Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.108 (TAT)
 2,389 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of vitronectin-thrombin-antithrombin III (VN.TAT) complex with endothelial cells (EC) was investigated. Binding was specific and time- and concentration-dependent. Kinetics revealed an apparent dissociation constant of 16 nM and 1.7 x 10(5) binding sites/endothelial cell. The binding determinant of the ternary complex was located on the VN moiety. Since the association of VN to TAT adds its specific properties to the VN.TAT complex, the involvement of the heparin binding domain and the cell attachment site of VN was investigated. Neither addition of RGD peptide nor blocking of the vitronectin receptor with a monoclonal antibody interfered with VN.TAT binding to EC. Addition of heparin, a VN-derived peptide comprising two heparin binding consensus sequences or a monoclonal antibody directed against the heparin binding domain on VN, completely inhibited VN.TAT binding to EC. These results indicate that the interaction is mediated through the heparin binding domain of VN. Digestion of heparan sulfate proteoglycans resulted in a decrease of VN.TAT binding to EC, indicating the involvement of heparin-like structures on the EC surface. Our findings point to an unrecognized mechanism by which VN may act as scavenger in order to enhance the clearance of end products of the clotting system via binding of the ternary VN.TAT complex to the luminal surface of EC.
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PMID:Binding of vitronectin-thrombin-antithrombin III complex to human endothelial cells is mediated by the heparin binding site of vitronectin. 137 Aug 15

A protein that binds to an oligonucleotide triplex, (dT)34.(dA)34.(dT)34 (TAT triplex), was purified from HeLa cells by a combination of conventional column chromatography and triplex DNA affinity chromatography. The protein has an apparent molecular mass of 55 kDa on sodium dodecyl sulfate/polyacrylamide gels. Although the protein has an affinity for AT duplex and TAT triplex, a higher affinity for TAT triplex was demonstrated by comparing the elution profiles from an AT duplex and a TAT triplex affinity column. The protein has a moderate affinity for poly(dA-dG).poly(dT-dC) and a very low affinity for single-strand (dA)34 or (dT)34 and various sources of duplex DNA including poly(dA-dT).poly(dA-dT). The possible biological function of this triplex DNA-binding protein is discussed.
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PMID:A triplex DNA-binding protein from human cells: purification and characterization. 196 9

We have recently demonstrated that many cancer cell lines produce a novel trypsinogen isoenzyme called tumor-associated trypsinogen 2 (TAT-2). It was found during a search of the target protease for tumor-associated trypsin inhibitor (TATI). We now show that degradation of subendothelial cell extracellular matrix (ECM) by four different cell lines (COLO 205 colon carcinoma, K-562 erythroleukemia, CAPAN-1 pancreatic carcinoma, and HT 1080 fibrosarcoma) can be partially inhibited by TATI or neutralizing trypsin antibodies. When cells were cultured in serum-free medium on ECM, TATI and trypsin antibodies inhibited the release of immunoreactive fibronectin fragments from ECM by 47-54 and 40%, respectively. Degradation of isotopically labeled ([3H]serine, [3H]proline, and [35S]sulfate) ECM was also significantly prevented by TATI. At its maximum, it exerted a 57% inhibition on the degradation of [3H]serine-labeled ECM. Plasminogen added exogenously to the culture medium further potentiated the proteolysis of ECM. Interestingly, addition of enteropeptidase, an activator of TAT-2, also enhanced cell-mediated proteolysis as assessed by degradation of purified fibronectin coated onto the surface of wells. Immunoblot analysis showed that enteropeptidase-mediated proteolysis generated a pattern of fibronectin fragments similar to that obtained by digestion of purified fibronectin by TAT-2. These results demonstrate the existence of a proteolytic system in tumor cells which is dependent on the activation of TAT-2. We suggest that TAT-2 is involved in a protease cascade-stimulating tumor cell invasion and degradation of extracellular matrix.
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PMID:Tumor-associated trypsin participates in cancer cell-mediated degradation of extracellular matrix. 200 30

A porcine strain of Pasteurella multocida (serotype D:3) produced a toxin causing turbinate atrophy (TA) in pigs. The toxin (TAT), processed on a high performance liquid chromatography size exclusion column, eluted as a single peak (molecular weight of about 160,000) containing trace amounts of endotoxin (lipopolysaccharide, LPS; protein:LPS, 85:1). The eluted fraction migrated on sodium dodecyl sulfate polyacrylamide gels as a single band. It could be prevented from dissociating into two prominent polypeptides by addition of a protease inhibitor. A single dose (2.0 to 79.0 micrograms/kg) of TAT given to pigs intravenously was lethal. Doses from 0.02 to 1.0 microgram/kg caused transient clinical signs of porcine systemic toxicosis with reduced appetite, generalized weakness, depression, lethargy, weight loss, and in some instances, death. Intradermal doses of TAT (greater than or equal to 0.1 microgram/site) produced hemorrhagic areas within four hours. Systemically, TAT causes bilateral TA, lymphopenia, liver dysfunctions, and possible renal impairment. Affinity of TAT for cells of epithelial origin was demonstrated in mice given 125I-TAT. In vitro, TAT stimulated DNA and protein syntheses of peripheral blood lymphocytes and suppressed syntheses in turbinate and kidney cell cultures without being cytolytic. Biological effects of TAT were eliminated by exposure to either heat, trypsin or anti-TAT antibody.
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PMID:Host response to Pasteurella multocida turbinate atrophy toxin in swine. 230 67

In search of the target protease for the tumor-associated trypsin inhibitor TATI we recently identified a trypsin-like protease in cyst fluid of mucinous ovarian tumors (Stenman, U.-H., Koivunen, E., and Vuento, M. (1988) Biol. Chem. Hoppe-Seyler 369, 9-14). We have now purified this protease and demonstrate that it represents isoenzyme forms of trypsinogen, here called tumor-associated trypsin(ogen)s (TAT). The purification procedure comprised batchwise anion exchange chromatography, immunoaffinity chromatography with antibodies to trypsin, and separation of the two isoenzymes by reverse phase chromatography. In sodium dodecyl sulfate (SDS)-gel electrophoresis, the TAT-1 and TAT-2 isoenzymes have relative molecular weights (Mr) of 25,000 and 28,000, respectively, TAT-2 being the major component. The amino-terminal amino acid sequences correspond to those of pancreatic trypsinogen-1 and -2, respectively, and activation of the zymogens results in cleavage of a NH2-terminal activation peptide of 8 residues characteristic of trypsinogen. Isoelectric focusing in the presence of urea gives pI values of about 5 and 4 for TAT-1 and -2, respectively. The substrate specificities of the two TAT isoenzymes are very similar to, but not identical with, those of trypsin-1 and trypsin-2, respectively, suggesting slight differences in substrate binding site. TAT was found to be an efficient activator of pro-urokinase. Hence, TAT could take part in the protease cascade associated with tumor invasion.
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PMID:Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase. 250 10

Mounting experimental evidence suggests that the TAT protein, released from human immunodeficiency virus-1 (HIV-1)-infected inflammatory cells, may genetically reprogram targeted cells within a localized environment to develop highly vascularized tumors of mesenchymal origin. The fibroblast growth factor (FGF) family of polypeptides has gained general acceptance as initiators of angiogenesis and functions as potent mitogens for mesoderm-derived cells. To evaluate a potential biological relationship between TAT and acidic FGF (FGF-1), primary murine embryonic fibroblasts either were transfected with the viral transactivator or were transduced (retrovirally mediated) with a secreted, chimeric form of the human polypeptide growth factor, human stomach tumor/Kaposi's sarcoma (hst/KS)FGF-1. Reverse transcriptase-polymerase chain reaction, Western blotting, in situ immunohistochemical, heparin affinity, DNA synthesis, and transient transfection techniques were used to confirm expression, localization, and functionality of the transgenes. Both transfected and transduced cells constitutively expressing either TAT or (hst/KS)FGF-1 adopted a transformed phenotype, maintained aggressive growth behavior, and demonstrated both induction of FGF-specific phosphotyrosyl proteins and nuclear association of FGF-1 and FGF-1 receptor. Increased levels of endogenous, murine FGF-1 mRNA (reverse transcriptase-polymerase chain reaction) and protein (immunoblot analysis) were apparent in both (hst/KS)FGF-1- and TAT-transformed cells. Medium conditioned by (hst/KS)FGF-1-transduced cells contained steady-state levels of biologically active FGF-1 which exhibited a representative molecular weight. Limited sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the conditioned medium from TAT-transformed cells demonstrated the appearance of FGF-1 as latent, high molecular weight complexes requiring reducing agents to activate full biological activity. Collectively, these results suggest that TAT induces the expression and secretion of FGF-1, which may be potentially relevant to the pathophysiological development of AIDS-Kaposi's sarcoma.
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PMID:The HIV-1 TAT protein induces the expression and extracellular appearance of acidic fibroblast growth factor. 754 39

Vascular heparan sulfate proteoglycan (vHSPG) is an important functional component of the microvasculature. Previous studies have demonstrated autoimmunity to vHSPG in systemic lupus erythematosus (SLE). In the current studies, we further investigated the immunospecificity of anti-vHSPG antibodies in SLE sera by enzyme-linked immunoassay (ELISA). In direct binding assays, SLE sera contained IgG antibodies reactive with native vHSPG and with heparan sulfate (HS) glycosaminoglycan in significantly higher titers than controls. Employing purified SLE IgG in liquid-phase competitive immunoinhibition ELISAs, SLE IgG anti-HS antibodies cross-reacted with heparin and DNA, but not with other glycosaminoglycans or anionic phospholipid antigens. Immunochemical studies demonstrated that the immunodominant site on HS recognized by SLE IgG contained 2-O-sulfated uronic acid. Removal of N-sulfated and 6-O-sulfated residues primarily on N-acetyl-glucosamine had no effect on antigenicity, further demonstrating that nonspecific charge interactions which are the result of sulfation do not solely account for the antigenicity of HS. SLE IgG from patients with active SLE was further affinity purified on DNA-cellulose and HS-Sepharose columns for immunospecificity studies. After affinity purification of both anti-DNA and anti-HS antibodies, significant enhancement of direct binding reactivity with HS was noted. In addition, anti-DNA and anti-HS IgG antibody reacted with the cell surface of endothelial cells by a cellular ELISA (CELISA). Immunoinhibition studies of CELISA reactivity confirmed that affinity-purified SLE IgG anti-DNA anti-HS antibody were reactive with endothelial cell surface HS antigens. Furthermore, SLE IgG anti-DNA antibody reactivity with endothelial cells was not reduced by DNase treatment of the cells, but was significantly reduced by heparitinase digestion. Since HS plays an important role in the maintenance of normal anticoagulation on the endothelial cell surface by binding antithrombin III, we investigated the inhibition of heparin-accelerated thrombin-antithrombin III complex formation by SLE IgG. Purified IgG from patients with active SLE, but not from normal controls, inhibited heparin-accelerated formation of TAT complexes. These studies demonstrate the presence of IgG autoantibodies to HS in patients with SLE. Anti-HS antibodies recognize an antigenic site also present in heparin, but not other glycosaminoglycans, bind to the endothelial cell surface, and inhibit the formation of TAT complexes. SLE IgG anti-HS antibodies recognize a sulfated uronic acid epitope containing 2-O-sulfate which is important in certain functions of HS, including antithrombin III binding. Thus, anti-HS antibodies may promote a procoagulant state at the endothelial cell surface.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Autoantibodies to vascular heparan sulfate proteoglycan in systemic lupus erythematosus react with endothelial cells and inhibit the formation of thrombin-antithrombin III complexes. 829 26

Protein transduction domains (PTDs), both naturally occurring and synthetic, have been increasingly utilized to deliver biologically active agents to a variety of cell types in vitro and in vivo. We report that in addition to previously characterized arginine-rich PTDs, including TAT, lysine homopolymers were able to mediate transduction of a wide variety of cell types, as measured by flow cytometric and enzymatic assays. The efficiency of PTD-mediated transduction was influenced by the cell type tested, although polylysine homopolymers demonstrate levels of internalization that consistently exceeded those of TAT and arginine homopolymers. Transduction of arginine/lysine-rich PTDs occurred at 4 degrees C and following depletion of cellular ATP pools, albeit generally at reduced levels. Although transduction was reduced in Chinese hamster ovary mutant lines deficient in either heparan sulfate or glycosaminoglycan synthesis, uptake was restored to wild-type levels by incubating target cells with dextran sulfate. The enhancement of transduction by dextran sulfate suggests that electrostatic interactions play an important first step in the process by which PTDs and their cargo traverse the plasma membrane.
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PMID:Efficiency of protein transduction is cell type-dependent and is enhanced by dextran sulfate. 1203 49

Protein transduction domains (PTDs) are short basic peptide sequences present in many cellular and viral proteins that mediate translocation across cellular membranes. PTDs have become widely used as tools for the delivery of high Mr polypeptides, polynucleotides, or nanoparticles to cells in culture; and even the transfer of cargo molecules to the tissue of live animals has been reported. These cell-permeable peptides are functional when fused in-frame to recombinant polypeptides or when chemically coupled to their cargo. The mechanism responsible for PTD-mediated membrane translocation is controversially discussed and may vary among the various PTDs reported in the literature. Thus direct physical interaction with membrane lipids resulting in vectorial delivery to cells has been proposed for the Antennapedia (Antp) PTD, whereas uptake by the retroviral TAT (transactivator of transcription) protein PTD seems to require cell surface-expressed glycosaminoglycans. The view that PTD-mediated cellular uptake is energy-independent has been dismissed recently as an artifact resulting from fixation of cells. The data reported here agree with and further extend this work. They support the idea that certain PTDs promote cellular uptake via endocytosis and require the expression of negatively charged glycosaminoglycans on the surface of the target cells. Uptake of Antp PTD conjugates or peptide-derivatized liposomes was blocked by heparan sulfate proteoglycans, whereas TAT-mediated uptake was inhibited by both heparin and dextran sulfate. Mutant cells defective for glycosaminoglycan synthesis showed dramatically reduced Antp- or TAT-mediated transmembrane transport confirming the role of these complex polysaccharides in PTD-mediated cellular uptake. The fact that PTDs selectively interact with distinct glycosaminoglycan species has implications for therapeutic applications and may allow targeting of selective tissues that differ in their surface-expressed glycosaminoglycan patterns.
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PMID:Antennapedia and HIV transactivator of transcription (TAT) "protein transduction domains" promote endocytosis of high molecular weight cargo upon binding to cell surface glycosaminoglycans. 1283 62

The positively charged protein transduction domain of the HIV-1 TAT protein (TAT-PTD; residues 47-57 of TAT) rapidly translocates across the plasma membrane of living cells. This property is exploited for the delivery of proteins, drugs, and genes into cells. The mechanism of this translocation is, however, not yet understood. Recent theories for translocation suggest binding of the protein transduction domain (PTD) to extracellular glycosaminoglycans as a possible mechanism. We have studied the binding equilibrium between TAT-PTD and three different glycosaminoglycans with high sensitivity isothermal titration calorimetry and provide the first quantitative thermodynamic description. The polysulfonated macromolecules were found to exhibit multiple identical binding sites for TAT-PTD with only small differences between the three species as far as the thermodynamic parameters are concerned. Heparan sulfate (HS, molecular weight, 14.2 +/- 2 kDa) has 6.3 +/- 1.0 independent binding sites for TAT-PTD which are characterized by a binding constant K0 = (6.0 +/- 0.6) x 10(5) M(-1) and a reaction enthalpy deltaHpep0 = -4.6 +/- 1.0 kcal/mol at 28 degrees C. The binding affinity, deltaGpep0, is determined to equal extent by enthalpic and entropic contributions. The HS-TAT-PTD complex formation entails a positive heat capacity change of deltaCp0 = +135 cal/mol peptide, which is characteristic of a charge neutralization reaction. This is in contrast to hydrophobic binding reactions which display a large negative heat capacity change. The stoichiometry of 6-7 TAT-PTD molecules per HS corresponds to an electric charge neutralization. Light scattering data demonstrate a maximum scattering intensity at this stoichiometric ratio, the intensity of which depends on the order of mixing of the two components. The data suggest cross-linking and/or aggregation of HS-TAT-PTD complexes. Two other glycosaminoglycans, namely heparin and chondroitin sulfate B, were also studied with isothermal titration calorimetry. The thermodynamic parameters are K0 = (6.0 +/- 0.8) x 10(5) M(-1) and kcal/mol for heparin and K0 = (2.5 +/- 0.5) x 10(5) M(-1) and kcal/mol for chondroitin sulfate B at 28 degrees C. The close thermodynamic similarity of the three binding molecules also implies a close structural relationship. The ubiquitous occurrence of glycosaminoglycans on the cell surface together with their tight and rapid interaction with the TAT protein transduction domain makes complex formation a strong candidate as the primary step of protein translocation.
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PMID:Interaction of the protein transduction domain of HIV-1 TAT with heparan sulfate: binding mechanism and thermodynamic parameters. 1469 67


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