MFAP4 (microfibrillar-associated protein 4) is an extracellular glycoprotein found in elastic fibers without a clearly defined role in elastic fiber assembly. In the present study, we characterized molecular interactions between MFAP4 and elastic fiber components. We established that MFAP4 primarily assembles into trimeric and hexameric structures of homodimers. Binding analysis revealed that MFAP4 specifically binds tropoelastin and fibrillin-1 and -2, as well as the elastin cross-linking amino acid desmosine, and that it co-localizes with fibrillin-1-positive fibers in vivo. Site-directed mutagenesis disclosed residues Phe 241 and Ser 203 in MFAP4 as being crucial for type I collagen, elastin, and tropoelastin binding. Furthermore, we found that MFAP4 actively promotes tropoelastin self-assembly. In conclusion, our data identify MFAP4 as a new ligand of microfibrils and tropoelastin involved in proper elastic fiber organization.Elastic fibers are key extracellular matrix structural elements of connective tissues that undergo repeated stretch, such as large arteries and the lung (1). The fibers consist of two major components: an amorphous elastin core surrounded by a sheath of fibrillin-rich microfibrils (2). Elastin is a highly hydrophobic polymer of the soluble precursor tropoelastin (3). Tropoelastin is known to undergo a self-assembly process known as coacervation (4), often believed to be a first step in the process of elastic fiber maturation. Because of the high content of lysine residues within the tropoelastin sequence, its assembly into a polymeric form is stabilized by formation of desmosine crosslinks, catalyzed by the lysyl oxidase (LOX) 3 enzyme family (5).Microfibrils, the other major component of elastic fibers, provide the structural scaffold for the deposition of elastin globules. They consist primarily of fibrillin-1 and fibrillin-2, large glycoproteins with a high degree of homology (6). Apart from fibrillins, numerous accessory proteins have been shown to associate with microfibrils or elastin and promote formation of mature fibers, including fibulins and microfibril-associated glycoproteins (MAGPs) (7-10). The importance of proper elastogenesis has been underscored by gene deficiency studies: mice lacking elastin, LOX, or fibrillin-1 die shortly after birth because of vascular abnormalities (11-13).MFAP4 (microfibrillar-associated protein 4) is an extracellular matrix protein belonging to the fibrinogen-related domain (FReD) family. The family includes several proteins engaged in tissue homeostasis and innate immunity, such as FIBCD1 (fibrinogen C domain-containing 1), ficolins, and angiopoietins (14 -16). The crystal structure of the FReD of several family members has been solved (17-19). The ligand-binding site, designated S1, is described in all the proteins and is located in close proximity to the calcium-binding site. MFAP4 has been reported to form homodimeric structures that further oligomerize, but its definite oligomerization pattern has not been established (20).MFAP4 is conside...

TGFB1 (transforming growth factor beta 1) is a potent cytokine playing a driving role in development, fibrosis and cancer. It is synthesized as prodomain-growth factor complex that requires tethering to LTBP (latent transforming growth factor beta binding protein) for efficient secretion into the extracellular space. Upon release, this large latent complex is sequestered by anchorage to extracellular matrix (ECM) networks, from which the mature growth factor needs to be activated in order to reach its receptors and initiate signaling. Here, we uncovered a novel intracellular secretion pathway by which the latent TGFB1 complex reaches the plasma membrane and is released from fibroblasts, the key effector cells during tissue repair, fibrosis and in the tumor stroma. We show that secretion of latent TGFB1, but not of other selected cytokines or of bulk cargo, is regulated by fibroblast-ECM communication through ILK (integrin linked kinase) that restricts RHOA activity by interacting with ARHGAP26/GRAF1. Latent TGFB1 interacts with GORASP2/GRASP55 and is detected inside MAP1LC3-positive autophagosomal intermediates that are secreted by a RAB8A-dependent pathway. Interestingly, TGFB1 secretion is fully abrogated in human and murine fibroblasts and macrophages that lack key components of the autophagic machinery. Our data demonstrate an unconventional secretion mode of TGFB1 adding another level of control of its bioavailability and activity in order to effectively orchestrate cellular programs prone to dysregulation as seen in fibrosis and cancer.

Bone morphogenetic proteins (BMPs) orchestrate key cellular events, such as proliferation and differentiation, in development and homeostasis. Extracellular antagonists, such as chordin, are essential regulators of BMP signaling. Chordin binds to BMPs blocking interaction with receptors, and cleavage by tolloid proteinases is thought to relieve this inhibition. A model has been previously proposed where chordin adopts a horseshoe-like arrangement enabling BMP binding cooperatively by terminal domains (1). Here, we present the nanoscale structure of human chordin using electron microscopy, small angle X-ray scattering, and solution-based biophysical techniques, which together show that chordin indeed has a compact horseshoe-shaped structure. Chordin variants were used to map domain locations within the chordin molecule. The terminal BMP-binding domains protrude as prongs from the main body of the chordin structure, where they are well positioned to interact with the growth factor. The spacing provided by the chordin domains supports the principle of a cooperative BMP-binding arrangement that the original model implied in which growth factors bind to both an N-and C-terminal von Willebrand factor C domain of chordin. Using binding and bioactivity assays, we compared full-length chordin with two truncated chordin variants, such as those produced by partial tolloid cleavage. Cleavage of either terminal domain has little effect on the affinity of chordin for BMP-4 and BMP-7 but C-terminal cleavage increases the efficacy of chordin as a BMP-4 inhibitor. Together these data suggest that partial tolloid cleavage is insufficient to ablate BMP inhibition and the C-terminal chordin domains play an important role in BMP regulation.

Twisted gastrulation (Tsg) and chordin are secreted glycoproteins that function together as BMP (bone morphogenetic protein) antagonists to regulate BMP growth factor signalling. Chordin binds to BMPs, preventing them from interacting with their receptors and Tsg is known to strengthen this inhibitory complex. Tsg also acts as a BMP agonist by promoting cleavage of chordin by tolloid-family proteinases. Here we explore the structural mechanism through which Tsg exerts this dual activity. We have characterized the nanoscale structure of human Tsg using in-solution biomolecular analysis and show that Tsg is a globular monomer with a flattened cross shape. Tsg has a high proportion of N-linked glycans, in relation to its molecular weight, which supports a role in solubilising BMPs. Tsg binds with high affinity to the C-terminal region of chordin and was also able to inhibit BMP-7 signalling directly but did not have an effect on BMP-4 signalling. Although both Tsg and mammalian tolloid are involved in chordin cleavage, no interaction could be detected between them using surface plasmon resonance. Together these data suggest that Tsg functions as a BMP-agonist by inducing conformational change in chordin making it more susceptible to tolloid cleavage and as a BMP-antagonist either independently or via a chordin-mediated mechanism. Following single cleavage of chordin by tolloids, Tsg continues to strengthen the inhibitory complex, supporting a role for partially cleaved chordin in BMP regulation.

Flotillin-1 and flotillin-2 are highly conserved, membrane-microdomainassociated proteins that have been shown to be involved in signal transduction, membrane trafficking and cell adhesion. Upon growth factor stimulation, flotillins are tyrosine phosphorylated and become endocytosed from the plasma membrane into endosomes from which they are recycled back to the plasma membrane. Although a role for flotillin-1 in the endocytosis of certain cargo proteins has been suggested, it is not known how the growthfactor-induced endocytosis of flotillins is regulated and which endocytosis pathway is used. However, this is likely to be different from the pathway used by flotillin-dependent cargo. In this study, we have addressed the mechanistic details of flotillin trafficking during growth factor signaling. We show that dynamin-2 activity is required for the uptake of flotillins from the plasma membrane upon epidermal growth factor stimulation, and inhibition of dynamin-2 GTPase activity impairs flotillin endocytosis. Surprisingly, recycling of flotillins from endosomes to the plasma membrane appears to require both dynamin-2 and clathrin. Upon overexpression of dynamin-2 mutants or depletion of clathrin heavy chain, flotillins are permanently trapped in endosomes. These data show that clathrin and dynamin are required for the endosomal sorting of flotillins, and the study provides a mechanistic dissection of the thus far poorly characterized endosomal trafficking of flotillins. Structured digital abstract• flotillin-1 and flotillin-2 colocalize by fluorescence microscopy (View interaction) • CHC and dyn2 colocalize by fluorescence microscopy (View interaction) • LAMP3 and flotillin-2 colocalize by fluorescence microscopy (View interaction)

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Bone morphogenetic proteins (BMPs) are essential signalling molecules involved in developmental and pathological processes and are regulated in the matrix by secreted glycoproteins. One such regulator is BMP-binding endothelial cell precursor-derived regulator (BMPER) which can both inhibit and enhance BMP signalling in a context and concentration-dependent manner. Twisted gastrulation (Tsg) can also promote or ablate BMP activity but it is unclear whether Tsg and BMPER directly interact and thereby exert a synergistic function on BMP signalling. Here, we show that human BMPER binds to Tsg through the N-terminal BMP-binding region which alone more potently inhibits BMP-4 signalling than full-length BMPER. Additionally, BMPER and Tsg cooperatively inhibit BMP-4 signalling suggesting a synergistic function to dampen BMP activity. Furthermore, full-length BMPER is targeted to the plasma membrane via binding of its C-terminal region to cell surface heparan sulphate proteoglycans but the active cleavage fragment is diffusible. Small-angle X-ray scattering and electron microscopy show that BMPER has an elongated conformation allowing the N-terminal BMP-binding and C-terminal cell-interactive regions to be spatially separated. To gain insight into the regulation of BMPER bioavailability by internal cleavage, a disease-causing BMPER point mutation, P370L, previously identified in the acid-catalysed cleavage site, was introduced. The mutated protein was secreted but the mutation prevented intracellular cleavage resulting in a lack of bioactive cleavage fragment. Furthermore, mutant BMPER was extracellularly cleaved at a downstream site presumably becoming available due to the mutation. This susceptibility to extracellular proteases and loss of bioactive N-terminal cleavage fragment may result in loss of BMPER function in disease.

Chordin-mediated regulation of bone morphogenetic protein (BMP) family growth factors is essential in early embryogenesis and adult homoeostasis. Chordin binds to BMPs through cysteine-rich von Willebrand factor type C (vWC) homology domains and blocks them from interacting with their cell surface receptors. These domains also self-associate and enable chordin to target related proteins to fine-tune BMP regulation. The chordin–BMP inhibitory complex is strengthened by the secreted glycoprotein twisted gastrulation (Tsg); however, inhibition is relieved by cleavage of chordin at two specific sites by tolloid family metalloproteases. As Tsg enhances this cleavage process, it serves a dual role as both promoter and inhibitor of BMP signalling. Recent developments in chordin research suggest that rather than simply being by-products, the cleavage fragments of chordin continue to play a role in BMP regulation. In particular, chordin cleavage at the C-terminus potentiates its anti-BMP activity in a type-specific manner.