Biophysical and Functional Characterization of Full-length, Recombinant Human Tissue Inhibitor of Metalloproteinases-2 (TIMP-2) Produced in Escherichia coli

Abstract

Matrix metalloproteinases (MMPs) function in the remodeling of the extracellular matrix that is integral for many normal and pathological processes. The tissue inhibitor of metalloproteinases family, including tissue inhibitor of metalloproteinases-2 (TIMP-2), regulates the activity of these multifunctional metalloproteinases. TIMP family members are proteinase inhibitors that contain six conserved disulfide bonds, one involving an amino-terminal cysteine residue that is critical for MMP inhibitor activity. TIMP-2 has been expressed in Escherichia coli, folded from insoluble protein, and functionally characterized. The wild type protein inhibited gelatinase A (MMP-2), whereas a variant with an alanine appended to the amino terminus (Ala؉TIMP-2) was inactive. Removal of amino-terminal alanine by exopeptidase digestion restored protease inhibitor activity. This confirms the mechanistic importance of the amino-terminal amino group in the metalloproteinase inhibitory activity, as originally suggested from the x-ray structure of a complex of MMP-3 with TIMP-1 and a complex of TIMP-2 with MT-1-MMP. The Ala؉TIMP-2 variant exhibited conformational, pro-MMP-2 complex formation and fibroblast growth modulating properties of the wild type protein. These findings demonstrate that Ala؉TIMP-2 is an excellent biochemical tool for examining the specific role of MMP inhibition in the multiple functions ascribed to TIMPs.Studies of disease states, such as arthritis and tumor progression, have demonstrated that the matrix metalloproteinases (MMPs) 1 and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are critical effectors of extracellular matrix turnover. In recent years, the role of MMPs and TIMPs in a variety of pathologic entities has greatly expanded. This suggests that therapeutic targeting of these proteases may be beneficial not only in cancer and arthritis but also in a wide spectrum of pathologic conditions, ranging from rather common pathologic entities, such as nonhealing wounds (e.g. decubitus ulcers), to rare conditions such as pulmonary lymphangiomyomatosis (1, 2). Although current attempts to modulate MMP activities are based principally on substrate analog approaches, an alternative strategy is to utilize analogs of the endogenous inhibitors, the TIMPs.The TIMPs form a family of at least four members, the best characterized of which are TIMP-1 and -2 (2, 3). TIMP-2 (21 kDa) is a nonglycosylated protein that shares a 66% sequence identity with the glycosylated TIMP-1 (28.5 kDa); each protein contains 12 conserved cysteine residues that form six disulfide bonds essential for maintaining functional and structural integrity. The initial stage of TIMP inhibition of MMPs involves the formation of binary complexes. The amino-terminal cysteine appears to play a key role in blocking catalytic activity. The crystal structure of an MMP-TIMP-1 complex between the catalytic domain of stromelysin-1 (MMP-3) and human TIMP-1 showed that Cys-1 bidentally coordinates the active site zinc atom, and the Thr-2 s...

Analysis of the MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells

“…ALA + TIMP-2 is a TIMP-2 mutant containing an additional amino terminal alanine, which renders it unable to inhibit MMPs (Wingfield et al 1999). As such, while ALA + TIMP-2 cannot inhibit MMPs directly, or activate proMMPs, it is still capable of binding to cell surface receptors, such as α3β1 integrin, and inducing integrin signaling (Seo et al 2003).…”

TIMP-2 upregulates RECK expression via dephosphorylation of paxillin tyrosine residues 31 and 118

“…As TIMP-2 signaling requires its selective interaction with integrin a3b1 (Seo et al, 2003;Oh et al, 2004;Perez-Martinez and Jaworski, 2005), we sought to determine the effects of TIMP-2 on the phosphorylation status of FAK and Src. Human microvascular endothelial cells (hMVECs) were treated with 100 nM recombinant human TIMP-2 (Wingfield et al, 1999) and cell lysates analysed by immunoblotting using phosphospecific antibodies. It was found that TIMP-2 increases the phosphorylation levels of FAK at both Tyr-397, the autophosphorylation site, and Tyr-925, known as a docking site for the adaptor Grb2 (Parsons, 2003) (Figure 1a).…”

Differential expression and localization of TIMP-1 and TIMP-4 in human gliomas

“…For example, TIMP-1 stimulates the proliferation of erythroid precursors (Gasson et al, 1985), and both TIMP-1 and TIMP-2 can positively influence the proliferation of numerous cell types (Hayakawa et al 1992(Hayakawa et al , 1994Wingfield et al, 1999). Additionally, TIMP-2 inhibits in vitro proliferation of human microvascular endothelial cells stimulated with bFGF (Murphy et al, 1993), and TIMP-3 promotes apoptosis (Ahonen et al, 1998;Baker et al, 1998), possibly through stabilization of TNF alpha receptors (Smith et al, 1997).…”