The glycosaminoglycan interactome 2.0.

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).

Identification of a two-component regulatory system involved in antimicrobial peptide resistance in Streptococcus pneumoniae.

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.

Structural Insights into the Catalytic Cycle of a Bacterial Multidrug ABC Efflux Pump.

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.

The IntAct database: efficient access to fine-grained molecular interaction data.

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.

Building Protein-Protein and Protein-Glycosaminoglycan Interaction Networks Using MatrixDB, the Extracellular Matrix Interaction Database.

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.

Computational and experimental characterization of the novel ECM glycoprotein SNED1 and prediction of its interactome.

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.

Glycosaminoglycan-Protein Interactions: The First Draft of the Glycosaminoglycan Interactome.

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.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

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.

Correction to "A 3D Model of Human Lysyl Oxidase, A Cross-Linking Enzyme".

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

The Interactome of Cancer-Related Lysyl Oxidase and Lysyl Oxidase-Like Proteins.

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 α5ß1 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.

Lysyl oxidases: from enzyme activity to extracellular matrix cross-links.

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.

A Three-Dimensional Model of Human Lysyl Oxidase, a Cross-Linking Enzyme.

Lysyl oxidase (LOX) is a cross-linking enzyme identified 50 years ago, but its 3D structure is still unknown. We have thus built a 3D model of human LOX by homology modeling using the X-ray structure of human lysyl oxidase-like 2 as a template. This model is the first one to recapitulate all known biochemical features of LOX, namely, the coordination of the copper ion and the formation of the lysine tyrosylquinone cofactor and the disulfide bridges. Furthermore, this model is stable during a 1 µs molecular dynamics simulation. The catalytic site is located in a groove surrounded by two loops. The distance between these loops fluctuated during the simulations, which suggests that the groove forms a hinge with a variable opening, which is able to accommodate the various sizes of LOX substrates. This 3D model is a pre-requisite to perform docking experiments with LOX substrates and other partners to identify binding sites and to design new LOX inhibitors specific for therapeutic purpose.

Functionality of membrane proteins overexpressed and purified from E. coli is highly dependent upon the strain.

Overexpression of correctly folded membrane proteins is a fundamental prerequisite for functional and structural studies. One of the most commonly used expression systems for the production of membrane proteins is Escherichia coli. While misfolded proteins typically aggregate and form inclusions bodies, membrane proteins that are addressed to the membrane and extractable by detergents are generally assumed to be properly folded. Accordingly, GFP fusion strategy is often used as a fluorescent proxy to monitor their expression and folding quality. Here we investigated the functionality of two different multidrug ABC transporters, the homodimer BmrA from Bacillus subtilis and the heterodimer PatA/PatB from Streptococcus pneumoniae, when produced in several E. coli strains with T7 expression system. Strikingly, while strong expression in the membrane of several strains could be achieved, we observed drastic differences in the functionality of these proteins. Moreover, we observed a general trend in which mild detergents mainly extract the population of active transporters, whereas a harsher detergent like Fos-choline 12 could solubilize transporters irrespective of their functionality. Our results suggest that the amount of T7 RNA polymerase transcripts may indirectly but notably impact the structure and activity of overexpressed membrane proteins, and advise caution when using GFP fusion strategy.

Fragments generated upon extracellular matrix remodeling: Biological regulators and potential drugs.

The remodeling of the extracellular matrix (ECM) by several protease families releases a number of bioactive fragments, which regulate numerous biological processes such as autophagy, angiogenesis, adipogenesis, fibrosis, tumor growth, metastasis and wound healing. We review here the proteases which generate bioactive ECM fragments, their ECM substrates, the major bioactive ECM fragments, together with their biological properties and their receptors. The translation of ECM fragments into drugs is challenging and would take advantage of an integrative approach to optimize the design of pre-clinical and clinical studies. This could be done by building the contextualized interaction network of the ECM fragment repertoire including their parent proteins, remodeling proteinases, and their receptors, and by using mathematical disease models.

MatrixDB: integration of new data with a focus on glycosaminoglycan interactions.

MatrixDB (http://matrixdb.univ-lyon1.fr/) is an interaction database focused on biomolecular interactions established by extracellular matrix (ECM) proteins and glycosaminoglycans (GAGs). It is an active member of the International Molecular Exchange (IMEx) consortium (https://www.imexconsortium.org/). It has adopted the HUPO Proteomics Standards Initiative standards for annotating and exchanging interaction data, either at the MIMIx (The Minimum Information about a Molecular Interaction eXperiment) or IMEx level. The following items related to GAGs have been added in the updated version of MatrixDB: (i) cross-references of GAG sequences to the GlyTouCan database, (ii) representation of GAG sequences in different formats (IUPAC and GlycoCT) and as SNFG (Symbol Nomenclature For Glycans) images and (iii) the GAG Builder online tool to build 3D models of GAG sequences from GlycoCT codes. The database schema has been improved to represent n-ary experiments. Gene expression data, imported from Expression Atlas (https://www.ebi.ac.uk/gxa/home), quantitative ECM proteomic datasets (http://matrisomeproject.mit.edu/ecm-atlas), and a new visualization tool of the 3D structures of biomolecules, based on the PDB Component Library and LiteMol, have also been added. A new advanced query interface now allows users to mine MatrixDB data using combinations of criteria, in order to build specific interaction networks related to diseases, biological processes, molecular functions or publications.

Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners.

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (Dmax: 11.7 nm, Rg: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway.

Expanding the Kinome World: A New Protein Kinase Family Widely Conserved in Bacteria.

Fine tuning of signaling pathways is essential for cells to cope with sudden environmental variations. This delicate balance is maintained in particular by protein kinases that control the activity of target proteins by reversible phosphorylation. In addition to homologous eukaryotic enzymes, bacteria have evolved some specific Ser/Thr/Tyr protein kinases without any structural resemblance to their eukaryotic counterparts. Here, we show that a previously identified family of ATPases, broadly conserved among bacteria, is in fact a new family of protein kinases with a Ser/Thr/Tyr kinase activity. A prototypic member of this family, YdiB from Bacillus subtilis, is able to autophosphorylate and to phosphorylate a surrogate substrate, the myelin basic protein. Two crystal structures of YdiB were solved (1.8 and 2.0Å) that display a unique ATP-binding fold unrelated to known protein kinases, although a conserved HxD motif is reminiscent of that found in Hanks-type protein kinases. The effect of mutations of conserved residues further highlights the unique nature of this new protein kinase family that we name ubiquitous bacterial kinase. We investigated the cellular role of YdiB and showed that a ∆ydiB mutant was more sensitive to paraquat treatment than the wild type, with ~13% of cells with an aberrant morphology. In addition, YdiE, which is known to participate with both YdiC and YdiB in an essential chemical modification of some specific tRNAs, is phosphorylated in vitro by YdiB. These results expand the boundaries of the bacterial kinome and support the involvement of YdiB in protein translation and resistance to oxidative stress in B. subtilis.

Decorin interacting network: A comprehensive analysis of decorin-binding partners and their versatile functions.

Decorin, a prototype small leucine-rich proteoglycan, regulates a vast array of cellular processes including collagen fibrillogenesis, wound repair, angiostasis, tumor growth, and autophagy. This functional versatility arises from a wide array of decorin/protein interactions also including interactions with its single glycosaminoglycan side chain. The decorin-binding partners encompass numerous categories ranging from extracellular matrix molecules to cell surface receptors to growth factors and enzymes. Despite the diversity of the decorin interacting network, two main roles emerge as prominent themes in decorin function: maintenance of cellular structure and outside-in signaling, culminating in anti-tumorigenic effects. Here we present contemporary knowledge regarding the decorin interacting network and discuss in detail the biological relevance of these pleiotropic interactions, some of which could be targeted by therapeutic interventions.

Matricryptins Network with Matricellular Receptors at the Surface of Endothelial and Tumor Cells.

The extracellular matrix (ECM) is a source of bioactive fragments called matricryptins or matrikines resulting from the proteolytic cleavage of extracellular proteins (e.g., collagens, elastin, and laminins) and proteoglycans (e.g., perlecan). Matrix metalloproteinases (MMPs), cathepsins, and bone-morphogenetic protein-1 release fragments, which regulate physiopathological processes including tumor growth, metastasis, and angiogenesis, a pre-requisite for tumor growth. A number of matricryptins, and/or synthetic peptides derived from them, are currently investigated as potential anti-cancer drugs both in vitro and in animal models. Modifications aiming at improving their efficiency and their delivery to their target cells are studied. However, their use as drugs is not straightforward. The biological activities of these fragments are mediated by several receptor families. Several matricryptins may bind to the same matricellular receptor, and a single matricryptin may bind to two different receptors belonging or not to the same family such as integrins and growth factor receptors. Furthermore, some matricryptins interact with each other, integrins and growth factor receptors crosstalk and a signaling pathway may be regulated by several matricryptins. This forms an intricate 3D interaction network at the surface of tumor and endothelial cells, which is tightly associated with other cell-surface associated molecules such as heparan sulfate, caveolin, and nucleolin. Deciphering the molecular mechanisms underlying the behavior of this network is required in order to optimize the development of matricryptins as anti-cancer agents.

Proteases decode the extracellular matrix cryptome.

The extracellular matrix is comprised of 1100 core-matrisome and matrisome-associated proteins and of glycosaminoglycans. This structural scaffold contributes to the organization and mechanical properties of tissues and modulates cell behavior. The extracellular matrix is dynamic and undergoes constant remodeling, which leads to diseases if uncontrolled. Bioactive fragments, called matricryptins, are released from the extracellular proteins by limited proteolysis and have biological activities on their own. They regulate numerous physiological and pathological processes such as angiogenesis, cancer, diabetes, wound healing, fibrosis and infectious diseases and either improve or worsen the course of diseases depending on the matricryptins and on the molecular and biological contexts. Several protease families release matricryptins from core-matrisome and matrisome-associated proteins both in vitro and in vivo. The major proteases, which decrypt the extracellular matrix, are zinc metalloproteinases of the metzincin superfamily (matrixins, adamalysins and astacins), cysteine proteinases and serine proteases. Some matricryptins act as enzyme inhibitors, further connecting protease and matricryptin fates and providing intricate regulation of major physiopathological processes such as angiogenesis and tumorigenesis. They strengthen the role of the extracellular matrix as a key player in tissue failure and core-matrisome and matrisome-associated proteins as important therapeutic targets.