Study of TIMP-1 interaction with its receptors

Le TIMP-1, inhibiteur naturel des métalloprotéinases matricielles, exerce des effets pléïotropes indépendants de l'inhibition des MMPs et participe au développement de certains cancers et maladies neurodégénératives. Ces effets cytokiniques du TIMP-1 impliquent sa liaison à des récepteurs membranaires dont certains sont caractérisés, la glycoprotéine CD63/intégrine beta 1 et le complexe pro MMP-9/CD44. Cependant les acides aminés ou les domaines du TIMP-1 se liant à ces récepteurs ne sont pas identifiés. Les travaux réalisés au cours de cette thèse mettent en évidence un nouveau récepteur du TIMP-1, la protéine LRP-1. Dans les neurones corticaux murins, le TIMP-1 se fixe aux domaines DII et DIV de LRP-1, est endocyté et induit une réduction de la taille des neurites ainsi qu'une augmentation du volume des cônes de croissance. Afin de caractériser cette interaction, nous avons utilisé une approche originale de modélisation moléculaire associant les analyses de modes normaux et la dynamique moléculaire. Ces analyses in silico ont permis d'identifier un mouvement de pince entre les domaines N et C-terminaux du TIMP-1. Nous avons muté trois résidus (F12, K47 et W105) localisés dans une région essentielle d'un point vue énergétique à l'exécution de ce mouvement. Ces trois mutants n'ont pas d'effet sur la longueur du réseau neuritique et ne sont pas endocytés par LRP-1. En revanche, ils interagissent avec les 2 autres récepteurs (CD63 et proMMP-9) et reproduisent les effets du TIMP-1 sauvage. De plus, nous avons identifié une séquence de 6 acides aminés localisée dans le domaine extracellulaire I de CD63 et essentielle à la liaison avec le TIMP-1. L'ensemble de ces travaux a permis l'identification de régions impliquées dans l'interaction du TIMP-1 avec ses différents récepteurs et pourrait permettre le développement de nouveaux outils pharmacologiques ciblant les activités cytokiniques du TIMP-1.TIMP-1, a natural inhibitor of matrix metalloproteinases, exerts pleiotropic effects independent of MMP inhibition and thus participates to the development of some cancers and neurodegenerative disorders. These cytokine-like activities require TIMP-1 binding to membrane receptors. Up to date two receptors, CD63/integrin beta 1 and proMMP-9/CD44, have been characterized. Nevertheless, TIMP-1 residues or regions binding these receptors remain unknown. In this work, we have identified the protein LRP-1 as a new receptor for TIMP 1. In mouse cortical neurons, TIMP-1 preferentially binds DII and DIV domains of LRP-1, is internalized via a LRP-1-dependent endocytosis, reduces neurite length and increases growth cone volume. To go deeper into TIMP-1/LRP-1 interaction, we used an original molecular modeling approach which combined normal mode analysis and molecular dynamic. These in silico studies allow us to point out a clamp movement between the N- and C-terminal domains of TIMP-1. Three residues localized in a region that seems essential for the movement have been mutated (F12, K47 and W105) and single mutants have been produced. These mutants do not reduce neurite outgrowth and are not internalized by LRP-1. In contrast, they interact with the two others receptors proMMP-9 and CD63 and induce associated biological effects. Furthermore, we have identified a sequence of six residues localized in the CD63 extracellular domain I and essential for TIMP 1 binding. The set of our data highlighted new regions of TIMP-1 interacting with its receptors and could lead to design novel therapeutic agents targeting the TIMP-1 cytokine like activities

LRP-1: A Checkpoint for the Extracellular Matrix Proteolysis

Low-density lipoprotein receptor-related protein-(LRP-1) is a large endocytic receptor that binds more than 35 ligands and exhibits signaling properties. Proteinases capable of degrading extracellular matrix (ECM), called matrix proteinases in this paper, are mainly serine proteinases: the activators of plasminogen into plasmin, tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, and the members of the matrix metalloproteinase (MMP) family. LRP-1 is responsible for clearing matrix proteinases, complexed or not with inhibitors. This paper attempts to summarize some aspects on the cellular and molecular bases of endocytic and signaling functions of LRP-1 that modulate extra- and pericellular levels of matrix proteinases

Intrinsic dynamics study identifies two amino acids of TIMP-1 critical for its LRP-1-mediated endocytosis in neurons

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Low-density lipoprotein receptor-related protein-1 mediates endocytic clearance of tissue inhibitor of metalloproteinases-1 and promotes its cytokine-like activities.

Tissue inhibitor of metalloproteinases-1 (TIMP-1) regulates the extracellular matrix turnover by inhibiting the proteolytic activity of matrix metalloproteinases (MMPs). TIMP-1 also displays MMP-independent activities that influence the behavior of various cell types including neuronal plasticity, but the underlying molecular mechanisms remain mostly unknown. The trans-membrane receptor low-density lipoprotein receptor-related protein-1 (LRP-1) consists of a large extracellular chain with distinct ligand-binding domains that interact with numerous ligands including TIMP-2 and TIMP-3 and a short transmembrane chain with intracellular motifs that allow endocytosis and confer signaling properties to LRP-1. We addressed TIMP-1 interaction with recombinant ligand-binding domains of LRP-1 expressed by CHO cells for endocytosis study, or linked onto sensor chips for surface plasmon resonance analysis. Primary cortical neurons bound and internalized endogenous TIMP-1 through a mechanism mediated by LRP-1. This resulted in inhibition of neurite outgrowth and increased growth cone volume. Using a mutated inactive TIMP-1 variant we showed that TIMP-1 effect on neurone morphology was independent of its MMP inhibitory activity. We conclude that TIMP-1 is a new ligand of LRP-1 and we highlight a new example of its MMP-independent, cytokine-like functions

TIMP-1 binding to LRP-1 reduces neurite length.

<p><b>A.</b> Cortical neurons from mouse embryos were cultured for 24-L-lysine-coated coverslips and then treated for 30 min with TIMP-1 (10 nM), RAP (500 nM), blocking LRP-1 polyclonal antibodies (R2629) or a combination of TIMP-1+RAP and TIMP-1+R2629. Untreated cells served as control (CTRL). Cells were labeled with anti-βIII-tubulin monoclonal antibody and observed under confocal microscopy. <b>B.</b> Quantification of neurite mean length per cell was performed using the ImageJ plugin NeuronJ and expressed as percent of untreated neurons (CTRL). Images in <b>A</b> are representative of results obtained in 3 independent experiments. Values in <b>B</b> represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** <i>p</i><0.01. Scale bar: 10 µm.</p

Inactive T2G mutant of TIMP-1 colocalizes with LRP-1 and exerts similar effects on the morphology of cortical neurons than wild-type TIMP-1.

<p><b>A.</b> Cortical neurons from mouse embryos were allowed to grow during 24-L-lysine-coated coverslips, and treated for 30 min with FLAG-TIMP-1 (10 nM) or FLAG-T2G (10 nM). Neurons were then stained with anti-LRP-1 antibody (Alexa Fluor 568, red) or anti-FLAG antibody (Alexa Fluor 488, green) and analyzed by confocal microscopy. Nuclei were counterstained with DAPI (blue). Images were treated with the AMIRA sofware. Fluorescent signals corresponding to LRP-1, FLAG and colocalization were shown by red (left), green (middle) and cyan (right) labeling. <b>B–C.</b> Neurons were treated as indicated in <b>A</b>, in the absence or presence of RAP. <b>B.</b> Neurites were labeled with anti-βIII-tubulin antibody and observed under confocal microscopy. The neurite mean length per cell was determined using the ImageJ plugin NeuronJ and expressed as percent of untreated neurons (CTRL). <b>C.</b> Actin-rich growth cones were visualized with Alexa Fluor 568-phalloidin, observed under confocal microscopy and quantified using the AMIRA software (right panel). Images in <b>A</b> are representative of results obtained in 3 independent experiments. Values in <b>B</b> and <b>C</b> represent the mean ± s.e.m. of 3 independent experiments. NS, not significant; ** <i>p</i><0.01. Scale bar: 5 µm.</p

Domains II and IV of the extracellular α-chain of LRP-1 are required to bind and promote TIMP-1 endocytosis in CHO cells.

<p><b>A.</b> Schematic representation of LRP-1-derived minireceptors carrying no-ligand-binding cluster (SPCT), extracellular binding-domain II (DII) or extracellular binding-domain IV (DIV). Each construct contains a HA tag at the amino-terminus of the α-chain. <b>B.</b> Transfected CHO cells stably express HA-tagged SPCT (SPCT), HA-tagged mini LRP-II (DII), or HA-tagged mini LRP-IV (DIV). Nontransfected cells served as control (CTRL). Biotinylation of cell-surface proteins was performed, followed by an immunoblot (IB) analysis using anti-HA tag. Bands correspond to the expected molecular weights of SPCT (106 kDa; arrowhead), DII (153 kDa; star), and DIV (164 kDa; double star). <b>C.</b> CHO cells overexpressing HA-tagged LRP-1-derived minireceptors (SPCT, DII, DIV) or not (CTRL) were transiently transfected with RFP-tagged TIMP-1 for 24 hours. Cell-surface proteins were subjected to immunoprecipitation (IP) assay with either anti-HA tag (left panel) or an anti-RFP tag (right panel). Then, immunoblot (IB) analysis was conducted using both anti-LRP-1 β-chain (5A6) and anti-RFP tag. <b>D.</b> Representative sensorgrams for TIMP-1 interacting with DII (left panel) and DIV (right panel). A set of concentrations (5–80 nM) of TIMP-1 or EGF was sequentially injected over immobilized Fc-DII and Fc-DIV. The solid black lines represent the specific binding of TIMP-1 obtained after double-subtraction of the signal obtained on the control flow cell and a blank run. The dotted black lines represent the fit of the data with a kinetic titration 1∶1 interaction model. The grey lines represent the specific binding of EGF obtained after double-subtraction of the signal obtained on the control flow cell and a blank run. Arrows indicate the beginning of each injection. The data illustrated are representative of three independent experiments. <b>E.</b> Binding and internalization of exogenous fluorescent TIMP-1 (fluo-TIMP-1) by CHO cells overexpressing minireceptor SPCT (left), DII (middle) or DIV (right). Binding was assessed by incubating fluo-TIMP-1 (10 nM) at 4°C for 2 hours. Cells were then transferred to 37°C for additional 2 h to allow internalization. All incubations were performed with or without RAP (500 nM), an antagonist of LRP-1-mediated binding and consequently, endocytosis. Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 10 A.U. are considered to be nonspecific. Images in <b>B–D</b> are representative of results obtained in 3 independent experiments. Values in <b>E</b> represent the means ± s.e.m. of 3 independent experiments. NS, not significant; * <i>p</i><0.05, as compared to untreated cells.</p

TIMP-1 binding to LRP-1 increases growth cone volume.

<p><b>A.</b> Cortical neurons from mouse embryos were treated after 24-L-lysine-coated coverslips for 30 min with TIMP-1 (10 nM), RAP (500 nM) or blocking LRP-1 polyclonal antibodies (R2629) or a combination of TIMP-1+RAP and TIMP-1+R2629. Untreated cells served as a control (CTRL). Neurons were incubated with Alexa Fluor 568-phalloidin to label F-actin structures and analyzed by confocal microscopy. <b>B.</b> 3D-quantification of growth cone volume was performed using the AMIRA software and expressed as percent of untreated neurons (CTRL). Images in <b>A</b> are representative of results obtained in 3 independent experiments. Values in <b>B</b> represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** <i>p</i><0.01. Scale bar: 5 µm.</p

Endogenous TIMP-1 interacts with LRP-1 in cortical neurons.

<p><b>A.</b> Cortical neurons from mouse embryos were plated onto poly-L-lysine-coated coverslips for 24 h at 37°C, fixed, washed and stained with anti-LRP1 antibody (Alexa Fluor 488, green) and anti-TIMP-1 antibody (Alexa Fluor 568, red) before confocal microscopy analysis. Nuclei were counterstained with DAPI (blue) and appropriate secondary antibody controls were performed. LRP-1 labeling (left), TIMP-1 labeling (middle), and a merged image (right) are shown. <b>B.</b> Biotinylation of cell-surface proteins was conducted at 4°C from cortical neurons previously treated for 24 h with or without RAP (500 nM). Proteins were affinity precipitated with avidin-agarose beads, then LRP-1-containing complexes were immunoprecipitated by either anti-LRP-1 β-chain (LRP-1 β; left panel) or anti-LRP-1 α-chain (LRP-1 α; middle panel) and analyzed by western-blot using anti-LRP-1 β-chain (5A6), anti-LRP-1 α-chain (8G1) and anti-TIMP-1 antibodies. Nonspecific IgGs were used as a negative control of immunoprecipitation. The presence of TIMP-1 in immunocomplexes was quantified by densitometric analysis relative to immunoprecipitated LRP-1-α-chain (histogram, right panel). <b>C.</b> Binding and internalization of exogenous fluorescent TIMP-1 (fluo-TIMP-1) by cortical neurons. Binding was determined by incubating fluo-TIMP-1 at 4°C for 2 h. After extensive washes, part of the cells was used to quantify total binding. The other part was incubated at 37°C for an additional 1 h to permit endocytosis. Experiments were carried out with or without RAP (500 nM). Fluorescence intensity was quantified by spectrophotometry and expressed as arbitrary units (A.U.). Values below 10 A.U. are considered to be nonspecific. Images in <b>A</b> and <b>B</b> are representative of results obtained in 3 independent experiments. Values in <b>B</b> and <b>C</b> represent the means ± s.e.m. of 3 independent experiments. NS, not significant; ** <i>p</i><0.01, as compared to untreated cells. Scale bar: 5 µm.</p

Kinetics of TIMP-1 binding to LRP-1 domains II and IV.

<p>Data are based on three measurements using five different concentrations for each measurement.</p><p>Mean values ± s.e.m. are presented.</p><p>The <i>k_on</i> values are in M⁻¹s⁻¹, and <i>k_off</i> values are in s⁻¹.</p><p>The equilibrium constants of dissociation (<i>K_D</i>) were calculated from the association (<i>k_on</i>) and dissociation (<i>k_off</i>) rate constants.</p