Tissue inhibitors of metalloproteinases (TIMPs) are widely distributed in the animal kingdom and the human genome contains four paralogous genes encoding TIMPs 1 to 4. TIMPs were originally characterized as inhibitors of matrix metalloproteinases (MMPs), but their range of activities has now been found to be broader as it includes the inhibition of several of the disintegrin-metalloproteinases, ADAMs and ADAMTSs. TIMPs are therefore key regulators of the metalloproteinases that degrade the extracellular matrix and shed cell surface molecules. Structural studies of TIMP–MMP complexes have elucidated the inhibition mechanism of TIMPs and the multiple sites through which they interact with target enzymes, allowing the generation of TIMP variants that selectively inhibit different groups of metalloproteinases. Engineering such variants is complicated by the fact that TIMPs can undergo changes in molecular dynamics induced by their interactions with proteases. TIMPs also have biological activities that are independent of metalloproteinases; these include effects on cell growth and differentiation, cell migration, anti-angiogenesis, anti- and pro-apoptosis, and synaptic plasticity. Receptors responsible for some of these activities have been identified and their signaling pathways have been investigated. A series of studies using mice with specific TIMP gene deletions has illuminated the importance of these molecules in biology and pathology.
The proteoglycan aggrecan is an important major component of cartilage matrix that gives articular cartilage the ability to withstand compression. Increased breakdown of aggrecan is associated with the development of arthritis and is considered to be catalyzed by aggrecanases, members of the ADAM-TS family of metalloproteinases. Four endogenous tissue inhibitors of metalloproteinases (TIMPs) regulate the activities of functional matrix metalloproteinases (MMPs), enzymes that degrade most components of connective tissue, but no endogenous factors responsible for the regulation of aggrecanases have been found. We show here that the N-terminal inhibitory domain of TIMP-3, a member of the TIMP family that has functional properties distinct from other TIMPs, is a strong inhibitor of human aggrecanases 1 and 2, with K i values in the subnanomolar range. This truncated inhibitor, which lacks the C-terminal domain that is responsible for interactions with molecules other than active metalloproteinases, is produced at high yield by bacterial expression and folding from inclusion bodies. This provides a starting point for developing a biologically available aggrecanase inhibitor suitable for the treatment of arthritis.Tissue inhibitors of matrix metalloproteinases (TIMPs) 1 are important regulators of matrix metalloproteinases (MMPs) that participate in the degradation of the extracellular matrix (1). To date, four isoforms of TIMP have been identified in humans that are designated TIMP-1, -2, -3, and -4 (2); these are homologous in sequence and have similar secondary and tertiary structures including six well conserved disulfide bonds. Structural and functional studies of TIMP-1 and TIMP-2 (3-6) have shown that the full inhibitory activity of TIMPs resides in the N-terminal domain that is stabilized by three disulfide bonds. Inhibition studies with recombinant TIMPs have shown that each TIMP binds to MMPs with varying degrees of affinity, implicating that they have distinct functions in vivo (2, 7).TIMP-3 was originally discovered as a transformation-induced protein in chicken fibroblasts (8), which was later shown to have inhibitory activity against MMPs (9). In addition to its function as an inhibitor of MMPs, TIMP-3 has been reported to inhibit the shedding of cell surface-anchored molecules such as tumor necrosis factor-␣ receptor (10), L-selectin (11), interleukin 6 receptor (12), and syndecans-1 and -4 (13). The release of these molecules is thought to be catalyzed by membrane-bound ADAMs (a disintegrin and a metalloproteinase domain), multidomain proteins containing an N-terminal propeptide, a metalloproteinase, a disintegrin-like, a transmembrane, and a cytoplasmic domain. The primary structures of the metalloproteinase domains of the MMPs and the ADAMs have little sequence similarity except near the catalytic Zn 2ϩ -binding motif, HEXXHXXGXXH (14). Direct evidence for the apparently unique ability of TIMP-3 to inhibit a broad spectrum of metalloproteinases is provided by the demonstration of its inhibitory actio...
Of the four known tissue inhibitors of metalloproteinases (TIMPs), TIMP-3 is distinguished by its tighter binding to the extracellular matrix. The present results show that glycosaminoglycans such as heparin, heparan sulfate, chondroitin sulfates A, B, and C, and sulfated compounds such as suramin and pentosan efficiently extract TIMP-3 from the postpartum rat uterus. Enzymatic treatment by heparinase III or chondroitinase ABC also releases TIMP-3, but neither one alone gives complete release. Confocal microscopy shows colocalization of heparan sulfate and TIMP-3 in the endometrium subjacent to the lumen of the uterus. Immunostaining of TIMP-3 is lost upon digestion of tissue sections with heparinase III and chondroitinase ABC. The N-terminal domain of human TIMP-3 was expressed and found to bind to heparin with affinity similar to that of full-length mouse TIMP-3. The A and B -strands of the N-terminal domain of TIMP-3 contain two potential heparin-binding sequences rich in lysine and arginine; these strands should form a double track on the outer surface of TIMP-3. Synthetic peptides corresponding to segments of these two strands compete for heparin in the DNase II binding assay. TIMP-3 binding may be important for the cellular regulation of activity of the matrix metalloproteinases.The extracellular matrix (ECM) 1 provides mechanical support to cells and regulates signals reaching the cell that govern cell localization, differentiation, proliferation, and apoptosis. Components of the ECM, particularly the glycosaminoglycans (GAGs), are able to sequester bioactive molecules such as growth factors (1), proteases (2), and inhibitors. Turnover of the ECM is a highly regulated process necessary for movement of cells and for release of growth factors. Matrix metalloproteases (MMPs) are believed to be key participants in this remodeling; there are at least 20 MMPs, all able to digest various ECM components (3, 4).The MMPs, in turn, are regulated by tissue inhibitors of metalloproteinases or TIMPs. The major function of the TIMPs is to inhibit MMPs; any imbalance in which the activities of MMPs outweigh the TIMP levels will favor tissue destruction and pathological processes (5, 6). The TIMPs also possess growth stimulatory and regulatory activities (7,8). The four members of the TIMP family all have similar secondary structures of six loops stabilized by six highly conserved disulfide bonds. The TIMPs all bind tightly, albeit with widely varying affinity, to the various MMPs. The x-ray structure (9) shows that the N-terminal cysteine chelates the active site zinc. TIMPs have N-and C-terminal domains, each with three loops. The N-terminal domain of TIMP-1 folds readily and displays full inhibitory activity (10).TIMP-3 has several features that distinguish it from the other TIMPs. First, it is the only TIMP to bind tightly to the ECM: it was first observed as a transformation-sensitive protein bound to the ECM of chick embryo fibroblasts (11) and extractable with SDS or guanidine. This protein was subsequently shown ...
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