At its best, it is exhilarating to make unexpected discoveries when addressing carefully formed scientific hypotheses. This review depicts my scientific journey in the field of extracellular matrix biology, and more specifically in collagen research, starting in 1978 and continuing with exciting findings up to the present day. While recounting my early work on the enzymes of collagen biosynthesis, the focus will be on our discoveries of new types of nonfibrillar collagen: type XIII collagen, belonging to the MACIT subgroup among the collagen family of proteins, and types XV and XVIII collagens, constituting the multiplexin subgroup. We have investigated these collagens through molecular biological approaches in order to define their primary structures, and through biochemical and cell biological work to understand their special molecular properties. Furthermore, the generation of many mouse models has led us to exciting studies of the roles of these collagens in adipose tissue, bone, eye, heart, kidney, liver, peripheral nerves, skin, and cancer models, although it has of course also been rather daunting in terms of choosing the correct approach for each tissue. The work on animal models has nevertheless resulted in a broad understanding of the in vivo significance of these collagens, forming a fruitful basis for studying their relevance to human diseases, including malignant processes. Our conclusions have been that these collagens can contribute to the stability of the extracellular matrix and tissue structures, e.g., the basement membrane and the adjacent fibrillar matrix in the case of the multiplexins and the motor synapse in the case of the MACIT type XIII collagen, and more unexpectedly, that they possess major roles as extrinsic regulators of the fates and functions of cells.
Decellularized extracellular matrix materials have been widely studied for tissue engineering and regenerative medicine (TERM) applications, largely because of their intrinsic bioactivity and immunomodulatory potentials. These properties confer decellularized extracellular matrix biomaterials a biological advantage over other biomaterials, especially synthetic ones, leading to several successful applications in TERM. While the complex composition of decellularized materials is well known and thought to play a role in providing the regenerative advantage, the fine mechanisms laying behind their bioactivity and immunomodulation were not fully understood yet. In the last decade, researchers have discovered a novel component in decellularized extracellular matrix materials: the matrix bound nanovesicle (MBV). This newly described type of extracellular vesicle is characterized by a tight relation to the extracellular matrix, differently from other liquid phase vesicles, and presents a unique tissue specific cargo, thought to be secreted by cells for specific cell signalling purposes. Although other extracellular vesicles subtypes have been extensively studied in past years, MBVs are different in many ways, making this research field noticeably young. Major bioactivity and immune modulating ability are key features of MBVs that were evident right from the first research works. However, to understand how MBVs can recapitulate and confer decellularized biomaterials with their signature biological performance, they are being characterized in depth. In particular, their rich and varied cargo is being explored, which has shown to play a fundamental role in MBVs' biological potential. This discovery not only revolutionized the look on decellularized extracellular matrix materials, but it also opened the way for research on a novel type of biomaterial, with plenty potential in therapeutical and regenerative applications. This review presents in detail what has been discovered up to now on MBVs, their properties, biological roles, and potential in TERM.
A subset of rheumatoid arthritis (RA) is the production of autoantibodies, including antibodies to citrullinated proteins (ACPA) and antibodies to type II collagen (AC2A). Type II collagen (COL2) is the major protein in joint cartilage and is a target of arthritogenic autoantibodies. We could confirm that sera from RA patients react with both citrullinated and native triple-helical COL2 epitopes. We examined the modulation of COL2 processing by matrix metalloproteinase 13 (MMP-13), the main collagenase responsible for degradation of articular cartilage. Anti-COL2 antibodies (AC2A) targeting the C1 epitope (residues 359-363) partially inhibited intact COL2 and fragment hydrolysis, resulting in two distinct fragments in the 25-30 kDa range. The AC2A targeting the E10 epitope (residues 777-783, the region where MMP-13 initially cleaves COL2) partially inhibited intact COL2 and fragment hydrolysis, resulting in a distinct fragment of ∼30 kDa. The AC2As targeting the F4 epitope (residues 932-936) partially inhibited collagen fragment hydrolysis, resulting in four distinct fragments in the 20-30 kDa range. Sequencing of isolated fragments revealed 14 terminated cleavage sites. Citrullination of the COL2 cleavage site reduced MMP-13 efficiency, which should further exacerbate fragment production rather than complete digestion. The results indicated that, under normal maintenance, MMP-13 cleaves COL2 initially at the 775-776 bond, followed by further digestion of COL2 fragments. Citrullination slows the initial processing of COL2 by MMP-13. In concert, AC2As inhibit the action of MMP-13 at different stages, resulting in production of collagen fragments differing in composition encountered under normal circumstances. The abnormal COL2 fragments could activate the immune system to be more pathogenic or regulatory as well as modify chondrocyte functions, and thereby play a role in the initiation of RA.
Collagen VI is a heterotrimeric, ubiquitously expressed microfibrillar collagen with a complex intracellular and extracellular assembly process. In addition to a short collagenous region, it is primarily composed of von Willebrand factor A (VWA) domains. Notably, only the C-terminal end of the α3 chain contains other domain types, including a Kunitz-like C5 domain, which has been reported to be necessary for microfibril formation, to function as a matrikine and exhibit biomarker properties. This region of the α3 chain undergoes proteolytic processing, with cleavage sites identified for proprotein convertases, matrix metalloproteinases (MMPs), and bone morphogenetic protein 1 (BMP1). Cleavage by furin-like convertases results in the generation of a mature collagen VI α3 chain lacking its 70 kDa C2-C5 domains. Here, we provide the first characterization of the functional significance of the furin-like cleavage site, demonstrating that while it is constitutively used, it is not essential for collagen VI assembly, microfibril formation, or skeletal muscle function under physiological conditions, likely due to the presence of redundant cleavage sites. We also present an initial characterization of the biological activity of the released fragments on myoblast cultures showing that they do not affect C2C12 myoblast behaviour or differentiation. These findings deepen our understanding of α3 chain processing and highlight its potential significance for collagen VI assembly and function, including the generation of peptides with potential biomarker and biological activity properties.
Collagens are fundamental components of extracellular matrices, requiring precise intracellular post-translational modifications for proper function. Among the modifications, prolyl 4-hydroxylation is critical to stabilise the collagen triple helix. In humans, this reaction is mediated by collagen prolyl 4-hydroxylases (P4Hs). While humans possess three genes encoding these enzymes (P4H⍺s), Drosophila melanogaster harbour at least 26 candidates for collagen P4H⍺s despite its simple genome, and it is poorly understood which of them are actually working on collagen in the fly. In this study, we addressed this question by carrying out thorough bioinformatic and biochemical analyses. We demonstrate that among the 26 potential collagen P4H⍺s, PH4⍺EFB shares the highest homology with vertebrate collagen P4H⍺s. Furthermore, while collagen P4Hs and their substrates must exist in the same cells, our transcriptomic analyses at the tissue and single cell levels showed a global co-expression of PH4⍺EFB but not the other P4H⍺-related genes with the collagen IV genes. Moreover, expression of PH4⍺EFB during embryogenesis was found to precede that of collagen IV, presumably enabling efficient collagen modification by PH4⍺EFB. Finally, biochemical assays confirm that PH4⍺EFB binds collagen, supporting its direct role in collagen IV modification. Collectively, we identify PH4⍺EFB as the primary and potentially constitutive prolyl 4-hydroxylase responsible for collagen IV biosynthesis in Drosophila. Our findings highlight the remarkably simple nature of Drosophila collagen IV biosynthesis, which may serve as a blueprint for defining the minimal requirements for collagen engineering.
Basement membranes (BM) are thin, nanoporous sheets of specialized extracellular matrix (ECM) that line epithelial tissues. They are dynamic structures that serve multiple key functions, as evidenced by numerous diseases, including cancer progression, that are associated with their alterations. Our understanding of the BM and its communication with adjoining epithelial cells remains highly fragmented due to the BM's complex molecular architecture, the lack of molecular tools, limitations in utilizing high-resolution imaging techniques to BMs assembled on tissues, and the difficulty of assessing their functional contributions in vivo. Here, by combining multiple -omics analyses and advanced microscopy methodologies, we characterized the BM from two normal human mammary epithelial cell lines, MCF10 and HMLE, grown as spheroids in 3D matrices. Our findings indicate that the spheroids autonomously assemble a BM exhibiting all the molecular, structural, and biophysical characteristics of physiological BM. Using these minimalist model systems, we provide evidence that collagen IV, laminins, perlecan, and hemidesmosomes all overlap in a shared porous lattice. Next, we demonstrate that the invasion-promoting PSD4/EFA6B knockout, found in patients with breast cancer, decreases the expression of BM components and their assembly on the spheroid surface. We then show that invasive spheroids develop enlarged pores in the BM via filopodia-like plasma membrane extensions, which further expand in a protease-dependent manner, thereby facilitating the passage of invasive cells.
Dystroglycan plays a crucial role for cell to extracellular matrix (ECM) adhesiveness in a plethora of different tissues and physio-pathological conditions. It belongs to the dystrophin-glycoprotein complex, whose overall structure has been recently solved, providing fundamental insight into the assembly of its various protein components, including the dystroglycan complex. This inspired us to embark in a timely "recollection journey" of our studies on the dystroglycan domain organization, mainly focusing on the targeted mutagenesis analysis of the α-dystroglycan's N-terminal domain (α-DGN) that we have carried out during the last 30 years. The account of such a journey also reinforces a crucial notion in protein biochemistry: a single amino acid substitution can lead to a significantly improved stability of the whole protein. Over-stabilizing matrix proteins, and proteins in general, has positive repercussions for the study of their structural and functional properties, and it is a crucial tool for developing biotechnological applications. Here we discuss newly emerged data along a series of yet unresolved points concerning the biochemical features and biological role of α-DGN, as well as the possible biomedical use recently emerged for a stabilized single site-directed variant of this protein domain.
The basal surface of epithelial tissues is attached to a thin network of macromolecules known as the basement membrane. The core components of the basement membrane - Collagen IV, Laminin, Perlecan, and Nidogen - are conserved extracellular matrix (ECM) proteins across species. However, the topography of basement membranes and the contribution of individual core components to its establishment remain poorly understood. Here, we used AFM-aided PeakForce tapping to analyze the topography of the basement membrane of Drosophila larval wing discs. We identified a self-affine surface topography, appearing structurally similar across multiple scales. Further, the topography is characterized by thin fiber-like structures that are intermittently aligned with a preferred orientation along the anterior-posterior axis. During larval development, the amplitude of surface patterns overall decreases, whereas the abundance of basement membrane components increases. Using targeted knockdown experiments, we show that Collagen IV is essential for the formation of fiber-like structures, while Laminin and Collagen IV appear to smooth or level out large-scale groove-like patterns. In contrast, Nidogen contributes to the maintenance of these grooves, and Perlecan increases surface pattern amplitudes at all length scales. Our findings reveal distinct topographical features in the basement membrane, whose amplitude and organization depend on its specific molecular composition.
The hallmark of amyloid diseases is deposition of misfolded proteins as amyloid fibrils in the interstitium of target organs. Amyloid deposits surround cells, distorting the micro and macro-architecture of the extracellular space and profoundly changing the physical and molecular properties of this compartment. In the heart, extracellular matrix (ECM) remodeling has a profound impact on the mechanical properties of this target organ and on the physiology and metabolism of resident cells. This review critically summarizes the available knowledge on ECM alterations in cardiac amyloidosis, with the goal of providing an overview on how biochemical, biophysical and anatomical modifications are interrelated, and how ECM remodeling participates in the pathophysiology of this unique type of cardiopathy.
Fibronectin is a key component of the extracellular matrix whose abundance and organization depend on both environmental conditions and intracellular signaling. In this study we show that oxygen tension modifies the response of extravillous trophoblasts to TGF-β1 and thereby controls fibronectin output and matrix dependent cell behavior. TGF-β1 increased fibronectin transcripts and protein through SMAD3, p38 and AKT, while hypoxia altered this response by reducing fibronectin protein despite preserved mRNA and by shifting downstream phosphorylation toward SMAD3, ERK and p38 with reduced AKT activity. These changes influenced functional outcomes: fibronectin rich conditions and TGF-β1 suppressed invasion and supported endothelial-like organization, and interference with fibronectin integrin binding preserved invasiveness and prevented network formation. Analysis of placental tissue showed that the spatial pattern of fibronectin expression differs in severe preeclampsia, where fibronectin appears earlier along the trophoblast trajectory compared with normal pregnancy. Together, these findings define how oxygen and TGF-β1 jointly regulate fibronectin and trophoblast behavior, while descriptive observations in human placental tissue provide histological context consistent with these cellular responses and suggest a potential role for matrix-associated signaling in severe preeclampsia.
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UDP-glucose 6-dehydrogenase (UGDH) is an essential enzyme that catalyzes the oxidation of UDP-glucose to UDP-glucuronate. UGDH is elevated in multiple cancers, including prostate cancer, and is functionally implicated in castration resistant recurrence. UGDH is composed of three dimeric units that associate stably as a hexamer in cellular conditions. The dynamic reorganization of noncovalent interactions at the dimer contact interfaces is essential for UGDH activity. In this study, we examined the functional relevance of a putative AGC kinase motif located at the dimer-dimer interface. We demonstrated that UGDH is phosphorylated in LNCaP cells, specifically at serine 316, by RSK2, p70S6K, and SGK1. To determine the functional implications of UGDH S316 phosphorylation, we generated and characterized phosphomimetic (S316D) and phosphodeficient (S316A) point mutations. Intrinsic properties of the purified recombinant proteins were only modestly affected by the substitutions. The stable overexpression of UGDH S316D in LNCaP cells significantly increased the rate of N- and O-glycan synthesis, as well as the production of hyaluronan and sulfated glycosaminoglycans, while reducing DHT glucuronidation, resulting in significant increases in growth of tumor spheroids, cell proliferation and motility, and resistance to enzalutamide. In contrast, UGDH S316A expression reduced the production of glycans and glycosaminoglycans, restored DHT glucuronidation, and impaired growth and motility. Overall, our results support UGDH phosphorylation as a point of control for intracellular and cell surface glycan production, thereby regulating cell proliferation, anchorage dependence, motility, and tumor cell therapeutic resistance.
Basement membranes are key mediators of many biological processes such as epithelial morphogenesis, kidney filtration, and muscle function among others. Basement membranes provide structural support to tissues so understanding their mechanical properties is important for determining how they contribute to tissue form and function. Further, basement membranes are altered in many diseases including cancer, diabetes, and fibrosis, and these changes may contribute to disease pathogenesis and progression. Understanding how basement membrane mechanics integrate with tissue function is the work of both biologists and engineers/material scientists, yet these disciplines have very different foundations. This review discusses basement membrane macromolecular structure with a view to illuminate how this structure confers basement membranes with unique mechanical properties adapted to resisting physiological stresses. The pathological implications of altered basement membrane mechanics are discussed in the context of different diseases. Additionally, we survey methods used to measure basement membrane mechanical properties, including atomic force microscopy, tensile stiffness assays, and non-quantitative assays such as cell bursting, assessing their strengths and limitations and their accessibility for different types of in vivo studies. We focus on explaining and illuminating the complexities of basement membrane material properties for biologists, and explaining the biological aspects for engineers, with the goal of making interdisciplinary science more accessible to experimentalists and readers.
Amelogenin, the most abundant protein in developing enamel, self-assembles into supramolecular structures that serve as templates for apatite growth. Recent studies revealed that amelogenin nanoribbons exhibit hallmark features of functional amyloids, yet the molecular mechanisms governing their formation remain incompletely understood. Here, we combine atomic force microscopy, transmission electron microscopy, and spectroscopic analyses to define the assembly pathways of full-length amelogenin (rH174) alongside its major proteolytic products generated by metalloproteinase-20 (MMP20). We demonstrate that both rH174 and the C-terminally truncated rH146 follow a nucleated conformational conversion mechanism, progressing from spherical oligomers through proto-ribbons to ordered β-sheet-rich nanoribbons. rH174 assembly progresses slowly, displaying an extended lag phase and delayed maturation, whereas rH146 nucleates rapidly, completing these stages within a shorter timeframe. Cross-seeding of rH146 into rH174 monomers (1:10) eliminates the delay in rH174 assembly, rapidly driving the system into elongation and leading to an earlier stabilization of the assembly system. C-terminus-driven interactions in rH174 trigger secondary nucleation that evolves into bundled nanoribbons resembling enamel organization, a process largely absent in rH146. Cross-seeding, therefore, exemplifies the in vivo mechanism whereby nascent amelogenin is immediately added to existing nanoribbon scaffolds, a cooperative strategy that generates a heterogeneous matrix, coupling the ability of rapid nucleation and spatial organization. Unexpectedly, the MMP20 cleavage product - TRAP, which comprises the cross-beta assembly domain, does not form nanoribbons and diverts from the assembly pathway full-length amelogenin takes when hydrolyzed at the C-terminal. Hence, a MMP20-driven mechanism exists that could contribute to an enamel matrix that acts as a spacer and prevents early crystal fusion during the secretory stage of amelogenesis. These findings offer insights into a proteolysis-triggered assembly pathway that may reconcile long-standing supramolecular models of amelogenin and establish amelogenin as a vertebrate example of a functional amyloid that can be tuned to enable ordered enamel biomineralization.
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Fibroblast cells are broadly distributed throughout the body and play instructive roles in tissue development and homeostasis. Defects in fibroblast function have been associated with developmental disorders, cancer and inflammatory disease. Our results reveal a previously unappreciated defect of fascial fibroblasts in patients with the upper limb congenital abnormality, Radial Dysplasia (RD). We identify compositional abnormalities in the extracellular matrix secreted by RD fascia-derived fibroblasts and provide an explanation for the consequent disorganisation and altered material properties of RD fascia and how this may impact disease pathology and patients' response to treatment. We show the abnormalities of RD fascial fibroblasts are reversible in vitro and identify a pathway with therapeutic potential to treat the soft tissue defects associated with RD. More broadly, our results have implications for understanding how heterogeneous tissue-resident fibroblast populations and the ECM they secrete contribute to normal tissue formation, homeostasis and disease.
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Inadequate collagen deposition is at the core of various pathologies, including non-healing wounds, fibrotic diseases, and tumour progression. It is therefore paramount to fully understand how central effectors affect collagen synthesis and turnover. Here, we will focus on two interconnected physiological effectors, vitamin C (ascorbic acid) and hypoxia (low oxygen levels), which both regulate collagen abundance and organisation. Ascorbic acid (AA) is known to act as a cofactor for collagen-modifying enzymes (collagen prolyl and lysyl hydroxylases) and is vital for collagen thermal stability, proper folding, and fibrillogenesis. Similarly, hypoxia has a strong impact on collagen synthesis, primarily through the stabilisation and activation of the hypoxia-inducible factors (HIFs), which, in turn, upregulate the expression of a range of collagen-modifying enzymes, including the collagen prolyl and lysyl hydroxylases. The consequence of both pathways is an enhanced collagen production, stability, maturation and cross-linking. Since AA and hypoxia both act on collagen hydroxylases, although by different mechanisms, i.e. AA increases the enzymatic activity whereas HIF increases the enzyme production, one can expect a possible synergistic effect of both factors. However, a more complex interplay exists between oxygen levels, AA and collagen as HIF affects AA intracellular uptake via the upregulation of one of its transporters while AA promotes HIF hydroxylation, thus reducing its stability and activity. A comprehensive understanding of this bidirectional control and the feedback loops between hypoxia, AA, and collagen is therefore critical to better tackle pathologies linked to abnormal collagen synthesis.
The myotendinous junction (MTJ) is a critical interface between muscle fibers and tendons, essential for force transmission between muscle and bone. Laminin-α2, a key extracellular matrix (ECM) component, is strongly enriched at this interface. Mutations in the LAMA2 gene cause LAMA2-related muscular dystrophy (LAMA2 MD), an early-onset severe congenital muscular dystrophy. Here, we examined the MTJ in dyW/dyW mice, a mouse model for LAMA2 MD. We find a strong disruption of MTJ morphology, including altered muscle fiber tips, collagen XXII mislocalization, and reduced muscle tendon interface. As MTJ loading is altered in dyW/dyW mice and MTJ maintenance requires loading and unloading, we also examined MTJ structures upon denervation-induced unloading. While muscle fiber tip morphology resembled that of dyW/dyW mice, collagen XXII distribution was not affected and the muscle-tendon interface was preserved. Finally, proteomic profiling via laser capture microdissection and mass spectrometry revealed significant regional and global shifts in MTJ protein composition in dyW/dyW and denervated mice. Across both models, we identified integrin-associated remodeling as a shared response linked to the perturbed muscle fiber tip morphology. These findings demonstrate that laminin-α2 is required for MTJ stability, and that mechanical unloading contributes to the observed phenotype. Importantly, our results suggest that disruptions in MTJ structure and protein composition may contribute to the pathology observed in LAMA2 MD.