A transient COS-7 cell expression system was used to investigate the functional domain arrangement of tissue inhibitor of metalloproteinases-3 (TIMP-3), specifically to assess the contribution of the amino-and carboxylterminal domains of the molecule to its matrix metalloproteinase (MMP) inhibitory and extracellular matrix (ECM) binding properties. Wild type TIMP-3 was entirely localized to the ECM in both its glycosylated (27 kDa) and unglycosylated (24 kDa) forms. A COOH-terminally truncated TIMP-3 molecule was found to be a non-ECM bound MMP inhibitor, whereas a chimeric TIMP molecule, consisting of the NH 2 -terminal domain of TIMP-2 fused to the COOH-terminal domain of TIMP-3, displayed ECM binding, albeit with a lower affinity than the wild type TIMP-3 molecule. Thus the functional domain arrangement of TIMP-3 is analogous to that seen in TIMP-1 and -2, namely that the NH 2 -terminal domain is responsible for MMP inhibition whereas the COOH-terminal domain is most important in mediating the specific functions of the molecule. A mutant TIMP-3 in which serine 181 was changed to a cysteine, found in Sorsby's fundus dystrophy, a hereditary macular degenerative disease, was also expressed in COS-7 cells. This gave rise to an additional 48-kDa species (possibly a TIMP-3 dimer) that retained its ability to inhibit MMPs and localize to the ECM. These data favor the hypothesis that the TIMP-3 mutations seen in Sorsby's fundus dystrophy contribute to disease progression by accumulation of mutant protein rather than by the loss of functional TIMP-3.The matrix metalloproteinases (MMPs) 1 are a family of zincdependent endopeptidases that exist in both secreted and membrane bound forms. The enzymes are initially expressed as inactive pro-enzymes becoming activated by proteolytic cleavage of their amino termini. The activity of MMPs is tightly regulated by the tissue inhibitors of metalloproteinases (TIMPs), a family of secreted proteins currently comprising four members (TIMP-1-TIMP-4) (1-4). The balance between MMPs and TIMPs regulates the integrity of the proteinacious extracellular matrix (ECM) and thus plays a key role in a wide range of physiological processes that include embryonic development, connective tissue remodeling, wound healing, glandular morphogenesis, and angiogenesis. An imbalance in MMP/ TIMP expression has been implicated in various diseases such as erosive joint disease, cardiovascular disease, and cancer (reviewed in Refs. 5-7).The TIMPs form high affinity 1:1 complexes with the active forms of most MMPs (reviewed in Ref. 8) but show varying specificity for different pro-MMPs allowing TIMPs to control the activation of specific MMPs (9 -12). Activities have also been ascribed to the TIMPs that are independent of their ability to inhibit MMPs; for example, anti-angiogenic and erythroid-potentiating activities have been described for TIMP-1 that are independent of MMP inhibition (13,14). Likewise TIMP-2 shows MMP independent inhibition of endothelial tube formation (15). These differences in TIMP ...

No abstract

The ADAMs (a disintegrin and metalloprotease) are membrane proteins containing both protease and adhesion domains and thus may be potentially important in cancer invasion and metastasis. The aim of our study was to investigate the distribution and potential clinical significance of ADAM-9 in breast cancer. ADAM-9 expression was measured using both reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. ADAM-9 mRNA was expressed more frequently in both breast carcinomas (72/110, 66%) and fibroadenomas (21/38, 55%) compared to normal breast tissue (6/25, 24%) (p ‫؍‬ 0.0004, p ‫؍‬ 0.028, respectively). Multiple forms of ADAM-9 protein were detected by Western blotting, i.e., at 124, 84 and 48 kDa under reducing conditions and at 115, 76, 55, 52 and 46 kDa under nonreducing conditions. The 84 and 55 kDa forms were detected more frequently in the primary cancers compared to normal breast tissue (p < 0.0001, p ‫؍‬ 0.0002, respectively). In addition, relative levels of the 84 kDa mature form were significantly higher in the primary cancers than in the fibroadenomas (p ‫؍‬ 0.003), while the reverse was found for the 124 kDa precursor form (p ‫؍‬ 0.026). In the carcinomas, the 84 kDa form of ADAM-9 protein was expressed at higher levels in node-positive than node-negative cancers (p ‫؍‬ 0.05) and correlated positively with HER-2/neu protein levels (r ‫؍‬ 0.313, p ‫؍‬ 0.016). This is the first report to describe expression of any ADAM in a large number of human carcinomas. © 2003 Wiley-Liss, Inc. Key words: breast cancer; ADAMs; ADAM-9, HER-2/neu; metastasisThe process of cancer invasion and metastasis is a multistep event that involves angiogenesis, local invasion, cell migration, intravasation, extravasation and growth at a secondary site (for review, see reference 1). Although multiple genes have been implicated in cancer dissemination, among the best characterised are those encoding matrix degrading proteases and adhesion proteins (for review, see reference 2). Proteases such as urokinase plasminogen activator (uPA) and specific matrix metalloproteinases (MMPs) degrade or remodel the extracellular matrix (ECM) allowing cancer cells to invade locally and ultimately form distant metastases. 3 Proteases may also promote metastasis by releasing or activating factors [e.g., fibroblast growth factor-2 (FGF-2), transforming growth factor-␤ (TGF-␤), vascular endothelial growth factor (VEGF)], which enhance cell growth, cell migration and angiogenesis. 4,5 Consistent with their role in metastasis, high levels of multiple proteases have been associated with adverse prognosis in different malignancies (for review, see reference 6).As with proteases, adhesion molecules are also involved at multiple stages during invasion and metastasis. 2,7 In the initial stages of the metastatic pathway, cells must detach themselves from their neighbouring cells and adhere to the basement membrane. As the invading cell migrates through the extracellular matrix (ECM), the leading edge undergoes consecutive cycles of adhesio...

No abstract

No abstract

No abstract

Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the retina, caused by mutations in exon 5 of the gene for tissue inhibitor of metalloproteinases-3 (TIMP-3). The mechanism by which these mutations give rise to the disease phenotype is unknown. In an attempt to identify common properties of these molecules that might underlie the disease phenotype, a range of SFD mutants were expressed from human retinal pigment epithelial (RPE) cells. This showed that resistance to turnover, resulting from intermolecular disulfide bond formation, was a common property of all the SFD mutants examined, providing a possible explanation for the increased deposition of the protein observed in eyes from SFD patients. In contrast, SFD mutants varied in their ability to inhibit cell-surface activation of matrix metalloproteinase-2 (MMP-2), a potent mediator of angiogenesis, ranging from being fully active to totally inactive. These data show that increased deposition of active TIMP-3, rather than dysregulation of metalloproteinase inhibition, is likely to be the primary, initiating event in SFD.

The linked structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been altered by site-directed mutagenesis and placed under the control of an inducible phage-T7-specific plasmid promoter in Escherichia coli. Conditions have been found under which both a-and fl-subunits are produced in soluble form, in near 1: 1 ratio, and assemble to form apo-mutase totalling about 5 % of the total cellular protein. Methylmalonyl-CoA mutase purified from these cells could be readily converted into the holoenzyme by addition of adenosylcobalamin. The active holoenzyme apparently crystallizes in the same space group as an inactive corrinoid-containing form of the enzyme obtained previously.