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

Matrix Biol. 2017 May;59:69-79. doi: 10.1016/j.matbio.2016.07.004. Epub 2016 Jul 29.

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.

Keywords: ADAMTSs; ADAMs; Extracellular matrix; Inflammation; Low-density lipoprotein receptor-related protein-1; Matrix metalloproteinases; Tissue inhibitor of metalloproteinase-3.

MeSH terms

  • Animals
  • Binding Sites
  • COS Cells
  • Cell Line, Tumor
  • Chlorocebus aethiops
  • Endocytosis
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism*
  • Extracellular Matrix / chemistry*
  • Extracellular Matrix / metabolism
  • Gene Expression Regulation
  • HEK293 Cells
  • Humans
  • Kinetics
  • Low Density Lipoprotein Receptor-Related Protein-1 / genetics
  • Low Density Lipoprotein Receptor-Related Protein-1 / metabolism*
  • Molecular Sequence Annotation
  • Neuroglia / cytology
  • Neuroglia / metabolism
  • Protein Binding
  • Protein Interaction Domains and Motifs
  • Protein Interaction Mapping
  • Protein Transport
  • Receptors, Artificial / genetics
  • Receptors, Artificial / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Solubility
  • Tissue Inhibitor of Metalloproteinase-3 / genetics
  • Tissue Inhibitor of Metalloproteinase-3 / metabolism*
  • Transfection

Substances

  • LRP1 protein, human
  • Low Density Lipoprotein Receptor-Related Protein-1
  • Receptors, Artificial
  • Recombinant Proteins
  • TIMP3 protein, human
  • Tissue Inhibitor of Metalloproteinase-3