TNF-K K converting enzyme (TACE; ADAM-17) is a membrane-bound disintegrin metalloproteinase that processes the membrane-associated cytokine proTNF-K K to a soluble form. Because of its putative involvement in inflammatory diseases, TACE represents a significant target for the design of specific synthetic inhibitors as therapeutic agents. In order to study its inhibition by tissue inhibitors of metalloproteinases (TIMPs) and synthetic inhibitors of metalloproteinases, the catalytic domain of mouse TACE (rTACE) was overexpressed as a soluble Ig fusion protein from NS0 cells. rTACE was found to be well inhibited by peptide hydroxamate inhibitors as well as by TIMP-3 but not by TIMP-1, -2 and -4. These results suggest that TIMP-3, unlike the other TIMPs, may be important in the modulation of pathological events in which TNF-K K secretion is involved.z 1998 Federation of European Biochemical Societies.

A recombinant soluble form of the catalytic domain of human ADAM-10 was expressed as an Fc fusion protein from myeloma cells. The ADAM-10 was catalytically active, cleaving myelin basic protein and peptides based on the previously described`metallosheddase' cleavage sites of tumour necrosis factor K K, CD40 ligand and amyloid precursor protein. The myelin basic protein degradation assay was used to demonstrate that hydroxamate inhibitors of matrix metalloproteinases (MMPs) were also inhibitors of ADAM-10. The natural MMP inhibitors, TIMP-2 and TIMP-4 were unable to inhibit ADAM-10, but TIMP-1 and TIMP-3 were inhibitory. Using a quenched fluorescent substrate assay and ADAM-10 we obtained approximate apparent inhibition constants of 0.1 nM (TIMP-1) and 0.9 nM (TIMP-3). The TIMP-1 inhibition of ADAM-10 could therefore prove useful in distinguishing its activity from that of TACE, which is only inhibited by TIMP-3, in cell based assays.z 2000 Federation of European Biochemical Societies.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor ␣ (TNF␣) cleavage site, a glutathione S-transferase-pro-TNF␣ fusion protein, and was found to shed pro-TNF␣ when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNF␣-converting activity suggests a role in inflammation.Matrix metalloproteinases (MMPs) 1 are a large family of structurally related zinc-dependent endopeptidases that are essential in tissue remodeling under physiological and pathological conditions. These include morphogenesis, angiogenesis, tissue repair, arthritis, tumor invasion, and migration of inflammatory leukocytes (1-5) A subset of the MMP family, the membrane type MMPs (MT-MMPs) have more recently been identified (6, 7). To date six such enzymes have been described, and MT1-, -2-, and -3-MMP have been shown to have a wide range of activities against extracellular matrix proteins (8 -13). The activity of MT-MMPs has been implicated in cell invasion of the ECM (4). Possibly more importantly, they are also activators of pro-MMP2 and pro-MMP13 which has been correlated with the invasiveness of tumor and other cells (7,9,14,15). Like other MMPs, the activity of MT-MMPs can be modulated by the tissue inhibitors of MMPs or TIMPs; however, TIMP1 was found to be a poor inhibitor of MT1-, -2-, and -5-MMP (9, 10, 16). TIMP2 and MT1-MMP can also associate to form a pro-MMP2 receptor at the cell surface, leaving the C terminus of TIMP2 free to bind pro-MMP2. This allows efficient activation of pro-MMP2 by adjacent TIMP2-free MT1-MMP and may be a common mechanism for pro-MMP2 activation by .The fourth member of the MT-MMP family, MT4-MMP, has the least degree of sequence identity to the other family members (7, 18). The human cDNA (hMT4-MMP) originally isolated from a human breast carcinoma cell line was found to lack the signal sequence and part of the pro-domain. Northern blot analysis of the tissue mRNA distribution showed hMT4-MMP to be localized in brain, colon, ovary...

Cleavage of amyloid precursor protein (APP) by the Alzheimer's β-secretase (BACE1) is a key step in generating amyloid β-peptide, the main component of amyloid plaques. Here we report evidence that heparan sulfate (HS) interacts with β-site APP-cleaving enzyme (BACE) 1 and regulates its cleavage of APP. We show that HS and heparin interact directly with BACE1 and inhibit in vitro processing of peptide and APP substrates. Inhibitory activity is dependent on saccharide size and specific structural characteristics, and the mechanism of action involves blocking access of substrate to the active site. In cellular assays, HS specifically inhibits BACE1 cleavage of APP but not alternative cleavage by α-secretase. Endogenous HS immunoprecipitates with BACE1 and colocalizes with BACE1 in the Golgi complex and at the cell surface, two of its putative sites of action. Furthermore, inhibition of cellular HS synthesis results in enhanced BACE1 activity. Our findings identify HS as a natural regulator of BACE1 and suggest a novel mechanism for control of APP processing.

Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied.Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition.Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays.Measurements and Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures.Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.

Optimization of lead compound 1, through extensive use of structure-based design and a focus on PI3Kδ potency, isoform selectivity, and inhaled PK properties, led to the discovery of clinical candidates 2 (GSK2269557) and 3 (GSK2292767) for the treatment of respiratory indications via inhalation. Compounds 2 and 3 are both highly selective for PI3Kδ over the closely related isoforms and are active in a disease relevant brown Norway rat acute OVA model of Th2-driven lung inflammation.

The ADAM family of proteases are type I transmembrane proteins with both metalloproteinase and disintegrin containing extracellular domains. ADAMs are implicated in the proteolytic processing of membrane-bound precursors and involved in modulating cell^cell and cell^matrix interactions. ADAM8 (MS2, CD156) has been identi¢ed in myeloid and B cells. In this report we demonstrate that soluble ADAM8 is an active metalloprotease in vitro and is able to hydrolyse myelin basic protein and a variety of peptide substrates based on the cleavage sites of membrane-bound cytokines, growth factors and receptors which are known to be processed by metalloproteinases. Interestingly, although ADAM8 was inhibited by a number of peptide analogue hydroxamate inhibitors, it was not inhibited by the tissue inhibitors of metalloproteinases (TIMPs). We also demonstrate that the activity of recombinant soluble ADAM9 (meltrin-Q Q, MDC9) lacks inhibition by the TIMPs, but can be inhibited by hydroxamate inhibitors. The lack of TIMP inhibition of ADAM8 and 9 contrasts with other membraneassociated metalloproteinases characterised to date in this respect (ADAM10, 12, 17, and the membrane-type metalloproteinases) which have been implicated in protein processing at the cell surface. ß 2002 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

ADAM (a disintegrin and metalloproteinases) are a recently discovered gene family of proteins with sequence similarity to the reprolysin family of snake venom metalloproteinases, and about one-third of the family members have the catalytic site consensus sequence in their metalloproteinase domains. We screened the mRNA expression of 11 different ADAM species with putative metalloproteinase activity in human non-small cell lung carcinomas by RT-PCR, and found that prototype membrane-anchored ADAM28 (ADAM28m) and secreted ADAM28 (ADAM28s) are predominantly expressed in the carcinoma tissues. Real-time quantitative PCR demonstrated that the expression levels of ADAM28m and ADAM28s are significantly 16.8-fold and 9.0-fold higher in the carcinomas than in the non-carcinoma tissues, respectively. In addition, the expression levels of ADAM28m and ADAM28s were significantly higher in the carcinomas with >30 mm in diameter than in those 530 mm. The expression levels were also significantly higher in the carcinomas with lymph node metastasis than in those without metastasis. MIB1-positive cell index of the carcinomas had a direct correlation with the expression levels of ADAM28m and ADAM28s (r 5 0.667, p < 0.001 and r 5 0.535, p < 0.01, respectively). In situ hybridization and immunohistochemistry demonstrated that ADAM28 is expressed predominantly in the carcinoma cells. Immunoblot analysis showed the activated form of ADAM28 in the carcinoma tissues. These data demonstrate for the first time that ADAM28 is overexpressed and activated in human non-small cell lung carcinomas, and suggest the possibility that ADAM28 plays a role in cell proliferation and progression of the human lung carcinomas. ' 2005 Wiley-Liss, Inc.Key words: ADAM; MMP; lung cancer; proliferation; metastasis ADAM (a disintegrin and metalloproteinases) are a gene family that have significant sequence similarity to the reprolysin/adamalysin family of snake venom metalloproteinases.1 ADAM are composed of several domains including propeptide, metalloproteinase, disintegrin, cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and cytoplasmic tail domains. More than 30 members of the ADAM gene family have been identified in a variety of animal species (see http://www.people.virginia.edu/ jw7g/ Table_of_the_ADAMs.html). Although the specific biological functions of ADAM are not clear, they may be involved in shedding of various membrane-anchored receptors and proteins, degradation of extracellular matrix (ECM), and cell adhesion and migration.2,3 About one-third of the ADAM members have the catalytic consensus sequence (HEXGHXXGXXHD) in their metalloproteinase domains, and are predicted to have catalytic activity. ADAM10 is reported to digest myelin basic protein 4 and type IV collagen.5 ADAM9 degrades insulin B chain, 6 and ADAM28 cleaves myelin basic protein 7 and insulin-like growth factor binding protein-3 (IGFBP-3). 8 In addition, the precursor of tumor necrosis factor-a (proTNF-a) is processed to the mature form by ADAM10 and ADAM17. 9 CD23 ...