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Abstract
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.
Highlights
From the ‡Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101 and the §Kennedy Institute of Rheumatology, Imperial College School of Medicine, 1 Aspenlea Road, Hammersmith, London W6 8LH United Kingdom
SDS-PAGE analysis under reducing conditions showed a single band with an apparent molecular weight of 16 kDa following staining with Coomassie Blue R250, in agreement with a molecular mass calculated for N-tissue inhibitors of metalloproteinases (TIMPs)-3, of 15,230
TIMPs expressed in connective tissues play important roles in the control of extracellular matrix metabolism, and the ability of TIMPs to inhibit the activities of matrix metalloproteinases (MMPs) in vitro has been well documented [2]
Summary
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 Ki 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. 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. This truncated protein, or variants engineered to enhance the specificity for aggrecanases, may represent a route for the treatment of arthritis
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