Similarity of copper and technetium binding sites in human IgG

Kits for direct labeling of IgG with ^99mTc were used without modification for the preparation of [⁶⁷Cu]IgG. The IgG was pre-treated to generate thiolate groups which would bind ⁶⁷Cu. The direct labeling of reduced IgG with ⁶⁷Cu was highly efficient, resulting in approx. 95% ⁶⁷Cu binding. Non-reduced IgG (negative control) had labeling efficiencies of less than 10%. IgG pre-exposed to Cu(II) had reduced amounts of ^99mTc bound to it. The results demonstrate a direct relationship between copper- and ^99mTc-binding sites in IgG.     

Similarity networks of protein binding sites

An increasing attention has been dedicated to the characterization of complex networks within the protein world. This work is reporting how we uncovered networked structures that reflected the structural similarities among protein binding sites. First, a 211 binding sites dataset has been compiled by removing the redundant proteins in the Protein Ligand Database (PLD) (http://www-mitchell.ch.cam.ac.uk/pld/). Using a clique detection algorithm we have performed all-against-all binding site comparisons among the 211 available ones. Within the set of nodes representing each binding site an edge was added whenever a pair of binding sites had a similarity higher than a threshold value. The generated similarity networks revealed that many nodes had few links and only few were highly connected, but due to the limited data available it was not possible to definitively prove a scale-free architecture. Within the same dataset, the binding site similarity networks were compared with the networks of sequence and fold similarity networks. In the protein world, indications were found that structure is better conserved than sequence, but on its own, sequence was better conserved than the subset of functional residues forming the binding site. Because a binding site is strongly linked with protein function, the identification of protein binding site similarity networks could accelerate the functional annotation of newly identified genes. In view of this we have discussed several potential applications of binding site similarity networks, such as the construction of novel binding site classification databases, as well as the implications for protein molecular design in general and computational chemogenomics in particular.     

Regulation of human monocyte matrix metalloproteinases by SPARC

SPARC (secreted protein, acidic and rich in cysteine), also called osteonectin or BM-40, is a collagen-binding glycoprotein secreted by a variety of cells and is associated with functional responses involving tissue remodeling, cell movement and proliferation. Because SPARC and monocytes/macrophages are prevalent at sites of inflammation and remodeling in which there is connective tissue turnover, we examined the effect of SPARC on monocyte matrix metalloproteinase (MMP) production. Treatment of human peripheral blood monocytes with SPARC stimulated the production of gelatinase B (MMP-9) and interstitial collagenase (MMP-1). Experiments with synthetic peptides indicated that peptide 3.2, belonging to the alpha helical domain III of SPARC, is the major peptide mediating the MMP production by monocytes. SPARC and peptide 3.2 were also shown to induce prostaglandin synthase (PGHS)-2 as determined by Western and Northern blot analyses. The increase in PGHS-2 stimulated by SPARC or peptide 3.2 correlated with substantially elevated levels of prostaglandin E₂ (PGE₂) and other arachidonic acid metabolites as measured by radioimmunoassay and high performance liquid chromatography (HPLC), respectively. Moreover, the synthesis of MMP was dependent on the generation of PGE₂ by PGHS-2, since indomethacin inhibited the production of these enzymes and their synthesis was restored by addition of exogenous PGE₂ or dibutyryl cAMP (Bt₂cAMP). These results demonstrate that SPARC might play a significant role in the modulation of connective tissue turnover due to its stimulation of PGHS-2 and the subsequent release of PGE₂, a pathway that leads to the production of MMP by monocytes. J. Cell. Physiol. 173:327–334, 1997. Published 1997 Wiley-Liss, Inc.

1 This article was prepared by a group of United States government employees and non-United States government employees, and as such is subject to 17 U.S.C. Sec. 105.

     

A unique substrate binding mode discriminates membrane type-1 matrix metalloproteinase from other matrix metalloproteinases

In our study, we characterized the substrate recognition properties of membrane type-1 matrix metalloproteinase (MT1-MMP; also known as MMP-14), a key enzyme in tumor cell invasion and metastasis. A panel of optimal peptide substrates for MT1-MMP was identified using substrate phage display. The substrates can be segregated into four groups based on their degree of selectivity for MT1-MMP. Substrates with poor selectivity for MT1-MMP are comprised predominately of the Pro-X-X- downward arrow-X(Hy) motif that is recognized by a number of MMPs. Highly selective substrates lack the characteristic Pro at the P(3) position; instead they contain an Arg at the P(4) position. This P(4) Arg is essential for efficient hydrolysis and for selectivity for MT1-MMP. Molecular modeling indicates that the selective substrates adopt a linear conformation that extends along the entire catalytic pocket of MT1-MMP, whereas non-selective substrates are kinked at the conserved P(3) Pro residue. Importantly, the selective substrates can be made non-selective by insertion of a proline kink at P(3), without significantly reducing overall k(cat)/K(m) values. Altogether the study provides a structural basis for selective and non-selective substrate recognition by MT1-MMP. The findings in this report are likely to explain several aspects of MT1-MMP biology.     

Increased production of matrix metalloproteinases in Helicobacter pylori-associated human gastritis

BACKGROUND AND AIMS: Helicobacter pylori infection results in an active, chronic inflammation of the gastric mucosa. Previous studies have highlighted the importance of matrix metalloproteinases (MMPs) in diseases involving mucosal inflammation, prompting us to investigate MMP activity in H. pylori-induced gastritis. METHODS: Gastric biopsies were obtained from H. pylori-infected and uninfected volunteers, and MMP activity was assessed using substrate gel electrophoresis. MMP production was also evaluated by immunohistochemistry and real time-polymerase chain reaction. In parallel, tissue inhibitors of MMPs (TIMP) levels and TIMP-MMP complexes were examined in corresponding tissues using enzyme-linked immunosorbent assays and Western blotting. Finally, MMP production by gastric macrophages was determined after stimulation with H. pylori. RESULTS: Antral mucosa of H. pylori-infected subjects demonstrated a 19-fold higher MMP-9 activity than that of uninfected individuals. MMP-2 was present at lower levels, but was also increased in H. pylori-infected individuals, while there was no difference in the total levels of TIMP-1 and TIMP-2 between the groups of volunteers. Significant numbers of MMP-9-containing cells were only found in the H. pylori-infected antral mucosa. Tissue-resident macrophages were significantly increased in H. pylori-infected individuals, and double-staining showed MMP-9 colocalized to macrophages. Furthermore, gastric macrophages secreted MMP-9 in response to H. pylori bacteria. A corresponding 10-fold increase of gene expression of MMP-9 was seen in patients infected with H. pylori compared to uninfected individuals. CONCLUSIONS: Helicobacter pylori infection results in a substantial increase in MMP-9 and MMP-2 activity in the gastric mucosa, probably contributed to in large part by tissue-resident macrophages, while no changes were seen in the TIMP levels. The net increase in gastric MMP activity is likely to contribute to tissue damage during H. pylori-associated gastritis.     

The effect of IL-1beta on the expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in human chondrocytes

Interleukin-1 (IL-1) plays key roles in altering cartilage matrix turnover. This turnover is regulated by matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). In the present study, we examined the effect of IL-1beta on cell proliferation, alkaline phosphatase (ALPase) activity, and the expression of MMPs, and TIMPs in chondrocytes derived from normal human femoral cartilage. The cells were cultured in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum and 0, 1, 10, or 100 U/ml of IL-1beta for up to 28 days. The level of expression of MMPs and TIMPs was estimated by determining mRNA levels using real-time PCR and by determining protein levels using an enzyme-linked immunosorbent assay. Cell proliferation decreased in the presence of IL-1beta after day 21 of culture. ALPase activity decreased significantly in the presence of IL-1beta after day 10 of culture. The expression of MMP-1, -2, and -3 increased markedly in the presence of IL-1beta after day 21 of culture. MMP-13 expression increased markedly in the presence of IL-1beta on day 1 of culture, but decreased markedly after day 7. The expression of TIMP-1 increased significantly after day 14 of culture. The expression of TIMP-2 decreased significantly on day 1, but increased significantly from day 3 to day 14 of culture. These results suggest that IL-1beta may stimulate cartilage matrix turnover by increasing mainly MMP-13 production by the cells.     

Regulation of matrix biology by matrix metalloproteinases

Matrix metalloproteinases (MMPs) are endopeptidases that contribute to growth, development and wound healing as well as to pathologies such as arthritis and cancer. Until recently, it has been thought that MMPs participate in these processes simply by degrading extracellular matrix (ECM) molecules. However, it is now clear that MMP activity is much more directed and causes the release of cryptic information from the ECM. By precisely cleaving large insoluble ECM components and ECM-associated molecules, MMPs liberate bioactive fragments and growth factors and change ECM architecture, all of which influence cellular behavior. Thus, MMPs have become a focal point for understanding matrix biology.     

Fibrillin degradation by matrix metalloproteinases: identification of amino- and carboxy-terminal cleavage sites.

Fibrillin molecules form the structural framework of elastic fibrillin-rich microfibrils of the extracellular matrix. We have investigated the proteolysis of recombinant fibrillin molecules by five matrix metalloproteinases. Cleavage sites were defined at the carboxy-terminal end of the fibrillin-1 proline-rich region and the corresponding fibrillin-2 glycine-rich region (exon 10), and within exon 49 towards the carboxy-terminus of fibrillin-1. Cleavage at these sites is predicted to disrupt the structure and function of the fibrillin-rich microfibrils.     

Inactivation of human plasma C1-inhibitor by human PMN leucocyte matrix metalloproteinases

Highly purified human polymorphonuclear (PMN) leucocyte matrix metalloproteinases, collagenase and gelatinase, cleaved human plasma C1-inhibitor at the carboxyl site of Ala⁴³⁹ (P₆). This led to a concomitant loss of C1-inhibitor activity. An additional cleavage site at the carboxyl site of Ser⁴⁴¹ (P₄), was observed during PMN leucocyte gelatinase-induced inactivation, and a minor fragment of the plasma C1-inhibitor was generated.     

Serotonin binding sites of human blood platelets

The possible use of formaldehyde-fixed platelets to characterize and enumerate the specific receptor sites for 5-hydroxytryptamine was investigated. Equilibrium, pH-dependent capacity and specificity of 5-hydroxytryptamine binding by formaldehyde-fixed platelets were demonstrated. Analysis of binding data revealed two different sites: (i) high affinity with low capacity, and (ii) low affinity with high capacity. The results of binding studies using nonfixed control platelets were comparable with those of formaldehyde-fixed platelets. The versatility of formaldehyde fixation for studies of surface receptors was also shown by demonstrating nearly equal binding affinity for PGE₁ in control and formaldehyde-treated platelets. Our results indicate that formaldehyde fixation is a useful tool for the study of membrane receptor sites especially when active transport of the ligand such as serotonin is a problem.     

Extracellular matrix protein fibronectin induces matrix metalloproteinases in human prostate adenocarcinoma cells PC-3

Abstract

Studies on interaction of tumor cells with ECM components showed increased extracellular protease activity mediated by the family of matrix metalloproteinases (MMPs). Here we studied the effect of human prostate adenocarcinoma PC-3 cells–fibronectin (FN) interaction on MMPs and the underlying signaling pathways. Culturing of PC-3 cells on FN-coated surface upregulated MMP-9 and MMP-1. This response is abrogated by the blockade of α₅ integrin. siRNA and inhibitor studies indicate possible involvement of phosphatidyl-inositol-3-kinase (PI-3K), focal adhesion kinase (FAK) and nuclear factor-kappaB (NF-κB) in FN-induced upregulation of MMPs. FN treatment also enhanced phosphorylation of FAK, PI3K, protein kinase B (PKB or Akt), nuclear translocation of NF-κB, surface expression of CD-44, and cell migration. Our findings indicate that, binding of PC-3 cells to FN, possibly via α_5β1 integrin, induces signaling involving FAK, PI-3K, Akt, NF-κB followed by upregulation of MMP-9 and MMP-1. CD-44 may have role in modulating MMP-9 activity.     

Clinical implications of matrix metalloproteinases

Matrix metalloproteinases (MMPs) are a family of neutral proteinases that are important for normal development, wound healing, and a wide variety of pathological processes, including the spread of metastatic cancer cells, arthritic destruction of joints, atherosclerosis, pulmonary fibrosis, emphysema and neuroinflammation. In the central nervous system (CNS), MMPs have been shown to degrade components of the basal lamina, leading to disruption of the blood brain barrier and to contribute to the neuroinflammatory responses in many neurological diseases. Inhibition of MMPs have been shown to prevent progression of these diseases. Currently, certain MMP inhibitors have entered into clinical trials. A goal to the future should be to design selective synthetic inhibitors of MMPs that have minimum side effects. MMP inhibitors are designed in such a way that these can not only bind at the active site of the proteinases but also to have the characteristics to bind to other sites of MMPs which might be a promising route for therapy. To name a few: catechins, a component isolated from green tea; and Novastal, derived from extracts of shark cartilage are currently in clinical trials for the treatment of MMP-mediated diseases.     

Structural basis of matrix metalloproteinases and tissue inhibitors of metalloproteinases

The matrix metalloproteinases (MMPs) constitute a family of secreted/cell-surface-anchored multidomain zinc endopeptidases, all of which exhibit a catalytic domain of a common metzincin-like topology, and which are involved in degradation of the extracellular matrix but also in a number of other biologic processes. Normally, the proteolytic activity of the MMPs is precisely regulated by their main endogenous protein inhibitors, in particular the tissue inhibitors of metalloproteinases (TIMPs). Disruption of this balance results in serious diseases such as arthritis, tumor growth, and tumor metastasis, rendering the MMPs attractive targets for inhibition therapy. Knowledge of their tertiary structures is crucial for a full understanding of their functional properties and their associations with dysfunctions. Since the reports of the first atomic structures of MMPs and TIMPs in 1994, considerable structural information has become available about both of these families of substances. Many of the MMP structures have been determined as complexes with synthetic inhibitors, facilitating knowledge-based drug design. This review focuses on the currently available 3D structural information about MMPs and TIMPs.     

Temporospatial expression of matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in mouse antigen-induced arthritis

Several lines of evidence speak for an important role of matrix metalloproteinases (MMPs) in the development of progressive joint destruction. To better understand the role of MMPs and their tissue inhibitors (TIMPs) in this process, we have used the antigen-induced arthritis model to study the temporospatial expression of several MMPs and TIMPs during the progression of arthritis. Arthritis was induced by a single intra-articular injection of methylated bovine serum albumin (mBSA) into one or both knee joints of adult mice previously immunised against mBSA. Samples were collected at 3, 7, 21 and 42 days after induction of arthritis for histology and RNA extraction, and analysed by Northern hybridisation, histochemistry and immunohistochemistry for production of several MMPs and TIMPs −1, −2 and −3. A systematic analysis of MMP and TIMP mRNA levels in mouse knee joints demonstrated a general upregulation of both MMPs and TIMPs during progression of arthritis. Upregulation of MMP-9, −13 and −14 coincided with the advancement of cartilage degeneration, but the expression patterns of MMP-9 and −13 also followed the course of synovial inflammation. TIMPs were steadily upregulated throughout the examination period. Immunohistochemical localisation of MMPs and TIMPs suggested the synovium to be the major source of MMP and TIMP production in arthritis, although articular cartilage chondrocytes also showed an increased production of both MMPs and TIMPs.     

基质金属蛋白酶及其组织抑制剂研究进展

基质金属蛋白酶家族是细胞外基质降解过程中的重要酶类,组织金属蛋白酶抑制剂是基质金属蛋白酶的天然抑制物。研究证实,细胞外基质中基质金属蛋白酶及其组织抑制剂的失衡与多种病理机制有关,尤其与肿瘤的侵袭和转移密切相关。本就基质金属蛋白酶及其组织抑制剂的性质、结构以及功能进行了综述。     

Effect of heparin on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts

The influence of heparin and a heparin fragment devoid of anticoagulant activity on the production of matrix metalloproteinases and tissue inhibitors of metalloproteinases by human dermal fibroblasts was studied. Doses (0.1-400 microg/ml) responses were performed and data obtained were similar whatever heparin or fragment was used. The basal expression of collagenase by fibroblasts decreased quasi-linearly with increasing doses of heparins from 1 to 400 microg/ml. TIMP-1 levels were not affected by supplementing serum free culture medium with heparins. On the contrary, at low concentration, i.e. 1-10 microg/ml, heparins stimulated the secretion of both 72-kDa gelatinase (1.4-1.6-fold) and particularly TIMP-2 (>4-fold). At high doses, MMP-2 and TIMP-2 production by fibroblasts returned to basal levels. These results suggested that the local concentration of heparin released by mast cells could be instrumental in modulating fibroblast growth and proteolytic phenotype.