title: Dissecting the interaction between tissue inhibitor of metalloproteinases-3 (TIMP-3) and low density lipoprotein receptor-related protein-1 (LRP-1): Development of a "TRAP" to increase levels of TIMP-3 in the tissue.
abstract: Tissue inhibitor of metalloproteinases 3 (TIMP-3) is a key regulator of extracellular matrix turnover for its ability to inhibit matrix metalloproteinases (MMPs), adamalysin-like metalloproteinases (ADAMs) and ADAMs with thrombospondin motifs (ADAMTSs). TIMP-3 is a secreted protein whose extracellular levels are regulated by endocytosis via the low-density-lipoprotein receptor-related protein-1 (LRP-1). In this study we developed a molecule able to "trap" TIMP-3 extracellularly, thereby increasing its tissue bioavailability. LRP-1 contains four ligand-binding clusters. In order to investigate the TIMP-3 binding site on LRP-1, we generated soluble minireceptors (sLRPs) containing the four distinct binding clusters or part of each cluster. We used an array of biochemical methods to investigate the binding of TIMP-3 to different sLRPs. We found that TIMP-3 binds to the ligand-binding cluster II of the receptor with the highest affinity and a soluble minireceptor containing the N-terminal half of cluster II specifically blocked TIMP-3 internalization, without affecting the turnover of metalloproteinases. Mass spectrometry-based secretome analysis showed that this minireceptor, named T3TRAP, selectively increased TIMP-3 levels in the extracellular space and inhibited constitutive shedding of a number of cell surface proteins. In conclusion, T3TRAP represents a biological tool that can be used to modulate TIMP-3 levels in the tissue and could be potentially developed as a therapy for diseases characterized by a deficit of TIMP-3, including arthritis.
authors.affiliation: Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan; Department of Neuroproteomics, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany; Neuroproteomics, Klinikum rechts der Isar and Institute for Advanced Study, Technische Universität München, Munich, Germany. Electronic address: simone.scilabra@dzne.de.
authors.forename: Simone D
authors.initials: SD
authors.lastname: Scilabra
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Yamamoto
authors.affiliation: Department of Neuroproteomics, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Pigoni
authors.affiliation: Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kazuma
authors.initials: K
authors.lastname: Sakamoto
authors.affiliation: Department of Neuroproteomics, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Müller
authors.affiliation: Department of Neuroproteomics, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany.
authors.forename: Alkmini
authors.initials: A
authors.lastname: Papadopoulou
authors.affiliation: Department of Neuroproteomics, Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Munich, Germany; Neuroproteomics, Klinikum rechts der Isar and Institute for Advanced Study, Technische Universität München, Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Stefan F
authors.initials: SF
authors.lastname: Lichtenthaler
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom.
authors.forename: Linda
authors.initials: L
authors.lastname: Troeberg
authors.affiliation: Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, United Kingdom.
authors.forename: Hideaki
authors.initials: H
authors.lastname: Nagase
authors.affiliation: Department of Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kadomatsu
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D004705:Endocytosis; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry* / Q000378:metabolism; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000235:genetics / Q000378:metabolism*; D058977:Molecular Sequence Annotation; D009457:Neuroglia / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D021381:Protein Transport; D062165:Receptors, Artificial / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D012995:Solubility; D019717:Tissue Inhibitor of Metalloproteinase-3 / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
chemical_list: C475476:LRP1 protein, human; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D062165:Receptors, Artificial; D011994:Recombinant Proteins; C088390:TIMP3 protein, human; D019717:Tissue Inhibitor of Metalloproteinase-3
keywords: ADAMTSs; ADAMs; Extracellular matrix; Inflammation; Low-density lipoprotein receptor-related protein-1; Matrix metalloproteinases; Tissue inhibitor of metalloproteinase-3