Studies on tissue transglutaminases: interaction of erythrocyte type-2 transglutaminase with GTP.
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ABSTRACT:
SUBMITTER: Bergamini CM
PROVIDER: S-EPMC1132477 | biostudies-other | 1993 Apr
REPOSITORIES: biostudies-other
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Project description:Calcium-dependent transglutaminases (TGs) are a family of enzymes that catalyze protein cross-linking and/or attachment of primary amines in a variety of organisms. Mammalian TGs are implicated in multiple biological events such as skin formation, blood coagulation, and extracellular matrix stabilization. Medaka (Oryzias latipes) has been used as a model fish to investigate the physiological functions of mammalian proteins. By analysis of the medaka genome, we found seven TGs orthologues, some of which apparently corresponded to the mammalian TG isozymes, TG1, TG2, and Factor XIII. All orthologues had preserved amino acid residues essential for enzymatic activity in their deduced primary structures. In this study, we analyzed biochemical properties of two orthologues (OlTGK1 and OlTGK2) of mammalian epithelium-specific TG (TG1) that are significantly expressed at the transcriptional level. Using purified recombinant proteins for OlTGK1 and OlTGK2, we characterized their catalytic reactions. Furthermore, immunohistochemical analyses of fish sections revealed higher expression in the pancreas (OTGK1), intervertebral disk (OlTGK2) and pharyngeal teeth (OlTGK2) as well as in the skin epidermis.
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Project description:Tissue transglutaminase (TG2) is a multifunctional protein with enzymatic, GTP-ase, and scaffold properties. TG2 interacts with fibronectin (FN) through its N-terminus domain, stabilizing integrin complexes, which regulate cell adhesion to the matrix. Through this mechanism, TG2 participates in key steps involved in metastasis in ovarian and other cancers. High-throughput screening identified several small molecule inhibitors (SMI) for the TG2/FN complex. Rational medicinal chemistry optimization of the hit compound (TG53) led to second-generation analogues (MT1-6). ELISA demonstrated that these analogues blocked TG2/FN interaction, and bio-layer interferometry (BLI) showed that the SMIs bound to TG2. The compounds also potently inhibited cancer cell adhesion to FN and decreased outside-in signaling mediated through the focal adhesion kinase. Blockade of TG2/FN interaction by the small molecules caused membrane ruffling, delaying the formation of stable focal contacts and mature adhesions points and disrupted organization of the actin cytoskeleton. In an in vivo model measuring intraperitoneal dissemination, MT4 and MT6 inhibited the adhesion of ovarian cancer cells to the peritoneum. Pretreatment with MT4 also sensitized ovarian cancer cells to paclitaxel. The data support continued optimization of the new class of SMIs that block the TG2/FN complex at the interface between cancer cells and the tumor niche.
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Project description:Tissue transglutaminase (TG2) mediates protein crosslinking through generation of ε-(γ-glutamyl) lysine isopeptide bonds and promotes cell adhesion through interaction with fibronectin (FN) and integrins. Cell adhesion to the peritoneal matrix regulated by TG2 facilitates ovarian cancer dissemination. Therefore, disruption of the TG2-FN complex by small molecules may inhibit cell adhesion and metastasis. A novel high throughput screening (HTS) assay based on AlphaLISA™ technology was developed to measure the formation of a complex between His-TG2 and the biotinylated FN fragment that binds TG2 and to discover small molecules that inhibit this protein-protein interaction. Several hits were identified from 10,000 compounds screened. The top candidates selected based on >70% inhibition of the TG2/FN complex formation were confirmed by using ELISA and bioassays measuring cell adhesion, migration, invasion, and proliferation. In conclusion, the AlphaLISA bead format assay measuring the TG2-FN interaction is robust and suitable for HTS of small molecules. One compound identified from the screen (TG53) potently inhibited ovarian cancer cell adhesion to FN, cell migration, and invasion and could be further developed as a potential inhibitor for ovarian cancer dissemination.
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Project description:PurposeAggressive pancreatic cancer is commonly associated with a dense desmoplastic stroma, which forms a protective niche for cancer cells. The objective of the study was to determine the functions of tissue transglutaminase (TG2), a Ca(2+)-dependent enzyme that cross-links proteins through transamidation and is abundantly expressed by pancreatic cancer cells in the pancreatic stroma.Experimental designOrthotopic pancreatic xenografts and coculture systems tested the mechanisms by which the enzyme modulates tumor-stroma interactions.ResultsWe show that TG2 secreted by cancer cells effectively molds the stroma by cross-linking collagen, which, in turn, activates fibroblasts and stimulates their proliferation. The stiff fibrotic stromal reaction conveys mechanical cues to cancer cells, leading to activation of the YAP/TAZ transcription factors, promoting cell proliferation and tumor growth. Stable knockdown of TG2 in pancreatic cancer cells leads to decreased size of pancreatic xenografts.ConclusionsTaken together, our results demonstrate that TG2 secreted in the tumor microenvironment orchestrates the cross-talk between cancer cells and stroma fundamentally affecting tumor growth. Our study supports TG2 inhibition in the pancreatic stroma as a novel strategy to block pancreatic cancer progression.
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Project description:Transglutaminases (TGs) are crosslinking enzymes best known for their vascular remodeling in hypertension. They require calcium to form an isopeptide bond, connecting a glutamine to a protein bound lysine residue or a free amine donor such as norepinephrine (NE) or serotonin (5-HT). We discovered that perivascular adipose tissue (PVAT) contains significant amounts of these amines, making PVAT an ideal model to test interactions of amines and TGs. We hypothesized that transglutaminases are active in PVAT. Real time RT-PCR determined that Sprague Dawley rat aortic, superior mesenteric artery (SMA), and mesenteric resistance vessel (MR) PVATs express TG2 and blood coagulation Factor-XIII (FXIII) mRNA. Consistent with this, immunohistochemical analyses support that these PVATs all express TG2 and FXIII protein. The activity of TG2 and FXIII was investigated in tissue sections using substrate peptides that label active TGs when in a catalyzing calcium solution. Both TG2 and FXIII were active in rat aortic PVAT, SMAPVAT, and MRPVAT. Western blot analysis determined that the known TG inhibitor cystamine reduced incorporation of experimentally added amine donor 5-(biotinamido)pentylamine (BAP) into MRPVAT. Finally, experimentally added NE competitively inhibited incorporation of BAP into MRPVAT adipocytes. Further studies to determine the identity of amidated proteins will give insight into how these enzymes contribute to functions of PVAT and, ultimately, blood pressure.
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Project description:Allosteric regulation is a fundamental mechanism of biological control. Here, we investigated the allosteric mechanism by which GTP inhibits cross-linking activity of transglutaminase 2 (TG2), a multifunctional protein, with postulated roles in receptor signaling, extracellular matrix assembly, and apoptosis. Our findings indicate that at least two components are involved in functionally coupling the allosteric site and active center of TG2, namely (i) GTP binding to mask a conformationally destabilizing switch residue, Arg-579, and to facilitate interdomain interactions that promote adoption of a compact, catalytically inactive conformation and (ii) stabilization of the inactive conformation by an uncommon H bond between a cysteine (Cys-277, an active center residue) and a tyrosine (Tyr-516, a residue located on a loop of the beta-barrel 1 domain that harbors the GTP-binding site). Although not essential for GTP-mediated inhibition of cross-linking, this H bond enhances the rate of formation of the inactive conformer.
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