Biomedical subjects

Gillian Murphy

Publications and source records attributed to Gillian Murphy.

68 records · Page 4Linked to original sources

PubMed2002

ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors.

ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan.

ADAMTS Proteins↗

PubMed2002

Investigation of the role of Endo180/urokinase-type plasminogen activator receptor-associated protein as a collagenase 3 (matrix metalloproteinase 13) receptor.

Procollagenase 3 can be activated by interaction with and cleavage by the cell-associated membrane type 1 metalloproteinase (MT1 MMP; MMP 14). It has also been shown to bind to a specific receptor, and is subsequently internalized via the low-density lipoprotein-related receptor by osteoblast cell lines. The receptor was identified as a recycling glycoprotein of the macrophage mannose receptor family, Endo180. In order to ascertain whether there is a relationship between Endo180 binding and procollagenase 3 activation, we have compared procollagenase 3 activation by an HT1080 fibrosarcoma cell line overexpressing MT1 MMP, without and with overexpression of Endo180. No difference in procollagenase 3 activation was observed, and neither was the enzyme bound to the cells or internalized. In contrast, the osteoblast cell lines, MG63 and UMR-106, both bound and internalized procollagenase 3. However, immunolocalization studies showed that the Endo180 abundantly expressed by these cells did not co-localize with the procollagenase 3. In further biochemical studies we confirmed that procollagenase 3 did not bind to Endo180, using both ligand- blotting and immunoprecipitation techniques. We conclude that Endo180 is unlikely to be a receptor for collagenase 3 in relation to either its activation or cell binding and internalization, and that other interaction partners must be sought.

Blotting, Western↗

PubMed2002

Tissue inhibitor of metalloproteinase-3 induces a Fas-associated death domain-dependent type II apoptotic pathway.

Tissue inhibitors of metalloproteinases (TIMPs) are important regulators of matrix metalloproteinase (MMP) and adamalysin metalloproteinase activity. We previously reported that overexpression of TIMP-3 inhibits MMPs and induces apoptotic cell death in a variety of cell types and demonstrated that apoptosis is mediated through the N terminus of TIMP-3, which harbors the MMP inhibitory domain. However, little is known about the mechanisms underlying TIMP-3-induced apoptosis. Here we demonstrate that overexpression of TIMP-3 induced activation of initiator caspase-8 and -9 and promoted caspase-mediated cleavage of the death substrates poly(ADP-ribose) polymerase and focal adhesion kinase. Furthermore, TIMP-3 induced mitochondrial activation as demonstrated by loss of mitochondrial membrane potential and release of cytochrome c. Intervention studies demonstrated that overexpression of Bcl-2, the anti-apoptotic mitochondrial membrane protein, or CrmA, a viral serpin inhibitor of caspase-8, completely inhibited TIMP-3-induced apoptosis. Furthermore, a dominant-negative Fas-associated death domain mutant inhibited TIMP-3-induced death substrate cleavage and apoptotic death. Taken together, these results indicate that TIMP-3 overexpression induces a type II apoptotic pathway initiated via a Fas-associated death domain-dependent mechanism.

Adenoviridae↗

PubMed2002

Family-based investigation of the C677T polymorphism of the methylenetetrahydrofolate reductase gene in ischaemic heart disease.

BACKGROUND: Elevated homocysteine is associated with ischaemic heart disease (IHD). The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene results in reduced MTHFR enzyme activity and reduced methylation of homocysteine to methionine resulting in mild hyperhomocysteinaemia. Case-control association studies of the role of the C677T MTHFR polymorphism in IHD have produced conflicting results. We therefore used newly described family-based association tests to investigate the role of this polymorphism in IHD, in a well-defined population. METHODS: A total of 352 individuals from 129 families (discordant sibships and parent-child trios) were recruited. Linkage disequilibrium between the polymorphism and IHD was tested for using the combined transmission disequilibrium test (TDT)/sib-TDT and pedigree disequilibrium test (PDT). Homocysteine levels were measured. RESULTS: Both the TDT/sib-TDT and PDT analyses found a significantly reduced transmission of the T allele to affected individuals (P=0.016 and P=0.021). There was no significant difference in homocysteine levels between affected and unaffected siblings. TT homozygotes had mean homocysteine levels significantly higher than those of TC heterozygotes (P<0.001) and CC homozygotes (P<0.001). CONCLUSIONS: These data suggest that in contrast to the conventional hypothesis the T allele may be protective against IHD, independent of homocysteine levels.

Adult↗

PubMed2002

Sorsby's fundus dystrophy tissue inhibitor of metalloproteinases-3 (TIMP-3) mutants have unimpaired matrix metalloproteinase inhibitory activities, but affect cell adhesion to the extracellular matrix.

The TIMP family of matrix metalloproteinase inhibitors consists of four members, of which TIMP-1, -2 and -4 are secreted, freely diffusible proteins, whereas TIMP-3 is ECM-associated. Mutations in the TIMP3 gene have been linked to Sorsby's fundus dystrophy (SFD), an autosomal dominant inherited retinal degenerative disease that leads to blindness. The SFD mutations characterized result in introduction of an unpaired cysteine residue in the C-terminal domain of TIMP-3. We have expressed four SFD mutant TIMP-3 proteins in baby hamster kidney (BHK) cells and evaluated their characteristics alongside wild-type TIMP-3. Analysis of the mutant proteins (Ser156Cys, Gly167Cys, Tyr168Cys and Ser181Cys) by SDS-PAGE and reverse zymography revealed that each of the mutants retained gelatinase A and gelatinase B inhibitory activity, and were localized to the ECM. Association rate constants for Ser156Cys TIMP-3 with gelatinase-A, gelatinase-B, stromelysin-1 and collagenase-3 were only moderately reduced compared to wild-type TIMP-3. However, all of the mutants displayed aberrant protein-protein interactions, resulting in the presence of additional proteins or complexes in ECM preparations. Two of the mutants (Ser156Cys and Ser181Cys) showed a marked propensity to form multiple higher molecular-weight complexes that retained TIMP activity on reverse zymography. Expression of the SFD mutant TIMP-3 (and to a lesser extent, wild-type TIMP-3) proteins in BHK cells conferred increased cell adhesiveness to the ECM. Our findings indicate that the pathogenesis of Sorsby's fundus dystrophy cannot be attributed to a failure to localize SFD TIMP-3 proteins to the ECM or defects in MMP inhibition, but may involve the formation of aberrant TIMP-3-containing protein complexes and altered cell adhesion.

Actin Cytoskeleton↗

PubMed2002

Differential expression of matrix metalloproteinases during impaired wound healing of the diabetes mouse.

A number of studies have shown elevated matrix metalloproteinase expression in chronic wound fluid compared to an acute wound; however, little has been done to characterize animal models in a similar manner and thus determine their usefulness. The diabetes mouse is an animal model of type II diabetes that shows impaired dermal wound healing and has been proposed as a model of human impaired wound healing. In this study we have determined the mRNA and protein expression profiles of matrix metalloproteinases 2, 3, and 9 during the first 10 d of dermal healing for the diabetes mouse and its normally healing littermate. Additionally, human wound fluid from diabetic chronic wounds and acute surgical wounds were studied to enable a comparison of the model to the human condition. We show that during the early stages of wound healing the diabetes mouse possesses significantly reduced protein levels of pro-matrix metalloproteinases 2 and 9 within the wound tissue and active matrix metalloproteinase 3 within the fluid. Pro-matrix metalloproteinase 3 levels are also significantly reduced in the diabetes mouse during the later stages of healing. These differences may be contributing to the impaired healing of the diabetes mouse; however, they differ from the human data presented here, which show elevated matrix metalloproteinase 2 and reduced matrix metalloproteinase 9 in human diabetic chronic wound fluid compared to acute wound fluid. Therefore, although clearly showing the importance of appropriate matrix metalloproteinase regulation for normal acute wound healing to occur, the diabetes mouse may not be an ideal model for study of matrix metalloproteinase involvement in human chronic wound healing.

Animals↗

PubMed2002

Mapping and characterization of the functional epitopes of tissue inhibitor of metalloproteinases (TIMP)-3 using TIMP-1 as the scaffold: a new frontier in TIMP engineering.

Tumor necrosis factor-alpha (TNF-alpha) converting enzyme (TACE/ADAM-17) is responsible for the release of TNF-alpha, a potent proinflammatory cytokine associated with many chronic debilitating diseases such as rheumatoid arthritis. Among the four variants of mammalian tissue inhibitor of metalloproteinases (TIMP-1 to -4), TACE is specifically inhibited by TIMP-3. We set out to delineate the basis for this specificity by examining the solvent accessibility of every epitope on the surface of a model of the truncated N-terminal domain form of TIMP-3 (N-TIMP-3) in a hypothetical complex with the crystal structure of TACE. The epitopes suspected of interacting with TACE were systematically transplanted onto N-TIMP-1. We succeeded in transforming N-TIMP-1 into an active inhibitor for TACE (K(i)(app) 15 nM) with the incorporation of Ser4, Leu67, Arg84, and the TIMP-3 AB-loop. The combined effects of these epitopes are additive. Unexpectedly, introduction of "super-N-TIMP-3" epitopes, defined in our previous work, only impaired the affinity of N-TIMP-1 for TACE. Our mutagenesis results indicate that TIMP-3-TACE interaction is a delicate process that requires highly refined surface topography and flexibility from both parties. Most importantly, our findings confirm that the individual characteristics of TIMP could be transplanted from one variant to another.

ADAM Proteins↗

PubMed2002

Total levels of tissue inhibitor of metalloproteinases 1 in plasma yield high diagnostic sensitivity and specificity in patients with colon cancer.

PURPOSE: The purpose of this study was to measure total levels of tissue inhibitor of metalloproteinases (TIMP-1) by ELISA in plasma from blood donors, patients with inflammatory bowel disease (IBD), and patients with cancer and to correlate the results to patient diagnosis. EXPERIMENTAL DESIGN: Total TIMP-1 plasma levels were measured by ELISA in blood samples from two different blood donor populations from IBD patients, and preoperative samples from patients with primary colon cancer (CC), rectal cancer (RC), or breast cancer. RESULTS: There were no significant differences in plasma TIMP-1 levels between healthy donors and IBD or breast cancer patients, whereas patients with CC or RC had significantly elevated TIMP-1 levels. Total TIMP-1 levels identified patients with CC with a sensitivity of 63% at 98% specificity, patients with early CC (Dukes' A+B) with a sensitivity of 56% at 98% specificity, and patients with right-sided CC with a sensitivity of 72% at 98% specificity. Combining carcinoembryonic antigen and TIMP-1 measurements increased the sensitivities obtained from TIMP-1 measurements alone. CONCLUSIONS: TIMP-1 was significantly elevated in plasma from CC and RC patients, including those with early-stage disease. Sensitivity and specificity were both sufficiently high to consider TIMP-1 as a marker for the early identification of CC patients, in particular, those with right-sided CC.

Adolescent↗

PubMed2002

Measurement of the noncomplexed free fraction of tissue inhibitor of metalloproteinases 1 in plasma by immunoassay.

BACKGROUND: We previously found differences in total concentrations of tissue inhibitor of metalloproteinases 1 (TIMP-1) in plasma from donors and cancer patients. Because TIMP-1 can exist in more than one molecular form, a new immunoassay to specifically detect free TIMP-1 was developed and concentrations were determined in plasma from healthy donors and colorectal cancer (CRC) patients. METHODS: We established and validated an immunoassay for the specific measurement of free TIMP-1 that uses a polyclonal anti-TIMP-1 antibody for capture and a monoclonal anti-TIMP-1 antibody that binds only free TIMP-1 for detection of antigen. Plasma samples from healthy donors and CRC patients were assayed for free TIMP-1. Total TIMP-1 was measured by our previously published assay. RESULTS: The mean (SD) concentrations of free TIMP-1 were similar in citrate [55.5 (11.5) microg/L] and EDTA plasma [58.9 (13.3) microg/L] from 76 donors (r(2) = 0.82). In 154 donors, the ratio of free TIMP-1 [mean (SD), 64.5 (18.0) microg/L] to total TIMP-1 [83.8 (19.8) microg/L] in EDTA plasma was 0.77. Plasma concentrations of free and total TIMP-1 correlated significantly to age (free, r(2) = 0.19; total, r(2) = 0.27; P <0.0001), increasing 50% over an age span of 45 years. Free and total TIMP-1 were significantly increased in CRC patients (P <0.0001), whereas the ratio of free to total TIMP-1 (mean, 0.58) was significantly lower than in donors. CONCLUSIONS: Most of the TIMP-1 in donor plasma is present in its free form, and free TIMP-1 increases with age. Free and total TIMP-1 are increased in CRC patient plasma, but the ratio of free to total TIMP-1 is significantly lower in these patients than in donors.

Adult↗

PubMed2002

Metalloproteases cleave the urokinase-type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in the intact soluble receptor.

Proteolytic cleavage of the urokinase plasminogen activator receptor (uPA(R)) prevents the binding of uPA and vitronectin while generating biologically active uPAR fragments. We have recently shown that matrix metalloproteinase-12 (MMP-12) releases cellular uPAR-antigen from stimulated human micro-vascular endothelial cells providing a novel feedback mechanism between the plasminogen activation and MMP systems. We now show that MMP-12 and other MMPs directly and efficiently cleave uPAR at the Thr86 paralal Tyr87 peptide bond located in the linker region connecting uPAR domains 1 and 2, releasing the major ligand binding domain 1 from the rest of the receptor. The possible biological importance of uPAR cleavage by MMPs is supported by the observation that also murine uPAR is cleaved by MMP-12 (at the Pro89 paralal Gln90 peptide bond), despite the limited sequence homology between the linker regions. Using an antibody raised against the human uPAR linker region we show that this region of uPAR, which contains the chemotactic SRSRY epitope, is exposed upon MMP cleavage.

Amino Acid Motifs↗

PubMed2001

Phosphorylation-dependent interactions between ADAM15 cytoplasmic domain and Src family protein-tyrosine kinases.

The adamalysins (ADAMs) are transmembrane glycoproteins involved in cell adhesion and proteolytic ectodomain processing of cytokines and adhesion molecules. Many ADAM cytoplasmic domains are proline-rich and have potential phosphorylation sites. We show here that the cytoplasmic domain of ADAM15, metargidin, can interact specifically with Src family protein-tyrosine kinases (PTKs) and the adaptor protein Grb2 in hematopoietic cells (Jurkat, THP-1, U937, and K562 cell lines). Src homology 3 domains from several Src family PTKs including Lck, Fyn, Abl, and Src associate with ADAM15 in vitro. Dephosphorylation of cell extracts resulted in decreased association of ADAM15 with Src family PTK SH3 domains, indicating that phosphorylation influences ADAM15 interactions with its binding partners. This was confirmed in vitro for Hck, Lck, and Grb2, which showed enhanced association with tyrosine-phosphorylated glutathione S-transferase-ADAM15 cytoplasmic domain compared with unphosphorylated protein. In contrast, binding of MAD2 to ADAM15 was slightly reduced by phosphorylation of the ADAM. Immunoprecipitation of ADAM15 from Jurkat cells confirmed the association with Lck in vivo, and upon PMA stimulation, the phosphorylation level of ADAM15 was increased. Cotransfection of ADAM15 and Hck showed Hck-dependent phosphorylation of ADAM15 in vivo. Hck, and to a lesser extent Lck, phosphorylated the ADAM15 cytoplasmic domain in vitro in immune complex kinase assays. Binding of ADAM15 cytoplasmic domain to Hck and Lck was also shown by Far Western analysis. In contrast to Hck, Lck activity was not required for binding to ADAM15, as shown by treatment of cells with PP1. Deletion and point mutation analysis of the ADAM15 cytoplasmic domain confirmed the importance of the proline-rich motifs for Grb2 and Lck binding and indicated the regulatory nature of Tyr(715) and Tyr(735). These data demonstrate selective, phosphorylation-dependent interactions of ADAM15 with Src family PTKs and Grb2, which highlight the potential for integration of ADAM functions and cellular signaling.

ADAM Proteins↗

PubMed2001

Expression of the type IV collagenase system during mouse kidney development and tubule segmentation.

Type IV collagenases matrix metalloproteinase-2 (MMP2) and MMP9 and their related proteins, MT1-MMP, tissue inhibitor of metalloproteinases 1 (TIMP1), TIMP2, and TIMP3, are expressed during kidney morphogenesis and nephrogenesis, but the renal ontogeny of these proteins is only partially known, and their persistence in the adult remains controversial. Their expression was analyzed from early metanephric stages to adulthood by Western blot semiquantitative analysis; laser confocal microscopy of whole-mount kidneys; and a two-step immunoperoxidase labeling procedure using specific markers of proximal tubule (megalin), ascending limb of Henle's loop (Tamm Horsfall protein), and collecting duct (Dolichos biflorus agglutinin lectin). By Western blot, all antigens were detected at day 11.5, peaked at day 16.5, and persisted in the adult at lower levels, although MMP2 was less modulated. All antigens were expressed in metanephric mesenchyme at embryonic day 11.5 and became concentrated in neural cell adhesion molecule-positive-induced mesenchymal cells at day 12.5. Only MT1-MMP and to a lesser extent MMP2 were detected in the ureter bud. At day 16.5, all antigens predominated in the cytoplasm of the proximal tubule, except TIMP1, which was mostly expressed in the ascending limb of Henle's loop and distal tubule. During tubule segmentation, components of the type IV collagenase system showed both spatial and temporal regulation. The distribution of gelatinases was not strictly superimposable to that of their natural inhibitors TIMP, especially for MMP9 and TIMP1. All components persisted in specific segments of the adult renal tubule, where MMP9, MMP2, and MT1-MMP showed an apical expression, suggesting that substrates for these enzymes should be in the tubule lumen or in the apical cell domain and not in the extracellular matrix. These results suggest that a regulated balance of gelatinase activity is required during kidney organogenesis and that gelatinases continue to play a role in adult renal tubule physiology.

Animals↗

PubMedDate not supplied

Matrix metalloproteinases in disease and repair processes in the anterior segment.

The pathogenesis of many anterior segment disorders and ocular complications following surgery are secondary to the wound healing response. The extent of clinical damage observed is closely related to the amount of scarring and tissue contraction. Matrix metalloproteinases (MMPs) are a family of enzymes that play a vital role in all stages of the wound healing process. They degrade all extracellular matrix components and also have the ability to synthesize collagen and extracellular matrix members, and are therefore important in the remodeling of a wound. Overexpression of MMPs results in excessive extracellular matrix degradation, leading to tissue destruction and loss of organ function. In the case of the anterior segment, this may mean the loss of visual function. This review focuses on the role MMPs have in the development of various anterior segment disorders. The importance of MMPs in the wound healing response and its potential modulation to manipulate the scarring response is being recognized, and current developments will be described.

Anterior Eye Segment↗

PubMedDate not supplied

Psoriasis: severity assessment in clinical practice. Conclusions from workshop discussions and a prospective multicentre survey of psoriasis severity.

Psoriasis treatment is highly individualized. Although a standardized assessment of psoriasis severity for clinical practice may be theoretically advantageous for the purposes of determining treatment, the relevance of currently available research tools in clinical practice is uncertain. Our objectives were to ascertain in workshop discussions and through a prospective survey the relevance of standard severity measures in clinical practice with regard to choice of treatment. Although there was agreement on the possible structure of an algorithm for the treatment-related definition of psoriasis severity, consensus on the cut-off levels for the PASI and %BSA that would indicate a switch in treatment mode could not be reached. The lack of agreement prompted a prospective survey of 112 patients with psoriasis from 10 countries. This survey used a formal questionnaire asking for the PASI and %BSA scores, the patient's self assessment score (VAS ranging from 0 to 10), location of the psoriatic lesions and disease phase. Severity scores from 20 patients pre-selected for inclusion in a trial of a biological agent were included for comparison. Severity scores were analysed in relation to the choice of treatment (topical or systemic, which included phototherapy and combination) suggested by the treating physician.PASI scores differed significantly between the treatment groups (topical vs systemic, p=0.009); however, there was large overlap in the range of PASI scores between the groups. The same was true for VAS scores (topical vs systemic, p=0.035). %BSA scores were not significantly different between treatment groups. There was a large overlap for both the topical and systemic treatment groups with the biologicals group for the range of both the PASI and %BSA scores. A standardized protocol for the evaluation of psoriasis severity based on established severity scores (PASI, %BSA) appears to be unrealistic in day-to-day clinical practice. In clinical practice, a host of factors must be evaluated alongside possible metric measures. This requires experience and the specialized medical education of those involved in the treatment of patients with psoriasis.

Humans↗