Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.23 (MMP)
 4,246 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Primary airway epithelial cell cultures can provide a faithful representation of the in vivo airway while allowing for a controlled nutrient source and isolation from other tissues or immune cells. The methods used have significant differences based on tissue source, cell isolation, culture conditions, and assessment of culture purity. We modified and optimized a method for generating tracheal epithelial cultures from Syrian golden hamsters and characterized the cultures for cell composition and function. Soon after initial plating, the epithelial cells reached a high transepithelial resistance and formed tight junctions. The cells differentiated into a heterogeneous, multicellular culture containing ciliated, secretory, and basal cells after culture at an air-liquid interface (ALI). The secretory cell populations initially consisted of MUC5AC-positive goblet cells and MUC5AC/CCSP double-positive cells, but the makeup changed to predominantly Clara cell secretory protein (CCSP)-positive Clara cells after 14 d. The ciliated cell populations differentiated rapidly after ALI, as judged by the appearance of beta tubulin IV-positive cells. The cultures produced mucus, CCSP, and trypsin-like proteases and were capable of wound repair as judged by increased expression of matrilysin. Our method provides an efficient, high-yield protocol for producing differentiated hamster tracheal epithelial cells that can be used for a variety of in vitro studies including tracheal cell differentiation, airway disease mechanisms, and pathogen-host interactions.
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PMID:Differentiated cultures of primary hamster tracheal airway epithelial cells. 1578 7

In contrast to the prevalent view in the literature hitherto, the present study shows that pancreatic trypsin can activate human promatrix metalloproteinase-2 (proMMP-2). It is shown that trypsin's ability to activate proMMP-2 is dependent on various environmental factors such as the level of exogenously added Ca(2+) and Brij-35, temperature, as well as trypsin concentration. The activation occurred as a sequential processing of the proenzyme, initially generating an active 62kDa species. This was followed by successive truncation of the C-terminal domain, giving rise to active species of 56kDa, 52kDa and 50kDa. Tissue inhibitor of matrix metalloproteinases-2 (TIMP-2) prevented the trypsin-mediated C-terminal truncation, without affecting the generation of the 62kDa species, while the presence of EDTA increased the rate of the trypsin-mediated activation of proMMP-2. MALDI-TOF MS analysis of the 50kDa form indicated that trypsin generated active forms with either Lys87 or Trp90 as the N-terminal residue and Arg538 as a C-terminal residue. The trypsin-activated MMP-2 was active in solution against both synthetic and physiologic substrates, and the steady-state kinetic coefficients k(cat), K(m) and k(cat)/K(m) were determined for the enzyme activated either by APMA, membrane-type 1 matrix metalloproteinase (MT1-MMP) or trypsin. The trypsin-activated MMP-2 exhibited slightly lower k(cat) and k(cat)/K(m) values as well as a slightly higher K(i) value against TIMP-1 compared to the enzyme activated by APMA or MT1-MMP. Docking studies of TIMP-1 revealed that the slightly weaker binding of the inhibitor to the trypsin-activated MMP-2 could be attributed to its shorter N terminus (Lys87/Trp90 versus Tyr81), as Phe83 and Arg86 interacted directly with the inhibitor. Our results suggest that the trypsin-activated MMP-2 possesses the catalytic and regulatory potential to be of significance in vivo.
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PMID:Pancreatic trypsin activates human promatrix metalloproteinase-2. 1595 Feb 41

We report the development and validation of a novel suite of programs, FITTED 1.0, for the docking of flexible ligands into flexible proteins. This docking tool is unique in that it can deal with both the flexibility of macromolecules (side chains and main chains) and the presence of bridging water molecules while treating protein/ligand complexes as realistically dynamic systems. This software relies on a genetic algorithm to account for the flexibility of the two molecules as well as the location of bridging water molecules. In addition, FITTED 1.0 features a novel application of a switching function to retain or displace key water molecules from the protein-ligand complexes. Two independent modules, ProCESS and SMART, were developed to set up the proteins and the ligands prior to the docking stage. Validation of the accuracy of the software was achieved via the application of FITTED 1.0 to the docking of inhibitors of HIV-1 protease, thymidine kinase, trypsin, factor Xa, and MMP to their respective proteins.
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PMID:Docking ligands into flexible and solvated macromolecules. 1. Development and validation of FITTED 1.0. 1730 29

Matrix metalloproteinase-9 (MMP-9) has been shown to be involved in the development of diabetic nephropathy (DNP). We studied the levels, molecular forms, and degree of activation of urinary MMP-8, -9, -14, trypsin-1 and -2, as well as tumor-associated trypsin inhibitor (TATI) of DNP patients and healthy controls. Urinary samples were analyzed for MMPs by Western blotting and gelatin zymography and for trypsin-1, -2, and TATI by time-resolved immunofluorometric assays. Total MMP-8 immunoreactivity, the proportion of active MMP-9, and gelatinolytic activity in urine were significantly higher in DNP patients than in controls. In urine of DNP patients the proportion of active polymorphonuclear neutrophil (PMN)-type (but not fibroblast-type) MMP-8 was increased. MMP-8 and MMP-9 were found to form high molecular weight complexes in DNP urine. Total immunoreactivity of soluble urinary MMP-14 and the levels of trypsin (TRY)-1 and TRY-2, but not of TATI, were also significantly increased in DNP. Zymography, Western blotting, and immunofluorometric analysis of DNP urine showed a significant association especially between activation of MMP-9 as well as PMN-type MMP-8 and TRY-2. Our findings suggest that a trypsin-MMP cascade is involved in the pathogenesis of DNP, which may offer new possibilities for diagnosis and treatment of DNP with MMP inhibitors.
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PMID:Urinary matrix metalloproteinase -8, -9, -14 and their regulators (TRY-1, TRY-2, TATI) in patients with diabetic nephropathy. 1842 24

The sulfamide moiety, similarly to the structurally related sulfonamide and sulfamate ones, is widely employed in medicinal chemistry for the design of biologically active compounds. Amongst the enzymes for which sulfamide-based inhibitors were designed are the carbonic anhydrases (CAs), and a large number of proteases belonging to the aspartic protease (HIV-1 protease, gamma-secretase), serine protease (elastase, chymase, tryptase and thrombin, among others) and metalloproteinase (carboxypeptidase A [CPA] and matrix metalloproteinase [MMP]) families. Some steroid sulfatase (STS) and protein tyrosine phosphatase inhibitors belonging to the sulfamide class of derivatives have also been reported. In all these compounds, many of which show low nanomolar affinity for the target enzymes for which they have been designed, the free or substituted sulfamide moiety plays an important role in the binding of the inhibitor to the active site cavity. This is achieved either by directly coordinating to the metal ion found in some metalloenzymes (CAs, CPA, STS), usually by means of one of the nitrogen atoms present in the sulfamide motif, or, as in the case of the cyclic sulfamides, acting as HIV protease inhibitors interacting with the catalytically critical aspartic acid residues of the active site by means of an oxygen atom belonging to the HN-SO(2)-NH motif that substitutes a catalytically essential water molecule. In other cases, the sulfamide moiety is important for inducing desired physicochemical properties to the drug-like compounds incorporating it, such as enhanced water solubility, better bioavailability etc., due to the intrinsic properties of this highly polarised moiety when attached to an organic scaffold. This interesting motif is, thus, of great value for the design of pharmacological agents with many applications.
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PMID:The sulfamide motif in the design of enzyme inhibitors. 2014 8

The goal of the present study was to devise an in vitro model suitable for investigations of the homing of mesenchymal stem cells to sites of injury. Such a model was designed on the basis of a "transwell" assay, with an insert seeded with human bone marrow stromal cells and a well with a desired cell type. To mimic physiological environment and to simulate "injury", cells in a well were maintained not only on tissue culture plastic but also on collagens I and IV, major matrix components in musculoskeletal and adipose tissues respectively, and subjected to a severe thermal stress. The results obtained showed a massive translocation of bone marrow stromal cells through the inserts' membrane toward the "injury" site. Unexpectedly, it emerged that collagen matrix is essential in producing such a migration. The results obtained suggest that upon injury cells secrete a substance which interacts with collagen matrix to produce a homing agent. The substance in question appears to be a protease and its interaction with the collagen matrix appears to be a digestion of the latter into fragments shown to be chemotactic. Both AEBSF, an inhibitor of serine proteases, and leupeptin, an inhibitor of cysteine proteases as well as of trypsin-like serine proteases, but not the broad spectrum MMP inhibitor marimastat, significantly inhibit the observed homing effect and this inhibition is not due to cytotoxicity. Moreover, immunoprecipitation of HTRA1, a trypsin-like serine protease known to be secreted by cells differentiating into all three major mesenchymal lineages and by stressed cells in general and shown to degrade a number of matrix proteins including collagen, significantly diminished the homing effect. The data suggest that this protease is a major contributor to the observed chemotaxis of bone marrow stromal cells. The present study indicates that collagen fragments can mediate the migration of bone marrow stromal cells. The results also suggest that, at least in musculoskeletal and in adipose tissues, matrix remodeling occurrences, usually closely associated with tissue remodeling, should also be regarded as potential stem cells recruitment events.
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PMID:Matrix remodeling as stem cell recruitment event: a novel in vitro model for homing of human bone marrow stromal cells to the site of injury shows crucial role of extracellular collagen matrix. 2082 13

We report the results of phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide (TBTO), an immunotoxic compound. After cell lysis, phosphoproteins were isolated using Phosphoprotein Purification Kit, separated by SDS-PAGE and subsequently digested with trypsin. Phosphopeptides were enriched employing titanium dioxide, and the obtained fractions were analyzed by nano-LC-MS/MS. A total of 160 phosphoproteins and 328 phosphorylation sites were identified in thymoma cells. Among the differentially phosphorylated proteins identified in TBTO-treated cells were key enzymes, which catalyze rate-limiting steps in pathways that are sensitive to cellular energy status. These proteins included acetyl-CoA carboxylase isoform 1, which catalyzes the rate-limiting step of fatty acid synthesis. Another enzyme was glutamine: fructose-6-phosphate amidotransferase, GFAT1, the first and rate-limiting enzyme for the hexoamine synthesis pathway. Pyruvate dehydrogenase (PDH), a multicomplex enzyme that catalyzes the rate-limiting step of aerobic oxidation of fuel carbohydrates, was identified in both TBTO-treated and control cells; however, phosphorylation at residue S293, known to inhibit PDH activity, was identified only in control cells. A lower expression level of ribosomal protein S6 kinase 1, a downstream kinase of the mammalian target of rapamycin signaling pathway implicated in protein synthesis through phosphorylation of 40 ribosomal S6, was observed in the treated cells. Giant kinases like AMP-activated protein kinase (AMPK) and cAMP-dependent protein kinase (PKAR1A), which are known to mediate the phosphorylation of these enzymes, were identified in TBTO-treated cells. Downregulation of proteins, such as MAPK, matrin-3 and ribonucleotide reductase, subunit RRM2, which are implicated in cell proliferation, was also observed in TBTO-treated cells. Together, the results show that TBTO affects proliferation and energy sensor pathways.
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PMID:Phosphoproteomic analysis of mouse thymoma cells treated with tributyltin oxide: TBTO affects proliferation and energy sensing pathways. 2217 45

Enhanced proteolysis and altered tight junction (TJ) proteins associate with carcinoma invasion. We hypothesized that trypsin-2, a tumor-associated serine proteinase, induces tongue carcinoma invasion by activating pro-membrane type-1 matrix metalloproteinase (MT1-MMP) and disturbing the TJs. The effects of invasion were analyzed using trypsin-2 over-expressing human tongue squamous cell carcinoma cells (Try2-HSC-3) in vitro and in vivo. The invasion of Try2-HSC-3 cells was increased in mouse xenografts and human organotypic model. Trypsin-2 activated proMT1-MMP, as well as altered the expression of TJ protein claudin-7. In conclusion, trypsin-2 over-expression enhanced tongue carcinoma cell invasion by various genetic and proteolytic mechanisms.
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PMID:Trypsin-2 enhances carcinoma invasion by processing tight junctions and activating ProMT1-MMP. 2290 50

Respiratory epithelial adenomatoid hamartoma (REAH) is regarded as a rare tumor of the nasal cavity. The mechanisms driving the development of REAH are unknown, and its nature as a benign tumor, hamartoma, or reactive inflammatory process is still open to discussion. A total of 150 consecutive patients operated on for nasal polyposis (NP) were extensively checked for the diagnosis of REAH. The profile of REAH occurring in association with NP was compared with solitary REAH in a series of 19 cases. The possible role of tryptase-producing mast cells (MC) and of metalloproteinases MMP2 and MMP9 in REAH development was investigated by immunohistochemistry. REAH lesions were identified in 35% of patients who had surgery for NP (53/150). The distribution of the lesions suggested that REAH originated in the olfactory cleft. Solitary REAH occurred about 20 times less frequently than those observed in an NP context but shared the same microscopic characteristics. Tryptase-producing MCs were recruited at high density in REAH (135/10 hpf), compared with inflammatory polyps (45/10 hpf; P<0.00005) and hypertrophied turbinates (51/10 hpf; P<0.0005). REAH also showed constant MMP9 expression and to a lesser degree MMP2 expression in epithelial cells. If solitary REAH is a relatively rare lesion, we demonstrated that an exhaustive sampling allows the detection of a high proportion of NP-associated REAH, sharing the same clinical and histologic characteristics with solitary REAH. Tryptase-producing MCs, possibly in association with MMP expression, may play a central role in REAH formation.
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PMID:Respiratory epithelial adenomatoid hamartoma: a poorly recognized entity with mast cell recruitment and frequently associated with nasal polyposis. 2412 Nov 71

Changes in barrier function of the gastrointestinal tract are thought to contribute to the inflammatory bowel diseases Crohn's disease and ulcerative colitis. Previous work in our lab demonstrated that apical exposure of intestinal epithelial cell lines to serine proteases results in an increase in transepithelial electrical resistance (TER). However, the underlying mechanisms governing this response are unclear. We aimed to determine the requirement for proteolytic activity, epidermal growth factor receptor (EGFR) activation, and downstream intracellular signaling in initiating and maintaining enhanced barrier function following protease treatment using a canine intestinal epithelial cell line (SCBN). We also examined the role of phosphorylation of myosin regulatory light chain on the serine protease-induced increase in TER through. It was found that proteolytic activity of the serine proteases trypsin and matriptase is required to initiate and maintain the protease-mediated increase in TER. We also show that MMP-independent EGFR activation is essential to the sustained phase of the protease response, and that Src kinases may mediate EGFR transactivation. PI3-K and ERK1/2 signaling were important in reaching a maximal increase in TER following protease stimulation; however, their upstream activators are yet to be determined. CK2 inhibition prevented the increase in TER induced by serine proteases. The bradykinin B(2) receptor was not involved in the change in TER in response to serine proteases, and no change in phosphorylation of MLC was observed after trypsin or matriptase treatment. Taken together, our data show a requirement for ongoing proteolytic activity, EGFR transactivation, as well as downstream PI3-K, ERK1/2, and CK2 signaling in protease-mediated barrier enhancement of intestinal epithelial cells. The pathways mediating enhanced barrier function by proteases may be novel therapeutic targets for intestinal disorders characterized by disrupted epithelial barrier function.
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PMID:Signaling pathways induced by serine proteases to increase intestinal epithelial barrier function. 2867 92


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