Heparan sulfate proteoglycans are central modulators of cell-cell communication, largely through the information encoded in their sulfation patterns. Among extracellular regulators of this 'heparan sulfate code', the endosulfatases SULF1 and SULF2 have emerged as unique enzymes with the capacity to selectively remove 6-O-sulfate groups from heparan sulfate (HS) glucosamine residues. This activity distinguishes them from canonical lysosomal sulfatases and positions them as critical editors of HS from the extracellular matrix and cell-surface. Recent biochemical studies have highlighted their distinctive domain organization, extensive post-translational modifications, and finely tuned substrate specificity, revealing that 6-O-desulfation is a non-random, highly regulated process. Functionally, SULFs influence major signaling pathways and thereby participate in diverse biological processes, such as development, tissue homeostasis, injury repair, inflammation, and tumor progression. Accumulating evidence also implicates SULF1 and SULF2 in disease pathogenesis and highlights them as promising, yet underexplored, therapeutic targets. In the present review, we provide an updated perspective on SULF biology, emphasizing recent advances in functional characterization, their roles as extracellular 'code editors', and the therapeutic opportunities that may arise from targeting their activity. We also address several key questions that remain unresolved and that are needed to understand this complex mechanism of regulation.

The expression of extracellular matrix protein domains, which exhibit biological activities of their own, is required in order to get a sufficient amount to perform experiments aiming at determining their structure, interactions, and functions, and for preclinical and clinical studies. We describe here the protocol we designed to express in human embryonic kidney cells a recombinant von Willebrand factor A domain of human collagen VI, and recombinant anti-angiogenic matrikines of human collagens VIII (vastatin) and XVIII (endostatin).

Growth factor signaling governs essential cellular processes, and its precision relies on interactions with heparan sulfate on the cell surface. The sulfation pattern of heparan sulfate dictates its capacity to bind specific growth factors and their receptors, thereby controlling the signaling strength and specificity. Extracellular endosulfatases, including sulfatase 1 and sulfatase 2, further modulate these interactions by selectively removing sulfate groups from defined regions of heparan sulfate. Although these enzymes are known to influence developmental- and disease-related signaling, their direct effects on growth factor recognition have remained unclear. Using a panel of bioengineered heparan sulfate conjugates with defined sulfation compositions, this study examines how the structural features of heparan sulfate govern its regulation by the sulfatases. By tracking enzyme binding and catalytic remodeling, we found that both enzymes rely on two coordinated mechanisms: catalytic desulfation of heparan sulfate and competitive binding that transiently prevents growth factor association. The balance between catalytic remodeling and competitive binding depends on the sulfation characteristics of the heparan sulfate substrate and the identity of the growth factor and differs between the two enzyme isoforms. These findings provide a new framework for understanding how extracellular sulfatases shape growth factor signaling in both development and disease.

Chondroitin sulfates are complex polysaccharide chains that regulate various biological processes at the cell surface and within the extracellular matrix. Here, we identify four heterodimeric complexes responsible for chondroitin sulfate chain polymerization in humans: CHSY1-CHPF, CHSY1-CHPF2, CHSY3-CHPF, and CHSY3-CHPF2. Using a custom-tailored in vitro glycosylation assay based on chemo-enzymatically synthesized fluorescent substrates, we demonstrate that all four complexes exhibit chain polymerization activity. The cryo-EM structure of the CHSY3-CHPF complex provides molecular insights into the chondroitin sulfate chain polymerization reaction. The architecture of the catalytic sites suggests that CHSY1 and CHSY3 are enzymatically active, while CHPF and CHPF2 primarily play a stabilizing role. Mutational analysis of purified enzyme complexes, combined with an in cellulo complementation assay, confirms that only CHSY1 and CHSY3 have bifunctional glycosyltransferase activities. Based on the spatial arrangement of the catalytic sites, we propose that chondroitin sulfate chain polymerization follows a non-processive, distributive mechanism.

MatrixDB, a member of the International Molecular Exchange consortium (IMEx), is a curated interaction database focused on interactions established by extracellular matrix (ECM) constituents including proteins, proteoglycans, glycosaminoglycans and ECM bioactive fragments. The architecture of MatrixDB was upgraded to ease interaction data export, allow versioning and programmatic access and ensure sustainability. The new version of the database includes more than twice the number of manually curated and experimentally-supported interactions. High-confidence predicted interactions were imported from the Integrated Interactions Database to increase the coverage of the ECM interactome. ECM and ECM-associated proteins of five species (human, murine, bovine, avian and zebrafish) were annotated with matrisome divisions and categories, which are used for computational analyses of ECM -omic datasets. Biological pathways from the Reactome Pathway Knowledgebase were also added to the biomolecule description. New transcriptomic and expanded proteomic datasets were imported in MatrixDB to generate cell- and tissue-specific ECM networks using the newly developed in-house Network Explorer integrated in the database. MatrixDB is freely available at https://matrixdb.univ-lyon1.fr. [GRAPHICS] .

Heparan sulfate (HS) is a linear polysaccharide composed of a glucuronic acid (GlcA)‐ N ‐acetyl‐glucosamine (GlcNAc) disaccharide repeat motif, polymerized by the EXT1–EXT2 complex. It is extensively modified by a series of Golgi localized enzymes, that generate distinct saccharide sequences involved in the binding and the regulation of numerous protein partners. N ‐deacetylase/ N ‐sulfotransferase (NDST), of which four isoforms have been identified in mammals, are involved in the first step of this process and catalyze both the N ‐deacetylation of the GlcNAc residues into GlcNH 2 and its re‐ N ‐sulfation into GlcNS residues. Further modifications of the HS chain depend on this first maturation event, NDST action is, therefore, key to HS biosynthesis. However, although the sulfotransferase domain of NDST1 has been characterized at the structural level some 20 years ago, information on the overall structure and activity of the enzyme are still lacking. Here, we report the characterization of the two most expressed NDSTs in humans, NDST1 and NDST2, and a model structure of NDST1 homodimer using cryoelectron microscopy combined with AlphaFold2 modeling. Structure‐driven mutagenesis along with two bioassays to follow the protein activities allowed us to characterize the kinetics of the deacetylation and sulfoaddition and to identify the residue H529 as necessary for N ‐deacetylation. These results shed light on a poorly understood family of enzymes and will help deciphering the molecular basis for HS and heparin maturation.

The IntAct molecular interaction database (https://www.ebi.ac.uk/intact) is a curated resource of molecular interactions, derived from the scientific literature and from direct data depositions. As of August 2021, IntAct provides more than one million binary interactions, curated by twelve global partners of the International Molecular Exchange consortium, for which the IntAct database provides a shared curation and dissemination platform. The IMEx curation policy has always emphasised a fine-grained data and curation model, aiming to capture the relevant experimental detail essential for the interpretation of the provided molecular interaction data. Here, we present recent curation focus and progress, as well as a completely redeveloped website which presents IntAct data in a much more user-friendly and detailed way.

Glycosaminoglycans (GAGs) are complex linear polysaccharides, which are covalently attached to core proteins (except for hyaluronan) to form proteoglycans. They play key roles in the organization of the extracellular matrix, and at the cell surface where they contribute to the regulation of cell signaling and of cell adhesion. To explore the mechanisms and pathways underlying their functions, we have generated an expanded dataset of 4,290 interactions corresponding to 3,464 unique GAG-binding proteins, four times more than the first version of the GAG interactome (Vallet, Clerc, and Ricard-Blum. J Histochem Cytochem 69: 93???104, 2021). The increased size of the GAG network is mostly due to the addition of GAG-binding proteins captured from cell lysates and biological fluids by affinity chromatography and identified by mass spectrometry. We review here the interaction repertoire of natural GAGs and of synthetic sulfated hyaluronan, the specificity and molecular functions of GAG-binding proteins, and the biological processes and pathways they are involved in. This dataset is also used to investigate the differences between proteins binding to iduronic acid-containing GAGs (dermatan sulfate and heparin/heparan sulfate) and those interacting with GAGs lacking iduronic acid (chondroitin sulfate, hyaluronan, and keratan sulfate).

ABC ("ATP-Binding Cassette") transporters of the type IV subfamily consist of exporters involved in the efflux of many compounds, notably those capable to confer multidrug resistance like the mammalian P-glycoprotein or the bacterial transporter BmrA. They function according to an alternating access mechanism between inward-facing (IF) and outward-facing (OF) conformations, but the extent of physical separation between the two nucleotide-binding domains (NBDs) in different states is still unsettled. Small Angle Neutron Scattering and hydrogen/deuterium exchange coupled to mass spectrometry were used to highlight different conformational states of BmrA during its ATPase cycle. In particular, mutation of the conserved Lysine residue of the Walker-A motif (K380A) captures BmrA in an ATP-bound IF conformation prior to NBD closure. While in the transition-like state induced by vanadate wild-type BmrA is mainly in an OF conformation, the transporter populates only IF conformations in either the apo state or in the presence of ADP/Mg. Importantly, in this post-hydrolytic step, distances between the two NBDs of BmrA seem to be more separated than in the apo state, but they remain shorter than the widest opening found in the related MsbA transporter. Overall, our results highlight the main steps of the catalytic cycle of a homodimeric bacterial multidrug transporter and underline structural and functional commonalities as well as oddities among the type IV subfamily of ABC transporters.

Two-component regulatory systems (TCS) are among the most widespread mechanisms that bacteria use to sense and respond to environmental changes. In the human pathogen Streptococcus pneumoniae , a total of 13 TCS have been identified and many of them have been linked to pathogenicity. Notably, TCS01 strongly contributes to pneumococcal virulence in several infection models. However, it remains one of the least studied TCS in pneumococci and its functional role is still unclear. In this study, we demonstrate that TCS01 cooperates with a BceAB-type ABC transporter to sense and induce resistance to structurally-unrelated antimicrobial peptides of bacterial origin that all target undecaprenyl-pyrophosphate or lipid II, which are essential precursors of cell wall biosynthesis. Even though tcs01 and bceAB genes do not locate in the same gene cluster, disruption of either of them equally sensitized the bacterium to the same set of antimicrobial peptides. We show that the key function of TCS01 is to upregulate the expression of the transporter, while the latter appears the main actor in resistance. Electrophoretic mobility shift assays further demonstrated that the response regulator of TCS01 binds to the promoter region of the bceAB genes, implying a direct control of these genes. The BceAB transporter was overexpressed and purified from E . coli . After reconstitution in liposomes, it displayed substantial ATPase and GTPase activities that were stimulated by antimicrobial peptides to which it confers resistance to, revealing new functional features of a BceAB-type transporter. Altogether, this inducible defense mechanism likely contributes to the survival of the opportunistic microorganism in the human host, in which competition among commensal microorganisms is a key determinant for effective host colonization and invasive path.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

The six mammalian glycosaminoglycans (GAGs), chondroitin sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronan, and keratan sulfate, are linear polysaccharides. Except for hyaluronan, they are sulfated to various extent, and covalently attached to proteins to form proteoglycans. GAGs interact with growth factors, morphogens, chemokines, extracellular matrix proteins and their bioactive fragments, receptors, lipoproteins, and pathogens. These interactions mediate their functions, from embryonic development to extracellular matrix assembly and regulation of cell signaling in various physiological and pathological contexts such as angiogenesis, cancer, neurodegenerative diseases, and infections. We give an overview of GAG–protein interactions (i.e., specificity and chemical features of GAG- and protein-binding sequences), and review the available GAG–protein interaction networks. We also provide the first comprehensive draft of the GAG interactome composed of 832 biomolecules (827 proteins and five GAGs) and 932 protein–GAG interactions. This network is a scaffold, which in the future should integrate structures of GAG–protein complexes, quantitative data of the abundance of GAGs in tissues to build tissue-specific interactomes, and GAG interactions with metal ions such as calcium, which plays a major role in the assembly of the extracellular matrix and its interactions with cells. This contextualized interactome will be useful to identify druggable GAG–protein interactions for therapeutic purpose:

The members of the lysyl oxidase (LOX) family are amine oxidases, which initiate the covalent cross-linking of the extracellular matrix (ECM), regulate ECM stiffness, and contribute to cancer progression. The aim of this study was to build the first draft of the interactome of the five members of the LOX family in order to determine its molecular functions, the biological and signaling pathways mediating these functions, the biological processes it is involved in, and if and how it is rewired in cancer. In vitro binding assays, based on surface plasmon resonance and bio-layer interferometry, combined with queries of interaction databases and interaction datasets, were used to retrieve interaction data. The interactome was then analyzed using computational tools. We identified 31 new interactions and 14 new partners of LOXL2, including the α5β1 integrin, and built an interactome comprising 320 proteins, 5 glycosaminoglycans, and 399 interactions. This network participates in ECM organization, degradation and cross-linking, cell-ECM interactions mediated by non-integrin and integrin receptors, protein folding and chaperone activity, organ and blood vessel development, cellular response to stress, and signal transduction. We showed that this network is rewired in colorectal carcinoma, leading to a switch from ECM organization to protein folding and chaperone activity.

The extracellular matrix (ECM) is a complex meshwork of proteins and an essential component of multicellular life. We have recently reported the characterization of a novel ECM protein, SNED1, and showed that it promotes breast cancer metastasis and regulates craniofacial development. However, the mechanisms by which it does so remain unknown. ECM proteins exert their functions by binding to cell surface receptors and interacting with other ECM proteins, actions that we can predict using knowledge of protein's sequence, structure, and post-translational modifications. Here, we combined in-silico and in-vitro approaches to characterize the physico-chemical properties of SNED1 and infer its putative functions. To do so, we established a mammalian cell system to produce and purify SNED1 and its N-terminal fragment, which contains a NIDO domain, and demonstrated experimentally SNED1's potential to be glycosylated, phosphorylated, and incorporated into an insoluble ECM. We also determined the secondary and tertiary structures of SNED1 and its N-terminal fragment and obtained a model for its NIDO domain. Using computational predictions, we identified 114 proteins as putative SNED1 interactors, including the ECM protein fibronectin. Pathway analysis of the predicted SNED1 interactome further revealed that it may contribute to signaling through cell surface receptors, such as integrins, and participate in the regulation of ECM organization and developmental processes. Last, using fluorescence microscopy, we showed that SNED1 forms microfibrils within the ECM and partially colocalizes with fibronectin. Altogether, we provide a wealth of information on an understudied yet important ECM protein with the potential to decipher its pathophysiological functions.

The interaction database MatrixDB reports protein-protein and protein-glycosaminoglycan interactions in human, mammalian, and model organisms, involving at least one extracellular matrix (ECM) constituent, namely full-length proteins, ECM multimeric proteins considered as stable complexes, proteoglycans, glycosaminoglycans (GAGs), and bioactive fragments called matricryptins, which are released upon limited proteolysis of ECM proteins. The current version of MatrixDB (as of October 2020) contains 106,543 experimentally supported interactions, with all types of biomolecules combined. MatrixDB is the only database focusing on the curation of ECM protein and GAG interactions. The iNavigator integrated in MatrixDB allows users to build interaction networks online and to filter them according to expression data, quantitative proteomics data, or interaction detection methods. MatrixDB belongs to the International Molecular Exchange (IMEx) consortium, and uses its curation rules to capture interaction data, which are available in standardized exchange formats according to the Human Proteome Organization-Proteomics Standards Initiative (HUPO-PSI). © 2021 Wiley Periodicals LLC. Basic Protocol 1: Browse MatrixDB Basic Protocol 2: Create a list of biomolecules of interest to build interaction networks Basic Protocol 3: Build and export interaction networks of selected biomolecules using the iNavigator Basic Protocol 4: Build specific interaction networks using the iNavigator widgets Basic Protocol 5: Generate 3D models of glycosaminoglycan oligosaccharides using the GAG Builder tool.

[This corrects the article DOI: 10.1021/acsomega.9b00317.].

ABSTRACT The extracellular matrix (ECM) protein SNED1 has been shown to promote breast cancer metastasis and control neural crest cell-specific craniofacial development, but the cellular and molecular mechanisms by which it does so remain unknown. ECM proteins exert their functions by binding to cell surface receptors, sequestering growth factors, and interacting with other ECM proteins, actions that can be predicted using knowledge of protein’s sequence, structure and post-translational modifications. Here, we combined in-silico and in-vitro approaches to characterize the physico-chemical properties of SNED1 and infer its putative functions. To do so, we established a mammalian cell system to produce and purify SNED1 and its N-terminal fragment, which contains a NIDO domain. We have determined experimentally SNED1’s potential to be glycosylated, phosphorylated, and incorporated into insoluble ECM produced by cells. In addition, we used biophysical and computational methods to determine the secondary and tertiary structures of SNED1 and its N-terminal fragment. The tentative ab-initio model we built of SNED1 suggests that it is an elongated protein presumably able to bind multiple partners. Using computational predictions, we identified 114 proteins as putative SNED1 interactors. Pathway analysis of the newly-predicted SNED1 interactome further revealed that binding partners of SNED1 contribute to signaling through cell surface receptors, such as integrins, and participate in the regulation of ECM organization and developmental processes. Altogether, we provide a wealth of information on an understudied yet important ECM protein with the potential to decipher its functions in physiology and diseases.

The lysyl oxidase family comprises five members in mammals, lysyl oxidase (LOX) and four lysyl oxidase like proteins (LOXL1-4). They are copper amine oxidases with a highly conserved catalytic domain, a lysine tyrosylquinone cofactor, and a conserved copper-binding site. They catalyze the first step of the covalent cross-linking of the extracellular matrix (ECM) proteins collagens and elastin, which contribute to ECM stiffness and mechanical properties. The role of LOX and LOXL2 in fibrosis, tumorigenesis, and metastasis, including changes in their expression level and their regulation of cell signaling pathways, have been extensively reviewed, and both enzymes have been identified as therapeutic targets. We review here the molecular features and three-dimensional structure/models of LOX and LOXLs, their role in ECM cross-linking, and the regulation of their cross-linking activity by ECM proteins, proteoglycans, and by inhibitors. We also make an overview of the major ECM cross-links, because they are the ultimate molecular readouts of LOX/LOXL activity in tissues. The recent 3D model of LOX, which recapitulates its known structural and biochemical features, will be useful to decipher the molecular mechanisms of LOX interaction with its various substrates, and to design substrate-specific inhibitors, which are potential antifibrotic and antitumor drugs.