Title

A tale of two families: The metzincins and their native inhibitors TIMPs

Date of Award

2003

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Keith Brew, Committee Chair

Abstract

The metzincin superfamily consists of a large number of zinc-dependent peptidases, including the matrix metalloproteinases (MMPs) and the disintegrin and metalloproteinase (ADAM) family proteases. Unregulated metzincin activities are linked to diseases such as cancers and arthritis, and inhibition of these proteolytic activities has been proposed as potential therapy for metzincin-related diseases. Many synthetic metzincin inhibitors were effective in pre-clinical studies but were withdrawn during clinical trials, because of the severe side effects presumably caused by non-specific inhibition of functional metzincins.The tissue inhibitors of metalloproteinases (TIMPS) are endogenous inhibitors that regulate the activities of MMPs and some ADAMS. This thesis describes studies of the structural basis of the specificity of TIMPs for different metzincins. These are aimed at developing a rational basis for engineering TIMPs into more specific inhibitors for possible future clinical use.We have established E. coli expression systems for the N-terminal inhibitory domains of three human TIMPs, namely N-TIMP-1, N-TIMP-2 and N-TIMP-3, and folded these proteins in vitro into native structures. However, the bacterially expressed N-TIMP-1 was found to be partially inactive due to a possible alpha-N-acetylation of the N-terminal amino group that is essential for the inhibitory activity. With a medium-pressure cation-exchange chromatography, we were able to separate the active form from the modified inactive form. Isothermal titration calorimetry with the catalytic domain of MMP-1 gave a large negative change in heat capacity, indicating that hydrophobic interactions make a significant contribution to the binding affinity.TIMPs strongly inhibit MMPs with little selectivity. In order to engineer N-TIMP-1 into selective MMP inhibitors, we used site-directed mutagenesis to study the roles of several residues in contact with MMPs in the crystal structures of MMP/TIMP complex. In addition to the previously characterized effects of residue 2, we found that residues 4 and 68 also affect the binding affinity and selectivity of TIMP-1 for MMPs. Combinations of mutations with similar effects result in mutants with higher selectivity. These selective TIMP variants may be useful for gene therapy of MMP-related diseases, and suggest the feasibility of finding more specific MMP inhibitors by combinatorial display of TIMPs.The elongated A-B loop in TIMP-2 has been proposed by some to be responsible for the much higher binding affinity of TIMP-2 for MT1-MMP than TIMP-1. However, we found that replacement of the A-B loop of N-TIMP-1 by that of TIMP-2 has no effect in inhibitory activity for MT1-MMP. (Abstract shortened by UMI.)

Keywords

Biology, Molecular; Chemistry, Biochemistry

Link to Full Text

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