Logue, Timothy

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a protein with multiple functions that include regulating the turnover of the extracellular matrix (ECM) by inhibiting members of the metzincin family. Extracellular levels of soluble TIMP-3 are low, reflecting its binding to components of the ECM including sulfated glycosaminoglycans (SGAGs) and its endocytosis by low density lipoprotein receptor-related protein 1. Because TIMP-3 inhibits ECM-degrading enzymes, the ability of SGAG mimetics to elevate extracellular concentrations of TIMP3 is of interest for osteoarthritis treatment. However, previous studies of such interactions have utilized immobilized forms of the protein or ligands. Here we have quantified the thermodynamics of the interactions of the inhibitory domain of TIMP-3 with chondroitin sulfate (CS), pentosan polysulfate (PPS) and suramin in solution using isothermal titration calorimetry. All three interactions are driven by a (favorable) negative enthalpy ychange combined with an unfavorable decrease in entropy. The heat capacity change (ΔCp) for the interaction of N-TIMP-3 with CS, PPS, or suramin is essentially zero, indicating an insignificant contribution from the hydrophobic effect. Based on the effects of ionic strength on the interaction of N-TIMP-3 with suramin, their interaction appears to be driven by electrostatic interactions. Modeling supports the view that the negatively charged sulfates of CS, PPS, and suramin interact with a cationic region on N-TIMP-3 that includes Lys -26, -27, -30, and -possibly 76 on the opposite face of TIMP-3 from its reactive site for metalloproteases.

Matrix metalloproteinases MMPs are one of the major families of proteinases that play
key roles in maintaining an appropriately assembled extracellular matrix ECM. MMPs are
essential to many biological processes, such as wound healing, embryo implantation, bone
remodeling, and organogenesis. Their biological antagonists, the tissue inhibitors of
metalloproteinases TIMPs, regulate the enzymatic activities of MMPs. Uncontrolled ECM
degradation occurs when the delicate balance between TIMPs and MMPs is disrupted, resulting
in many diseases such as rheumatoid and osteoarthritis, cancer cell metastasis, and heart disease.
There are currently no effective treatments for osteoarthritis OA except for joint replacement
surgery. Therefore, gaining the knowledge about the structures and molecular mechanisms of
these key enzymes in order to uncover new ways to specifically inhibit these proteinases are an
opportunity for the development of therapeutics and treatments to prevent the joint destruction
seen in OA.
Our goal is to understand the biophysical interaction of catalytic domain of MMP-1 with NTIMP-
3 using isothermal calorimetry ITC. The ITC determines if binding between the proteins is
entropy or enthalpy driven and heat capacity will indicate the hydrophobic or hydrophilic
contributions of the interaction. The information from the ITC results in combination with the
known structures of N-TIMP-3/cd_MMP-1 will provide a more complete picture of the
interaction. This is in particular of relevance with respect clinical application by engineering
TIMPs for targeted inhibition of particular MMPs to treat diseases such as: cancer and arthritis.