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Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).

ABSTRACT: Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library was generated containing 2 × 10(10) variants of TIMP-2. Five clones were isolated after five rounds of selection with MMP-1, using MMP-3 as a competitor. The enriched phages selectively bound MMP-1 relative to MMP-3 and contained mutations only in L1. The most selective variant (TM8) was used to generate a second library in which residues Cys(1)-Gln(9) were soft-randomized. Four additional clones, selected from this library, showed a similar affinity for MMP-1 as wild-type TIMP-2 but reduced affinity for MMP-3. Variants of the N-terminal domain of TIMP-2 (N-TIMP-2) with the sequences of the most selective clones were expressed and characterized for inhibitory activity against eight MMPs. All were effective inhibitors of MMP-1 with nanomolar K(i) values, but TM8, containing Ser(2) to Asp and Ser(4) to Ala substitutions, was the most selective having a nanomolar K(i) value for MMP-1 but no detectable inhibitory activity toward MMP-3 and MMP-14 up to 10 ?M. This study suggests that phage display and selection with other MMPs may be an effective method for discovering tissue inhibitor of metalloproteinase variants that discriminate between specified MMPs as targets.

SUBMITTER: Bahudhanapati H

PROVIDER: S-EPMC3173135 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).

Bahudhanapati Harinath H Zhang Yingnan Y Sidhu Sachdev S SS Brew Keith K

The Journal of biological chemistry 20110629 36

Tissue inhibitor of metalloproteinases-2 (TIMP-2) is a broad spectrum inhibitor of the matrix metalloproteinases (MMPs), which function in extracellular matrix catabolism. Here, phage display was used to identify variants of human TIMP-2 that are selective inhibitors of human MMP-1, a collagenase whose unregulated action is linked to cancer, arthritis, and fibrosis. Using hard randomization of residues 2, 4, 5, and 6 (L1) and soft randomization of residues 34-40 (L2) and 67-70 (L3), a library wa ...[more]

PMID: 21715326

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Project description:Matrix metalloproteinases (MMPs) are involved in the pathology of numerous inflammatory retinal degenerations, including retinitis pigmentosa (RP). Our previous work revealed that intravitreal injections with tissue inhibitor of metalloproteinases 1 (TIMP-1) reduce the progression of rod cell death and inhibit cone cell remodeling that involves reactive gliosis in retinal Müller glial cells (MGCs) in rodent models. The underlying cellular and molecular mechanisms of how TIMP-1 functions in the retina remain to be resolved; however, MGCs are involved in structural homeostasis, neuronal cell survival and death. In the present study, MMP-9 and TIMP-1 expression patterns were investigated in a human MGC line (MIO-M1) under inflammatory cytokine (IL-1β and TNF-α) and oxidative stress (H2O2) conditions. First, both IL-1β and TNF-α, but not H2O2, have a mild in vitro pro-survival effect on MIO-M1 cells. Treatment with either cytokine results in the imbalanced secretion of MMP-9 and TIMP-1. H2O2 treatment has little effect on their secretion. The investigation of their intracellular expression led to interesting observations. MMP-9 and TIMP-1 are both expressed, not only in the cytoplasm, but also inside the nucleus. None of the treatments alters the MMP-9 intracellular distribution pattern. In contrast to MMP-9, TIMP-1 is detected as speckles. Intracellular TIMP-1 aggregation forms in the cytoplasmic area with IL-1β treatment. With H2O2 treatments, the cell morphology changes from cobbles to spindle shapes and the nuclei become larger with increases in TIMP-1 speckles in an H2O2 dose-dependent manner. Two TIMP-1 cell surface receptors, low density lipoprotein receptor-related protein-1 (LRP-1) and cluster of differentiation 82 (CD82), are expressed within the nucleus of MIO-M1 cells. Overall, these observations suggest that intracellular TIMP-1 is a target of proinflammatory and oxidative insults in the MGCs. Given the importance of the roles for MGCs in the retina, the functional implication of nuclear TIMP-1 and MMP-9 in MGCs is discussed.

Dataset Information

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).

Publications

Phage display of tissue inhibitor of metalloproteinases-2 (TIMP-2): identification of selective inhibitors of collagenase-1 (metalloproteinase 1 (MMP-1)).

Similar Datasets

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