Tissue inhibitor of metalloproteinase (TIMP)-4 binds pro-matrix metalloproteinase (MMP)-2 and efficiently inhibits MT1-MMP, but unlike TIMP-2 neither forms a trimolecular complex nor supports pro-MMP-2 activation. To investigate the structural and functional differences between these two TIMPs, the C-terminal domains (C-TIMP-4 and C-TIMP-2) were expressed independently from their N domains and mutations were introduced into the C-terminal tails. Myoglobin was used as a novel recombinant fusion protein partner because spectroscopic measurement of the heme Soret absorbance at 408 nm readily enabled calculation of the molar equivalent of the red-colored recombinant protein, even in complex protein mixtures. Both C-TIMP-4 and C-TIMP-2 bound pro-MMP-2 and blocked concanavalin A-induced cellular activation of the enzyme. Measurement of k on rates revealed that the inhibition of MMP-2 by TIMP-4 is preceded by a C domain docking interaction, but in contrast to TIMP-2, this is not enhanced by a C-terminal tail interaction and so occurs at a slower rate. Indeed, the binding stability of C-TIMP-4 was unaltered by deletion of the C-terminal tail, but replacement with the tail of TIMP-2 increased its affinity for pro-MMP-2 by ϳ2-fold, as did substitution with the TIMP-2 C-terminal tail acidic residues in the tail of C-TIMP-4 (V193E/ Q194D). Conversely, substitution of the C-terminal tail of C-TIMP-2 with that of TIMP-4 reduced pro-MMP-2 binding by ϳ75%, as did reduction of its acidic character by mutation to the corresponding TIMP-4 amino acid residues (E192V/D193Q). Together, this shows the importance of Glu 192 and Asp 193 in TIMP-2 binding to pro-MMP-2; the lack of these acidic residues in the TIMP-4 C-terminal tail, which reduces the stability of complex formation with the MMP-2 hemopexin C domain, probably precludes TIMP-4 from supporting the activation of pro-MMP-2.Matrix metalloproteinases (MMPs) 1 are a family of important processing enzymes that can cleave and regulate the activity of an expanding degradome of bioactive molecules (1-5) as well as degrade extracellular matrix proteins in pathology (6). Gelatinase A (MMP-2) has been implicated in numerous biological processes, including the activation of cytokines such as tumor necrosis factor-␣ (7), transforming growth factor-␤1 (8), and interleukin-1␤ (9). MMP-2 has also been shown to have anti-inflammatory actions by converting monocyte chemokine agonists to antagonists (10, 11) and causes loss of protection of CD4 ϩ cells from human immunodeficiency virus-1 infection by processing stromal cell-derived factor-1␣ (12).2 MMP-2 cleavage of type IV collagen, a major component of basement membranes, is important for tumor cell metastasis and angiogenesis (14 -16). In view of these diverse and biologically important functions, it is not surprising that MMPs are under tight regulatory control, both at the transcriptional and post-transcriptional levels (17-19). Post-translational regulation is also pivotally important in regulating proteolytic activity in the pericellul...

Secretin is the most potent regulator of pancreatic bicarbonate, electrolyte and volume secretion. In this report, the organization of the human secretin receptor (hSR) gene was characterized by overlapping genomic phage clones. The hSR gene consists of 13 exons and 12 introns with all the splice donor and acceptor sites conforming to the canonical GT/AG rule. By transient reporter gene assays, the wild-type promoter, containing 3.0 kb of the hSR gene 5P P flanking region, was able to drive 5.8 þ 0.6 and 6.6 þ 0.2-fold (P 6 0.01) increases in luciferase activities in pancreatic ductule-derived PANC-1 and BPD-1 cells, respectively. By subsequent 5P P and 3P P deletion analysis, a promoter element was identified within 3 3408 to 3 3158, relative to the ATG codon. This promoter element was found to be cellspecific since it could drive reporter gene expression in PANC-1 and BPD-1 cells but not in Hs 262.St, Hs 746T and K KT3-1 cells. The study of the transcriptional control of human secretin and its receptor should shed light on the pathological developments of pancreatic cancer and autism in the future.z 1999 Federation of European Biochemical Societies.

No abstract

cotranscribed genes, termed the mercury resistance mer operon, allows them to survive in environments contain mercurial compounds. The mer operon encodes proteins capable of converting inorganic (Hg(II)) and organiomercurial compounds (such as methylmercury, MeHg) to less toxic form (Hg(0)). The mer operon transcription is activated by MerR family protein. MerR family protein turns into a transcription activator upon Hg(II) binding. To understand how MerR family protein regulates the transcription of mer operon, we have determined the structure of MerR family protein from Gram-positive bacteria Bacillus megaterium MB1 by the multiwavelength anomalous diffraction method. The MerR family protein monomer contains a DNA-binding domain, a dimerization helix and a metal-binding motif. Like most other transcription activators, dimerization of MerR family protein is required for function. A total of four MerR family protein dimers are present in the asymmetric unit, all exhibiting similar quaternary structure. The N-terminal DNA-binding domain contains three helices which form a helix-turn-helix motif. The motif is followed by an 8-residue loop and the dimerization domain that is composed of an 8.5-turn alfa-helix. Dimerization of MerR family protein is mediated by packing the two long helices as an antiparallel coiled-coil. The C-terminal metal-binding motif is quite small, consisted of two 3 10 helices and two connecting residues. A total of four MerR family protein dimers are present in the asymmetric unit, all exhibiting similar quaternary structure. Compared with the structures of other MerR family members, our structure suggests that Hg(II) binding may alter the quaternary structure of MerR family protein. Such a structural transition may reposition the two DNA-binding domains, thus allows the promoter DNA to interact productively with the RNA polymerase to turn on transcription.