and 0.91-0.95 for bacteria and fungi, respectively-showed soil biome dissimilarities driven primarily by soil chemistry-pH and effective cation exchange capacity (ECEC)-and cycles of soil temperature-land surface temperature (LST-phase and LST-amplitude). Regionally, the spatial patterns of microbes parallel the distribution of soil classes (e.g., Vertosols) beyond spatial distances and rainfall, for example. Soil classes can be valuable discriminants for monitoring approaches, for example pedogenons and pedophenons. Ultimately, cultivated soils exhibited lower richness due to declines in rare microbes which might compromise soil functions over time., (© 2023 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Genetic mutations in fibrillin microfibrils cause serious inherited diseases, such as Marfan syndrome 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. Here we describe a detailed structural analysis of native fibrillin microfibrils from mammalian tissue by cryogenic electron microscopy. The major bead region showed pseudo eightfold symmetry where the amino and carboxy termini reside. On the basis of this structure, we show that a WMS deletion mutation leads to the induction of a structural rearrangement that blocks interaction with latent TGFβ-binding protein-1 at a remote site. Separate deletion of this binding site resulted in the assembly of shorter fibrillin microfibrils with structural alterations. The integrin α v β 3 -binding site was also mapped onto the microfibril structure. These results establish that in complex extracellular assemblies, such as fibrillin microfibrils, mutations may have long-range structural consequences leading to the disruption of growth factor signaling and the development of disease., (© 2023. The Author(s).)

The ADAMTS superfamily is composed of secreted metalloproteases and structurally related non-catalytic ADAMTS-like proteins. A subset of this superfamily, including ADAMTS6, ADAMTS10 and ADAMTSL2, are involved in elastic fiber assembly and bind to fibrillin and other matrix molecules that regulate the extracellular bioavailability of the potent growth factor TGFβ. Fibrillinopathies, that can also result from mutation of these ADAMTS/L proteins, have been linked to disrupted TGFβ homeostasis. ADAMTS6 and ADAMTS10 are homologous metalloproteases with poorly characterized substrates where ADAMTS10 is thought to process fibrillin-2 and ADAMTS6 latent TGFβ-binding protein (LTBP)-1. In order to understand the contribution of ADAMTS6, and these other members of the ADAMTS/L family, to TGFβ homeostasis, we have analyzed the effects of ADAMTS6, ADAMTS10 and ADAMTSL2 expression on TGFβ activation. We found that their expression increases TGFβ activation in a dose dependent manner, following stimulation with mature TGFβ1. For ADAMTS6, the catalytically active protease is required for effective TGFβ activation, where ADAMTS6 cleaves LTBP3 as well as LTBP1, and binds to the large latent TGFβ complexes of LTBP1 and LTBP3. Furthermore, ADAMTS6 expression increases the mechanotension of cells which results in inactivation of the Hippo Pathway, resulting in an increased translocation of YAP/TAZ complex to the nucleus. Together these findings suggest that when the balance of TGFβ is perturbed ADAMTS6 can influence TGFβ activation via two mechanisms. It directly cleaves the latent TGFβ complexes and also acts indirectly, along with ADAMTS10 and ADAMTSL2, by altering the mechanotension of cells. Together this increases activation of TGFβ from large latent complexes which may contribute to disease pathogenesis., Competing Interests: Declaration of Competing Interest Authors declare that they have no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Aims: To evaluate, in UK acute hospitals, the early implementation of the Recommended Summary Plan for Emergency Care and Treatment (ReSPECT), which embeds cardiopulmonary resuscitation (CPR) recommendations within wider emergency treatment plans. To understand for whom and how the process was being used and the quality of form completion., Methods: A retrospective observational study evaluating emergency care and treatment planning approaches used in acute UK hospitals (2015-2019), and in six English hospital trusts the extent of ReSPECT use, patient characteristics and completion quality in a sample 3000 patient case notes., Results: The use of stand-alone Do Not Attempt Cardiopulmonary Resuscitation forms fell from 133/186 hospitals in 2015 to 64/186 in 2019 (a 38% absolute reduction). ReSPECT accounted for 52% (36/69) of changes. In the six sites, ReSPECT was used for approximately 20% of patients (range 6%-41%). They tended to be older, to have had an emergency medical admission, to have cognitive impairment and a lower predicted 10 year survival. Most (653/706 (92%)) included a 'not for attempted resuscitation' recommendation 551/706 (78%) had at least one other treatment recommendation. Capacity was not recorded on 13% (95/706) of forms; 11% (79/706) did not record patient/family involvement., Conclusions: ReSPECT use accounts for 52% of the change, observed between 2015 and 2019, from using standalone DNACPR forms to approaches embedding DNACPR decisions within in wider emergency care plans in NHS hospitals in the UK. Whilst recommendations include other emergencies most still tend to focus on recommendations relating to CPR. Completion of ReSPECT forms requires improvement., Study Registration: https://www.isrctn.com/ISRCTN11112933., Competing Interests: Declaration of Competing Interest CH, GDP and ZF are members of the ReSPECT natioal working group. CB is Clinical Lead for ReSPECT at the Resuscitation Council UK. CH, GDP, ZF, AMS, FG, MU, RL and DG revieced grants from the UK National Insititue for Health Research during the study. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier B.V.)

Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.

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. This article has an associated First Person interview with the first author of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

The embryonic extracellular matrix (ECM) undergoes transition to mature ECM as development progresses, yet few mechanisms ensuring ECM proteostasis during this period are known. Fibrillin microfibrils are macromolecular ECM complexes serving structural and regulatory roles. In mice, Fbn1 and Fbn2, encoding the major microfibrillar components, are strongly expressed during embryogenesis, but fibrillin-1 is the major component observed in adult tissue microfibrils. Here, analysis of Adamts6 and Adamts10 mutant mouse 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 discovery of ADAMTS6-cleaved sites, identifies a proteostatic mechanism contributing to postnatal fibrillin-2 reduction and fibrillin-1 dominance. The lack of ADAMTS6, alone and in combination with ADAMTS10 led to excess fibrillin-2 in perichondrium, with impaired skeletal development defined by a drastic reduction of aggrecan and cartilage link protein, impaired BMP signaling in cartilage, and increased GDF5 sequestration in fibrillin-2-rich tissue. 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 the defects in Adamts6 -/- embryos by genetic reduction of Fbn2 , but not Fbn1 ., Competing Interests: TM, DM, LW, SC, CG, EC, JM, DR, CL, CB, SA No competing interests declared, (© 2022, Mead et al.)

As essential components of our connective tissues, elastic fibres give tissues such as major blood vessels, skin and the lungs their elasticity. Their formation is complex and co-ordinately regulated by multiple factors. In this review, we describe key players in elastogenesis: fibrillin-1, tropoelastin, latent TGFβ binding protein-4, and fibulin-4 and -5. We summarise their roles in elastogenesis, discuss the effect of their mutations on relevant diseases, and describe their interactions involved in forming the elastic fibre network. Moreover, we look into their roles in wound repair for a better understanding of their potential application in tissue regeneration.

TGFβ superfamily members are potent growth factors in the extracellular matrix with essential roles in all aspects of cellular behaviour. Latent TGFβ binding proteins (LTBPs) are co-expressed with TGFβ, essential for correct folding and secretion of the growth factor, to form large latent complexes. These large latent complexes bind extracellular proteins such as fibrillin for sequestration of TGFβ in the matrix, essential for normal tissue function, and dysregulated TGFβ signalling is a hallmark of many fibrillinopathies. Transglutaminase-2 (TG2) cross-linking of LTBPs is known to play a role in TGFβ activation but the underlying molecular mechanisms are not resolved. Here we show that fibrillin is a matrix substrate for TG2 and that TG2 cross-linked complexes can be formed between fibrillin and LTBP-1 and -3, and their latent TGFβ complexes. The structure of the fibrillin-LTBP1 complex shows that the two elongated proteins interact in a perpendicular arrangement which would allow them to form distal interactions between the matrix and the cell surface. Formation of the cross-link with fibrillin does not change the interaction between latent TGFβ and integrin αVβ6 but does increase TGFβ activation in cell-based assays. The activating effect may be due to direction of the latent complexes to the cell surface by fibrillin, as competition with heparan sulphate can ameliorate the activating effect. Together, these data support that TGFβ activation can be enhanced by covalent tethering of LTBPs to the matrix via fibrillin., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Although carpal tunnel syndrome (CTS) is the most common form of peripheral entrapment neuropathy, its pathogenesis remains largely unknown. An estimated heritability index of 0.46 and an increased familial occurrence indicate that genetic factors must play a role in the pathogenesis., Methods and Results: We report on a family in which CTS occurred in subsequent generations at an unusually young age. Additional clinical features included brachydactyly and short Achilles tendons resulting in toe walking in childhood. Using exome sequencing, we identified a heterozygous variant (c.5009T>G; p.Phe1670Cys) in the fibrillin-2 ( FBN2 ) gene that co-segregated with the phenotype in the family. Functional assays showed that the missense variant impaired integrin-mediated cell adhesion and migration. Moreover, we observed an increased transforming growth factor-β signalling and fibrosis in the carpal tissues of affected individuals. A variant burden test in a large cohort of patients with CTS revealed a significantly increased frequency of rare (6.7% vs 2.5%-3.4%, p<0.001) and high-impact (6.9% vs 2.7%, p<0.001) FBN2 variants in patient alleles compared with controls., Conclusion: The identification of a novel FBN2 variant (p.Phe1670Cys) in a unique family with early onset CTS, together with the observed increased frequency of rare and high-impact FBN2 variants in patients with sporadic CTS, strongly suggest a role of FBN2 in the pathogenesis of CTS., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Latent TGFβ binding protein-4 (LTBP4) is a multi-domain glycoprotein, essential for regulating the extracellular bioavailability of TGFβ and assembly of elastic fibre proteins, fibrillin-1 and tropoelastin. LTBP4 mutations are linked to autosomal recessive cutis laxa type 1C (ARCL1C), a rare congenital disease characterised by high mortality and severely disrupted connective tissues. Despite the importance of LTBP4, the structure and molecular consequences of disease mutations are unknown. Therefore, we analysed the structural and functional consequences of three ARCL1C causing point mutations which effect highly conserved cysteine residues. Our structural and biophysical data show that the LTBP4 N- and C-terminal regions are monomeric in solution and adopt extended conformations with the mutations resulting in subtle changes to their conformation. Similar to LTBP1, the N-terminal region is relatively inflexible, whereas the C-terminal region is flexible. Interaction studies show that one C-terminal mutation slightly decreases binding to fibrillin-1. We also found that the LTBP4 C-terminal region directly interacts with tropoelastin which is perturbed by both C-terminal ARCL1C mutations, whereas an N-terminal mutation increased binding to fibulin-4 but did not affect the interaction with heparan sulphate. Our results suggest that LTBP4 mutations contribute to ARCL1C by disrupting the structure and interactions of LTBP4 which are essential for elastogenesis in a range of mammalian connective tissues., Competing Interests: AW is the founder of Elastagen which was sold to Allergan. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Alanazi, Lockhart-Cairns, Cain, Jowitt, Weiss and Baldock.)

Elastic tissues owe their functional properties to the composition of their extracellular matrices, particularly the range of extracellular, multidomain extensible elastic fibre and microfibrillar proteins. These proteins include elastin, fibrillin, latent TGFβ binding proteins (LTBPs) and collagens, where their biophysical and biochemical properties not only give the matrix structural integrity, but also play a vital role in the mechanisms that underlie tissue homeostasis. Thus far structural information regarding the structure and hierarchical assembly of these molecules has been challenging and the resolution has been limited due to post-translational modification and their multidomain nature leading to flexibility, which together result in conformational and structural heterogeneity. In this review, we describe some of the matrix proteins found in elastic fibres and the new emerging techniques that can shed light on their structure and dynamic properties., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Authors.)

Bone morphogenic proteins (BMPs) are important growth regulators in embryogenesis and postnatal homeostasis. Their tight regulation is crucial for successful embryonic development as well as tissue homeostasis in the adult organism. BMP inhibition by natural extracellular biologic antagonists represents the most intensively studied mechanistic concept of BMP growth factor regulation. It was shown to be critical for numerous developmental programs, including germ layer specification and spatiotemporal gradients required for the establishment of the dorsal-ventral axis and organ formation. The importance of BMP antagonists for extracellular matrix homeostasis is illustrated by the numerous human connective tissue disorders caused by their mutational inactivation. Here, we will focus on the known functional interactions targeting BMP antagonists to the ECM and discuss how these interactions influence BMP antagonist activity. Moreover, we will provide an overview about the current concepts and investigated molecular mechanisms modulating BMP inhibitor function in the context of development and disease., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

Background: The Recommended Summary Plan for Emergency Care and Treatment (ReSPECT) is an emergency care and treatment planning (ECTP) process, developed to offer a patient-centred approach to deciding about and recording treatment recommendations. Conversations between clinicians and patients or their representatives are central to the ReSPECT process. This study aims to understand why, when, and how ReSPECT conversations unfold in practice., Methods: ReSPECT conversations were observed in hospitals within six acute National Health Service (NHS) trusts in England; the clinicians who conducted these conversations were interviewed. Following observation-based thematic analysis, five ReSPECT conversation types were identified: resuscitation and escalation; confirmation of decision; bad news; palliative care; and clinical decision. Interview-based thematic analysis examined the reasons and prompts for each conversation type, and the level of detail and patient engagement in these different conversations., Results: Whereas resuscitation and escalation conversations concerned possible futures, palliative care and bad news conversations responded to present-tense changes. Conversations were timed to respond to organisational, clinical, and patient/relative prompts. While bad news and palliative care conversations included detailed discussions of treatment options beyond CPR, this varied in other conversation types. ReSPECT conversations varied in doctors' engagement with patient/relative preferences, with only palliative care conversations consistently including an open-ended approach., Conclusions: While ReSPECT supports holistic, person-centred, anticipatory decision-making in some situations, a gap remains between the ReSPECT's aims and their implementation in practice. Promoting an understanding and valuing of the aims of ReSPECT among clinicians, supported by appropriate training and structural support, will enhance ReSPECT conversations., (Copyright © 2021. Published by Elsevier B.V.)

Since their discovery as pluripotent cytokines extractable from bone matrix, it has been speculated how bone morphogenetic proteins (BMPs) become released and activated from the extracellular matrix (ECM). In contrast to TGF-βs, most investigated BMPs are secreted as bioactive prodomain (PD)-growth factor (GF) complexes (CPLXs). Recently, we demonstrated that PD-dependent targeting of BMP-7 CPLXs to the extracellular fibrillin microfibril (FMF) components fibrillin-1 and -2 represents a BMP sequestration mechanism by rendering the GF latent. Understanding how BMPs become activated from ECM scaffolds such as FMF is crucial to elucidate pathomechanisms characterized by aberrant BMP activation and ECM destruction. Here, we describe a new MMP-dependent BMP-7 activation mechanism from ECM-targeted pools via specific PD degradation. Using Edman sequencing and mutagenesis, we identified a new and conserved MMP-13 cleavage site within the BMP-7 PD. A degradation screen with different BMP family PDs and representative MMP family members suggested utilization of the identified site in a general MMP-driven BMP activation mechanism. Furthermore, sandwich ELISA and solid phase cleavage studies in combination with bioactivity assays, single particle TEM, and in silico molecular docking experiments provided evidence that PD cleavage by MMP-13 leads to BMP-7 CPLX disintegration and bioactive GF release., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Elastic fibers are an important component of the extracellular matrix, providing stretch, resilience, and cell interactivity to a broad range of elastic tissues. Elastin makes up the majority of elastic fibers and is formed by the hierarchical assembly of its monomer, tropoelastin. Our understanding of key aspects of the assembly process have been unclear due to the intrinsic properties of elastin and tropoelastin that render them difficult to study. This review focuses on recent developments that have shaped our current knowledge of elastin assembly through understanding the relationship between tropoelastin's structure and function., Competing Interests: AW is the Scientific Founder of Elastagen Pty. Ltd., which was sold to Allergan, now a division of AbbVie. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ozsvar, Yang, Cain, Baldock, Tarakanova and Weiss.)

Protochlorophyllide oxidoreductase (POR) catalyses reduction of protochlorophyllide (Pchlide) to chlorophyllide, a light-dependent reaction of chlorophyll biosynthesis. POR is also important in plant development as it is the main constituent of prolamellar bodies in etioplast membranes. Prolamellar bodies are highly organised, paracrystalline structures comprising aggregated oligomeric structures of POR-Pchlide-NADPH complexes. How these oligomeric structures are formed and the role of Pchlide in oligomerisation remains unclear. POR crystal structures highlight two peptide regions that form a 'lid' to the active site, and undergo conformational change on binding Pchlide. Here, we show that Pchlide binding triggers formation of large oligomers of POR using size exclusion chromatography. A POR 'octamer' has been isolated and its structure investigated by cryo-electron microscopy at 7.7 Å resolution. This structure shows that oligomer formation is most likely driven by the interaction of amino acid residues in the highly conserved lid regions. Computational modelling indicates that Pchlide binding stabilises exposure of hydrophobic surfaces formed by the lid regions, which supports POR dimerisation and ultimately oligomer formation. Studies with variant PORs demonstrate that lid residues are involved in substrate binding and photocatalysis. These highly conserved lid regions therefore have a dual function. The lid residues position Pchlide optimally to enable photocatalysis. Following Pchlide binding, they also enable POR oligomerisation - a process that is reversed through subsequent photocatalysis in the early stages of chloroplast development., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Elastic fibres are essential components of all mammalian elastic tissues such as blood vessels, lung and skin, and are critically important for the mechanical properties they endow. The main components of elastic fibres are elastin and fibrillin, where correct formation of elastic fibres requires a fibrillin microfibril scaffold for the deposition of elastin. It has been demonstrated previously that the interaction between fibrillin and tropoelastin, the elastin precursor, increases the rate of assembly of tropoelastin. Furthermore, tropoelastin and fibrillin can be cross-linked by transglutaminase-2, but the function of cross-linking on their elastic properties is yet to be elucidated. Here we show that transglutaminase cross-linking supports formation of a 1:1 stoichiometric fibrillin-tropoelastin complex. SAXS data show that the complex retains features of the individual proteins but is elongated supporting end-to-end assembly. Elastic network models were constructed to compare the dynamics of tropoelastin and fibrillin individually as well as in the cross-linked complex. Normal mode analysis was performed to determine the structures' most energetically favourable, biologically accessible motions which show that within the complex, tropoelastin is less mobile and this molecular stabilisation extends along the length of the tropoelastin molecule to regions remote from the cross-linking site. Together, these data suggest a long-range stabilising effect of cross-linking that occurs due to the covalent linkage of fibrillin to tropoelastin. This work provides insight into the interactions of tropoelastin and fibrillin and how cross-link formation stabilises the elastin precursor so it is primed for elastic fibre assembly., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

PSD-95 is a member of the membrane-associated guanylate kinase class of proteins that forms 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 (PDZ) domains of PSD-95 have been shown to be the key component in the formation of channel clusters. We report crystal structures of this dual domain in both apo- and ligand-bound form: thermodynamic analysis of the 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 three-dimensional scaffold that can be described by a space group. The concentration of the components in this study is comparable with those found in compartments of excitable cells such as the postsynaptic density and juxtaparanodes of Ranvier. These in vitro experiments inform the basis of the scaffolding function of PSD-95 and provide a detailed model for scaffold formation by the PDZ domains of PSD-95., (Copyright © 2020 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Inter-α-inhibitor is a proteoglycan essential for mammalian reproduction and also plays a less well-characterized role in inflammation. It comprises two homologous "heavy chains" (HC1 and HC2) covalently attached to chondroitin sulfate on the bikunin core protein. Before ovulation, HCs are transferred onto the polysaccharide hyaluronan (HA) to form covalent HC·HA complexes, thereby stabilizing an extracellular matrix around the oocyte required for fertilization. Additionally, such complexes form during inflammatory processes and mediate leukocyte adhesion in the synovial fluids of arthritis patients and protect against sepsis. Here using X-ray crystallography, we show that human HC1 has a structure similar to integrin β-chains, with a von Willebrand factor A domain containing a functional metal ion-dependent adhesion site (MIDAS) and an associated hybrid domain. A comparison of the WT protein and a variant with an impaired MIDAS (but otherwise structurally identical) by small-angle X-ray scattering and analytical ultracentrifugation revealed that HC1 self-associates in a cation-dependent manner, providing a mechanism for HC·HA cross-linking and matrix stabilization. Surprisingly, unlike integrins, HC1 interacted with RGD-containing ligands, such as fibronectin, vitronectin, and the latency-associated peptides of transforming growth factor β, in a MIDAS/cation-independent manner. However, HC1 utilizes its MIDAS motif to bind to and inhibit the cleavage of complement C3, and small-angle X-ray scattering-based modeling indicates that this occurs through the inhibition of the alternative pathway C3 convertase. These findings provide detailed structural and functional insights into HC1 as a regulator of innate immunity and further elucidate the role of HC·HA complexes in inflammation and ovulation., (© 2020 Briggs et al.)